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Genetic analysis reveals a key role for actin in tip growth in Neurospora crassa Virag, Aleksandra 2004

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GENETIC A N A L Y S I S R E V E A L S A K E Y R O L E FOR A C T I N IN TIP G R O W T H IN Neurospora crassa by ALEKSANDRA VIRAG B.Sc, The University of Belgrade, 1996 M . S c , The University of British Columbia, 1999 A THESIS SUBMITTED IN P A R T I A L F U L F I L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Botany) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A February 2004 © Aleksandra Virag, 2004  ABSTRACT  The body of a fdamentous fungus, the mycelium, is comprised of elongated cells called hyphae. During hyphal morphogenesis the elongated shape of a hypha is formed by polarized growth. Once initiated, polarized growth is restricted to hyphal tips and branching sites. Although some aspects of polarized growth, such as structural participants, are known, many others, such as control mechanisms, remain unclear. The goal of this study is to establish the position that actin maintains in the hierarchy of events leading to polarized growth in Neurospora crassa. The position of the mutation in the previously isolated mutant ccr-100 was identified by functional complementation, and the actin gene of the mutant was cloned and sequenced. The mutation occurred in the actin gene, and the ccr-100 strain was renamed act . The act mutant had a different actin distribution, Spitzenkorper size and 1  1  behaviour, and nuclear distribution. Additional actin mutants were obtained by taking advantage of the RIP (Repeat Induced Point mutations) mechanism. The actin genes of the actin RIP mutants were cloned and sequenced, and the mutants were analyzed in the same way as the act strain. The actin RIP mutants differed from the wild type in their colony and 1  hyphal morphologies, and actin distributions. Interactions between act and seven other 1  genes involved in polarized growth were demonstrated by the presence of epistasis. Based on the obtained results and existing models of tip growth, the Calcium-Actin-Phosphoinositide (CAP) model of tip growth is proposed. Actin has a central role in this model, and is anticipated to control the tip-high C a  2+  concentration gradient.  ii  TABLE OF CONTENTS  Abstract  ii  Table of Contents  iii  List of Tables  vi  List of Figures  ix  List of Abbreviations  xxix  Acknowledgements  xxx  CHAPTER I Introduction  1  1.1 Participants of the Hyphal Tip Growth Process  1  1.1.1  Actin  2  1.1.2  Plasma Membrane, Cell Wall and the Spitzenkorper  6  1.1.3  Microtubules  8  1.1.4  Turgor  9  1.1.5  Tip-High Calcium Gradient  10  1.2 Models of Tip Growth and Branching  11  1.3 Control Mechanisms in the Growth Process..;  17  1.4 The Objective of This Study  22  CHAPTER II Materials and Methods  28  2.1 Strains and Their Maintenance  28  2.2 Cloning and Subcloning  29  2.3 Determining the Presence of Actin in the act Strain  30  2.4 RIP (Repeat Induced Point Mutations) of act  31  1  iii  2.5 Hyphal Staining  32  2.6 Image Capturing  33  C H A P T E R III Cloning of act and Some Characteristics of the act Mutant Strain 35 1  1  3.1 Introduction  35  3.2 Results  38  3.2.1 Cloning and Sequencing of act  38  3.2.2 Cytology of act  40  1  1  3.2.3 Loss of Polarity in act at Threshold Temperatures 1  3.3 Summary  45 46  C H A P T E R IV RIP-Generated Actin Mutants  64  4.1 Introduction  64  4.2 Results  66  4.2.1 Category A Apolar Growth Mutants, and Category B Mutants With Germination Tube Defects  68  4.2.2 Category C Mutants With Delayed Germination  69  4.2.3 Category D Early Abortion Mutants  71  4.2.4 Category E Mutants With Directional Growth Defects  71  4.2.5 Category F Mutants With Bursting Hyphae  73  4.2.6 Category G Mutants With a Colonial Phenotype  74  4.2.7 Category H Mutants With Frequently Branched Hyphae  76  4.2.8 Category I Mutants With a Reduction in the Frequency of Branching, Category J Mutants With a One-Sided Curl and Category K Mutants With Wavy Hyphal Growth  iv  78  4.2.9 Category L Mutants With Aberrant Growth at Low Temperatures  79  4.3 Summary  80  C H A P T E R V Gene Interactions  130  5.1 Introduction  130  5.2 Results  131  5.2.1 The col-16; act Double Mutant  132  5.2.2 The fr; act Double Mutant  133  5.2.3 The gran act Double Mutant  134  5.2.4 The col-15; act Double Mutant  135  5.2.5 The col-8; act Double Mutant  135  5.2.6 The act ; spco-4 Double Mutant  136  5.2.7 The mdact Double Mutant  137  1  1  1  1  1  1  1  5.3 Summary  138  C H A P T E R V I Discussion  169  6.1 Conclusions Based on act Characteristics 1  6.1.1 The Calcium-Actin-Phosphoinositide (CAP) Model  169 170  6.2 Conclusion Based on the Actin RIP Mutant Characteristics  178  6.3 Conclusion Based on act Double Mutant Characteristics  188  6.4 Future Work  189  1  BIBLIOGRAPHY  195  v  LIST O F T A B L E S  Table 1.1. Some members of signaling pathways in N. crassa.  Page 25.  Table 2.1. List of strains from the FGSC used in this study, including morphological mutants used to generate double mutants with act .  Page 34.  1  Table 3.1. Amount of actin in bands from Fig. 3.4 expressed through the peak area measured using the Alphalmagerl220 ver.4.0 system. The lanes correspond to the lanes in Fig. 3.4. The amount of total protein is the amount of protein in the cytoplasmic fraction that is loaded in the appropriate lane.  Page 50.  Table 4.1. Phenotype of germinated ascospores, number of germinated ascospores and viability percentage of ascospores from crosses between wild type and nine transformants. The transformants contain at least one extra copy of the wild type actin gene. Tr = transformant. U C = transforming plasmid was circular (uncut). C = transforming plasmid was linear (cut).  Page 83.  Table 4.2. Mating type, hygromycin resistance and number of actin copies in actin RIP mutants. Actin RIP mutant names: e.g. 4.2C, 4 = serial number of mutant progeny from the RIP-generating cross, 2 = number of transformant used as one of the parents in the R e generating cross, C = in generating the transformant that was later used as one of the parents in the RIP-generating cross, a linear plasmid was used. U C = in generating the transformant that was later used as one of the parents in the RIP-generating cross, a circular plasmid was used. ? = indicates that progeny could not be scored with confidence. (+#) = indicates number of octads for which the phenotype could not be clearly scored).  vi  Pages 84-85.  Table 4.3. Actin RIP mutants grouped in categories based on their phenotypes. Mutant name abbreviations: e.g. 1.4UC, 1 = serial number of mutant progeny from the RIP-generating cross, 4 = number of transformant used as one of the parents in the RIP-generating cross, U C (uncut plasmid) = in generating the transformant that was later used as one of the parents in the RIP-generating cross, a circular plasmid was used. C (cut plasmid) = in generating the transformant that was later used as one of the parents in the RIP-generating cross, a linear plasmid was used. Name in brackets indicates that the actin gene of the mutant has been sequenced and named.  Page 86.  Table 4.4. Mutations in the actin alleles of the actin gene and corresponding amino-acid changes in the actin protein. The nucleotides are numbered according to the corresponding nucleotide number in the genomic actin D N A sequence from the N C B I web site, against which the obtained mutant actin sequence was compared. S = silent change, NS = non-silent change, N C = non-coding region, C = coding region, I = intron, E = exon, PS = Post-stop. Mutant act is added for comparison.  Pages 87-88.  1  Table 4.5. Pattern of actin distribution at the hyphal tip of actin RIP mutants and controls. The range of patterns in all the actin RIP mutant strains was higher than in the wild type strain.  Page 89.  Table 4.6. Predicted phenotypes of actin mutants with defects in different stages of polarized growth. Green font = observed phenotypes. Red font = phenotypes that were not observed. Page 90. Table 5.1 Actin distribution in hyphal tips of morphological mutants that affect tip growth and branching, double mutants in which one mutated gene is act , and controls. Two parental 1  strains,^- and col-15, are not included, because of problems with sample preparation. Bold  vii  blue font indicates the most common category of actin distribution in each mutant. A l l the parental strains and the double mutants showed more variation in the number of represented distribution categories than wild type. The shift was most radical in the double mutants, with the maximum number of categories in fr. A l l the double mutants showed more similarity to the act parent than the non-act parent, both in the range of actin distribution patterns, and 1  1  the most represented pattern. In most of the double mutants the category 3 distribution was most common, as in the act parent. The most common category of distribution in the non1  act parents was category 1, as in wild type.  Page 141.  1  Table 5.2. Colony, hyphal morphology, branching pattern and actin distribution in the double mutants. Brackets indicate partial similarity; two phenotypes connected with a "+" indicate an intermediate phenotype; "new" indicates the presence of novel characteristics. Page 142. Table 6.1. List of amino acid residues substituted in the actin RIP mutants and their possible ligands. The actin gene of mutants from categories A , B, D, I, J and K were not sequence, and are not included in this table.  Page 192.  viii  LIST OF FIGURES  Figure 1.1. Terminology used to describe position and movement of components within a hypha. The hyphal tip region is the region in which the cell wall is still expanding. The part of this region closest to the apex (tip) is the apical region, while the region immediately behind, but still in the hyphal tip region, is the subapical region. Distal refers to a position closer to the tip. Proximal refers to a position further away from the apex. Retrograde movement is in the direction away from the apex. Anterograde movement is in the direction towards the apex. Also see Fig. 6.1.  Page 26.  Figure 1.2. Signal transduction cascades regulating polarized growth in fungi, with upstream and downstream components.  Page 27.  Figure 3.1. (A) Three-day-old wild type colony. (B) Three-day-old act colony. (C) Six-day1  old act colony. Hyphal morphology of (D, E, F, G) act and (H) wild type. (D) Initial stage. 1  1  (E) Intermediate stage. (F) Conidiating stage. (G) Constant stage. A l l bars =100 u m (I) The median of lengths between branching sites in strain act on solid V M containing 10 u.g/ml 1  cytochalasin A (VM+10), and without cytochalasin A (VM). The bars represent the standard error. White columns show the results from the initial stage, light gray columns show results from the intermediate stage, and dark grey columns show the results from the constant stage. Page 51. Figure 3.2. ORFs on cosmid G17B12 from the Orbach/Sachs cosmid library of N. crassa genomic D N A sequences.  Page 52.  Figure 3.3. (A) Three-dimensional model of actin. Arrow and red colour in ribbon indicate the position of C374 at the carboxyl terminal. IA-IIB indicate the subdomains on the actin ix  molecule. The model was retrieved from Swiss-Model, a protein structure homology-server, and the program DeepView-spdv 3.7. (B) Position of actin domains in an F-actin fdament. Page 53. Figure 3.4. Actin presence in total protein extracts of wild type and act . Lanes (A-D) show 1  wild type actin. Lanes (E-G) show act actin. Amount of total protein loaded into the wells of 1  the SDS-PAGE gel were 70 pg (lanes A and E), 50 pg (lanes B and F), 30 ug (lanes C and G), and 10 pg (lanes D and H). The arrows indicate the positions of recombinant proteins from the full-range molecular weight markers (see Chapter II).  Page 54.  Figure 3.5. (A) Brightfield image, (B) apical actin distribution, and (C) nuclear distribution of a wild type hyphal tip. (D) Head-on view of the apical actin distribution in a wild type hyphal tip. Actin distribution in act : (E) subapical actin cap, (F) large spherical subapical 1  actin accumulation, (G) two subapical actin accumulations, (H) multiple small subapical actin accumulations, and (I) small apical actin accumulation. (J) Brightfield image, (K) peripherally displaced subapical actin accumulation, and (L) nuclear distribution of an act  1  hyphal tip. Arrowheads indicate the position of the extreme apex. A l l bars =10 pm. Bar in image (A) applies to images (B, C). Bar in image (E) applies to the rest of the images without bars.  Page 55.  Figure 3.6. Co-localization of actin with some nuclei in wild type. (A) Merged image of actin distribution and nuclear distribution. (B) Actin distribution. (C) Nuclear distribution. (D) A different hyphal segment of wild type with ring-like actin structures. Bar =10 pm. Colour was added using the program Photoshop 5.0.  x  Page 56.  Figure 3.7. Several act hyphal tips. (A-L) Successive images of longitudinal planes 1  0.45 u.m apart. Subapical and apical accumulations are present in hyphal tips. Bar =10 u m Page 57. Figure 3.8. FM.4-64 staining of (A) wild type and (B) act strains. The far right hypha in 1  image (B) is branching dichotomously. Arrowheads indicate less stained areas that correspond to the position of subapical actin accumulations in act . Note the smaller size 1  of the Spitzenkdrper in act . Bar =10 urn  Page 58.  1  Figure 3.9. (A-Y) Growth of two act hyphae stained with FM4-64 viewed at 15 s intervals. 1  (F) Two Spitzenkdrpers are visible at the apex before the emergence of two new branches in the hypha on the left. (O) Two Spitzenkdrpers are visible at the apex before the emergence of two new branches in the hypha on the right. Bar =19 urn.  Page 59.  Figure 3.10. Wild type hyphae stained with 8 u M FM4-64. (A-Y) Successive images collected 15s apart. (L) The Spitzenkdrper disappears. (O) The triangular arrowhead indicates the emergence of the first lateral branch. (P) The triangular arrowhead indicates the emergence of the second lateral branch. (V) The Spitzenkdrper reappears. (L-V) The growth rate is slower. Bar = 20 u m  Page 60.  Figure 3.11. Growth of wild type hyphae stained with 32 U.M FM4-64. (A-L) Images taken 5 s apart. (F) The two triangular arrowheads indicate the emergence of two new apical branches with Spitzenkdrpers. Bar = 20 um.  Page 61.  Figure 3.12. FM4-64 stained (A-E) wild type and (F-L) act strains with GFP-tagged nuclei. 1  (A) A leading hypha with abundant nuclei and two lateral branches without nuclei. Each tip has a Spitzenkdrper. (B) A leading hypha with three branches. The two longer branches have nuclei, and all tips have a Spitzenkdrper. (C, D) Leading hyphae with round and elongated xi  nuclei. (E) Primary lateral branch with a smaller diameter and less nuclei. (F) Leading hypha, (G) small dichotomous branch, (H) small lateral branch, and (I) an unbranched and branched hypha. Arrows indicate the region that often has a compartment not stained with FM4-64. (J) Hyphal tips with fewer nuclei per diameter than wild type. (K) A n early dichotomous branch. (L) Hyphal tips with nuclei closer to the tip than in wild type. Bar in image (A) = 20 pm and applies to images (A, B , J, K , L). Bar in image (C) = 10 pm, and applies to the rest of the images.  Page 62.  Figure 3.13. (A, C, E, G , I) DIC images and (B, D, F, H, I) actin distribution in (A-F) act  1  and (G-J) wild type after temperature downshift. (A, B) Initial absence of actin accumulation at hyphal tip. (C, D) Loss of polarity and tip swelling. (E, F) Regained polarity with an increase in hyphal diameter and a subapically displaced enlarged actin accumulation. (G, H) Absence of actin accumulations in early stages of apical branching. (I, J) Presence of apical actin accumulations in later stages of apical branching. Bars = 10 pm. Bar in image (A) applies to images (C-F). Bar in image (G) applies to images (I-J). Page 63. Figure 4.1. A category A isotropic germination mutant ascospore unable to polarize growth. Bar = 30 pm.  Page 91.  Figure 4.2. The category C delayed germination actin RIP mutant 3.4UC (act ). (A) A 16  three-day-old colony. (B) Hyphal morphology of a one-day-old colony. Bar =100 pm. (C) Branching pattern of a one-day-old colony. Bar = 1 pm. (D) Branching pattern of a four-dayold colony. Bar = 200 pm.  Page 92.  Figure 4.3. (A) Germination of the wild type strain after one day. (B) Germination of the category C delayed germination actin RIP mutant 1.4UC after one day, (C) after two days, xii  (D, E) after three days, (F, G) after four days. A l l bars =100 nm. Bar in image (D) applies to images (E, F, G).  Page 93.  Figure 4.4. Actin and nuclear distributions in hyphal tips of the category C actin mutant act (3.4UC) with delayed germination. (A, B) Images of the same hyphal tip. (A) One 16  apical actin accumulation in median plane. (B) Peripheral actin accumulations in peripheral plane. (C) Three actin accumulations. (D) Four apical actin accumulations. (E) Multiple actin accumulations at a hyphal tip. (F, G, H) Images of the same hyphal tip. (F) DIC image. (G) Nuclear distribution. (H) Absence of actin accumulations. Subapical co-localization of actin with some nuclei. Bar =10 pm and applies to all images.  Page 94.  Figure 4.5. FM4-64-stained hyphal tips of the category C actin mutant 1.4UC with delayed germination. (A, E , G) DIC images. (B, C, D) Time series of images of an FM4-64 stained hyphal tip illustrating bleaching of the vital stain. (B, C) The Spitzenkorper is visible at the tip. (F) A diffuse Spitzenkorper. (H) A compact Spitzenkorper. Bar =10 pm, and applies to all images.  Page 95.  Figure 4.6. Hyphal morphology of the category E actin RIP mutant 37.9UC (act ) with a 15  directional growth defect. (A) A long unbranched hypha. (B) A n unbranched hypha with enlarged segments at the tip, indicating loss of polarity. (C) A hyphal tip that temporarily lost polarity, and resumed polarized growth at more than one point. (D) Hyphae with disrupted polarity resulting in a meandering appearance, and dichotomous branching. (E) Hyphae with a less pronounced polarity problem that retain the lateral branching pattern. Bar =100 pm, and applies to all images.  Page 96.  Figure 4.7. Hyphal tips of the category E actin mutant 36.2C (act ) with a directional 14  growth defect. (A, B, C) Images of the same hyphal tip. (A) DIC image. (B) Nuclear xiii  distribution. (C) One apical actin accumulation. (D, E, F) Images of the same hyphal tip. (D) DIC image. (E) Nuclear distribution. (F) Almost no visible actin staining. (G, H , I) Images of the same irregularly swollen hyphal tip. (G) DIC image. (H) Nuclear distribution. (I) Diffuse actin staining. Bar =10 fim, and applies to all images.  Page 97.  Figure 4.8. Hyphal tips of the category E actin mutant 36.2C (act ) with a directional 14  growth defect. (A, B, C) Images of the same swollen hyphal tip. (A) DIC image. (B) Nuclear distribution. (C) One small apical actin accumulation. (D, E, F) Images of the same contorted hyphal tip. (D) DIC image. (E) Nuclear distribution. (F) Several peripheral actin plaques. (G, H, I) Images of the same swollen sections of a hypha. (G) DIC image. (H) Nuclear distribution. (I) Actin accumulations at the septae. Bar =10 u.m and applies to all images. Page 98. Figure 4.9. Eight-day-old hyphae of act (36.2C), a category E actin RIP mutant with a 14  directional growth defect. (A) A dichotomous branch and two unbranched hyphae that cannot maintain direction well. (B) A combination of dichotomous and lateral branching. Bar =100 um, and applies to both images.  Page 99.  Figure 4.10. Eight-day-old hyphae of the category E mutant act (31.9UC) with a 13  directional growth defect. (A) Long unbranched hypha. (B) Hypha with regions close to the tip that lost polarity. (C) Wavy growth of hypha and loss of polarity at the tip. (D) A lateral branch that lost polarity. (E) Wavy growth, with bead-like regions that lost polarity. Bar = 100 u.m, and applies to all images.  Page 100.  Figure 4.11. Hyphae of the category E actin mutant 33.9UC with a directional growth defect. (A, C, E; and B, D, F) Images of the same group of hyphae, respectively. (A, B) DIC images. (C, D) Nuclear distributions. (E, F) Mainly diffuse actin distribution distributed xiv  around groups of nuclei. Bar = 30 pm in image (A) applies to images (C, D). Bar = 10 pm in image (B) and applies to images (D, F).  Page 101.  Figure 4.12. Actin and nuclear distributions in hyphal tips of the category E actin.mutant 37.9UC (act ) with a directional growth defect. (A, B) Images of the same hyphal tip. (A) 15  One apical actin accumulation. (B) Nuclear distribution. (C, F) Images of the same hyphal tip. (C) DIC image. (D, E) Images of the same hyphal tip. (D) Two apical actin accumulations at the tip of a leading hypha and one apical actin accumulation in the lateral branch. (E) Nuclear distribution. (F) No prominent actin accumulations. (G, H, I) Images of the same hyphal tip. (G) DIC image. (H) Nuclear distribution. (I) Peripheral actin patches at a bulbous enlargement. Bar =10 pm and applies to all images.  Page 102.  Figure 4.13. Hyphal tips of the category E actin mutant 33.9UC with a directional growth defect. (A, B, C; D, E, F; and G, H, I) Three sets of images of three different hyphae. (A, D, G) DIC images. (B, E, H) Nuclear distributions. (C) Small apical actin accumulation. (F) Apical actin cap of peripheral plaques. (I) Two small subapical actin accumulations. Bar =10 pm, and applies to all images.  Page 103.  Figure 4.14. (A, B) Hyphae of a subcategory of mutants with a directional growth defect (category E), unable to maintain growth direction. Magnification = 100X. Figure 4.15. The category F actin RIP mutant act  11  Page 104.  (1.2C) with bursting hyphae. (A) A  three-day old colony. (B) Hyphal morphology of a one-day-old colony. Bar =100 pm. (C) Branching pattern of a two-day-old colony. Bar = 1 mm. (D) Two-day-old colony with burst hyphae. Bar = 1 mm.  Page 105.  Figure 4.16. Actin and nuclear distribution in hyphal tips of the category F actin mutant act  11  (1.2C) with bursting hyphae. (A, B, C) Images of the same hyphal tip. (A) DIC image. xv  (B) Nuclear distribution. (C) One apical actin accumulation. (D) One large apical actin accumulation. (E) Multiple small actin accumulations. (F) Three apical actin accumulations. (G) Few apical actin accumulations. (H) Hyphal tip without apical actin accumulations. A l l bars = 10 u.m. Bar in image (E) applies to images (G) and (H). Bar in image (A) applies to the rest of the images.  Page 106.  Figure 4.17. The category F actin RIP mutant act (2.5UC) with bursting hyphae. (A) A 5  three-day-old colony. (B) A n eight-day-old colony. (C) Branching pattern of a two-day-old colony. Bar = 1 mm. (D) Hyphal morphology of a two-day-old colony. Bar = 200 urn. Page 107. Figure 4.18. The category G actin RIP mutant act (7.4UC) with colonial growth. (A) A 17  three-day-old colony. (B, C) Hyphal morphology and branching pattern of a one-day-old colony. Bar =100 urn. (D) Branching pattern and colony density of a four-day-old colony. Bar = 1 mm.  Page 108.  Figure 4.19. Actin and nuclear distribution in hyphal tips of the category G actin mutant act (7.4UC) with colonial growth. (A, B , C) Images of the same hyphal tip. (A) DIC image. 17  (B) Nuclear distribution. (C) One apical actin accumulation. (D) Two apical actin accumulations. (E) Three actin accumulations at apex. (F) One subapical actin accumulation. (F) Multiple weakly stained actin accumulations. (H) Absence of actin accumulations. A l l bars = 10 urn. Bar in image (D) applies to images (D) and (E). Bar in image (A) applies to the rest of the images.  Page 109.  Figure 4.20. Actin and nuclear distribution in hyphal tips of the category G actin mutant act (1.5UC) with colonial growth. (A, B , C) Images of the same hyphal tip. (A) DIC image. 3  (B) Nuclear distribution. (C) One apical actin accumulation. (D, E) Two apical actin xvi  accumulations. (F) Multiple weakly stained actin accumulations. (G) One subapical actin accumulation. (H) Hyphal tips with diffuse actin caps. Bar = 20 pm. Bar in image (A) = 10 pm, and applies to all images except image (H).  Page 110.  Figure 4.21. Actin distribution in hyphal tips of actin RIP mutant act (5.5UC). (A) One 4  apical actin accumulation in a hyphal tip that is not branching. (B) Dichotomous branching before the emergence of two new tips. There are two apical actin accumulations, one for each new branch. (C) Dichotomous branching after the emergence of two new tips. There are two apical actin accumulations at the tip of each new branch. (D) A dichotomous branch with longer branches. The apical actin accumulation persists at each branch tip. Bar =10 pm, and applies to all images.  Page 111.  Figure 4.22. FM4-64-stained hyphae of category G actin RIP mutants (A, B) 17.4UC and (C-H) act (5.5UC) with colonial growth. Images are presented in pairs, with the 4  DIC image first, followed by the fluorescence image. (A, B) Multiple small accumulations of membranous material replacing the Spitzenkorper at the hyphal tip. (C, D) Hypha without prominent staining. (E, F) Multiple small membranous accumulations. (G, H) Multiple small membranous accumulations at dichotomous branch. Bar in image (C) = 20 pm and applies to image (D). Bar in image (A) = 10 pm, and applies to the rest of the images.  Page 112.  Figure 4.23. FM4-64-stained hyphal tip of category G actin RIP mutant act (1.5UC) 3  with colonial growth. (A-C) Time sequence of the growth of a hyphal tip. A diffuse stronger stained area is visible in the apical region instead of a Spitzenkorper. (D) DIC image of the same hyphal tip. (E, G) Multiple small stained accumulations in the apical region. (F, H) DIC images of the appropriate hyphae. Bars = 10 pm. xvii  Bar in image (A) applies to images (B-D). Bar in image (E) applies to the rest of the images.  Page 113.  Figure 4.24 The category H actin RIP mutant act (4.2C) with high branching frequency. 2  (A) A three-day-old colony. (B) Hyphal morphology of a one-day-old colony. Bar = 100 um. (C) Branching pattern of a one-day-old colony. Bar = 1 mm. (D) Branching pattern of a one and a half-day-old colony. Bar = 1 mm.  Page 114.  Figure 4.25 Actin and nuclear distribution in the category H actin RIP mutant act (4.2C) 2  with high branching frequency. (A, B, C) Images of the same hyphal tip. (A) DIC image. (B) Nuclear distribution. (C) One apical actin accumulation. (D) Peripheral actin network. (E) Multiple weakly stained actin accumulations at hyphal tip. (F) Two apical actin accumulations. (G) Multiple strongly stained actin accumulations at a hyphal tip. (H) Absence of actin accumulations. A l l bars =10 urn. Bar in image (D) applies to image (G). Bar in image (A) applies to the rest of the images.  Page 115.  Figure 4.26. Growth of an FM4-64-stained hyphal tip of the category H mutant act (4.2C) 2  with a high branching frequency. (A-K) Sequence of images following the growth and dichotomous branching event. (A-D) Before branching the tip has one Spitzenkdrper. (E) Before the two new branches emerge, the Spitzenkdrper is divided, and each of the two Spitzenkdrpers is assigned to a new branch. The emergence of a lateral branch is also visible, but at a different focal plane. (L) DIC image. Ba r = 10 um, and applies to all images. Page 116. Figure 4.27. The category H actin RIP mutant act (52.9UC) with a high branching 18  frequency. (A) A three-day-old colony. (B) Hyphal morphology of a one-day-old colony. Bar  xvm  = 100 pm. (C) Branching pattern of a one-day-old colony. Bar = 1 mm. ( D ) Branching pattern and colony density of a four-day-old colony. Bar = 1 mm.  Page 117.  Figure 4.28. The category H actin RIP mutant act (2.8C) with a high branching frequency. 6  (A) A three-day-old colony. (B) Hyphal morphology of a one-day-old colony. Bar = 200 pm. Page 118. Figure 4.29. The category H actin RIP mutant 6.2C with high branching frequency. (A) A three-day-old colony. (B) Hyphal morphology of a one-day-old colony. Bar =100 pm. (C) Branching pattern and colony density of a one-day-old colony. Bar = 1 mm. ( D , E) The category H actin RIP mutant 8.2C, hyphal morphology and branching pattern. (D) Bar =100 pm. (E) Bar = 1 mm.  Page 119.  Figure 4.30. FM4-64 stained hyphal tip of the category H actin RIP mutant act (2.8C) with 6  high branching frequency. (A-E) Time series of images of a growing hyphal tip. A Spitzenkorper is visible at the apex of the leading hypha, but not at the apex of the new lateral branch. (F) DIC image of the same hyphal tip. Growth of a second lateral branch subapically in the z-plane is visible. Bar in image (A) = 10 pm, and applies to all images. Page 120. Figure 4.31. Growth and Spitzenkorper position in an FM4-64-stained hyphal tip of the category H actin RIP mutant act (52.9UC) with a high branching frequency. (A-D) Time 18  sequence of hyphal tip growth. The Spitzenkorper is at the apex during growth. In image (D), the hypha grew out of the initial plane. Images (E, F) are DIC images of different planes of the same hypha. Bar =10 pm and applies to all the images.  Page 121.  Figure 4.32. The category I actin RIP mutant 18.4UC with low branching frequency.  xix  (A) Branching pattern of a one-day-old colony. Bar = 1 mm. (B) Hyphal morphology of a one-day-old hyphae. Bar = 200 urn. (C) Branching pattern of a two-day-old colony. Bar from image (A) applies to this image. (D) Hyphal morphology of two-day-old hyphae. Bar =100 u.m.  Page 122.  Figure 4.33. The category I actin RIP mutant 38.4UC with a low branching frequency. (A) A three-day-old colony. (B) Hyphal morphology of one-day-old hyphae. Bar =100 um. (C) Branching pattern of a two-day-old colony. Bar = 1 mm. (D) Branching pattern of a fourday-old colony. Bar from image (C) applies to this image.  Page 123.  Figure 4.34. The category I actin RIP mutant 1.6C with a low branching frequency. (A) A three-day-old colony. (B) Branching pattern of a three-day-old colony with an anticlockwise turn. Bar = 1mm. (C) Anticlockwise turn of a two-day-old hypha. Bar = 200 urn (D) A oneday-old hypha with a mild direction maintenance problem. Bar =100 u.m. (E) Hyphae that lost polarity in some regions. Bar =100 um. (F) A long unbranched hypha. Bar =100 mm. Page 124. Figure 4.35. A category J actin RIP mutant with curling hyphae. (A) Branching pattern of colony. Bar = 1 mm. (B) Hyphal morphology and branching of colony. Bar = 200 u.m.  Page 125.  Figure 4.36. The category K mutant 2.9UC with wavy hyphal growth. (A) Wavy hyphal morphology of a one-day-old colony. Bar =100 um. (B) Hyphae of a two-day-old colony. Bar = 200 um. (C) Hyphal of a four-day-old colony. Bar = 200 urn (D) Branching pattern of a four-day-old colony. Bar = 1mm. Figure 4.37. The category L actin RIP mutant act  20  Page 126.  (20.2C) with aberrant growth at low  temperatures. (A) Branching pattern of colony at 7°C. Bar = 1mm, and also applies to xx  image (C). (B) Hyphal morphology at 7°C. Bar = 200 pm, and also applies to image (D). Wild type control. ( C ) Branching pattern of colony at 7°C. (D) Hyphal morphology of wild type. A l l strains were grown at 25°C for one day then shifted to 7°C for five days. Page 127. Figure 4.38. Steps in the life cycle of N. crassa where defects due to actin modification light occur. Green = observed defects. Red = predicted defects that were not observed. * = for more details about defects in polarized growth, see Table 4.6. Drawings of the life cycle are taken from Perkins et al. (2001).  Page 128.  Figure 4.39. Position of point mutations along the actin gene in the actin RIP mutants. (A) Mutants from categories C, E, F, G and non-classified mutants (NC). (B) Mutants from categories H and L. Black lines indicate a RIP G-C to A-T point mutation. Grey lines indicate a non-RIP point mutation. The upper number indicates the position of the mutation relative to the 3'-end of the gene. The lower number indicates the position of the amino acid that the codon translates to relative to the NH2-terminal of the actin polypeptide. Brackets indicate that the point mutation does not result in the change in the amino acid that the codon encodes. For more information about mutants from different categories, refer to Tabs. 4.3 and 4.4.  Page 129.  Figure 5.1. Colony morphology, hyphal morphology and branching pattern of col-16 and col-16; act . (A) A three-day-old colony of col-16. (B) A n eight-day-old colony of col-16. 1  ( C ) Hyphal morphology and branching pattern of a one-day-old colony of col-16. (D) A three-day-old colony of col-16; act . (E) A n eight-day-old colony of col-16; act . 1  1  (F) Hyphal morphology and branching pattern of a one-day-old-colony of col-16; act . Bars 1  = 100 pm.  Page 143. xxi  Figure 5.2. Actin and nuclear distribution in hyphal tips of col-16. (A, B, C) Images of the same hyphal tip. (A) DIC image (B) Nuclear distribution. (C) One apical actin accumulation. (D, E) Two apical actin accumulations. (F) Three stronger stained dots in an apical actin accumulation. (G) Multiple small actin accumulations in a hyphal tip. (H) Absence of actin accumulations in a hyphal tip. A l l bars = 10 um. Bar in image (G) applies to image (H). Bar in image (A) applies to the rest of the images. White triangles indicate the position of the hyphal apex.  Page 144.  Figure 5.3. Actin and nuclear distribution in hyphal tips of col-16; act . (A, B, C) Images of 1  the same hyphal tip. (A) One large subapical actin accumulation. (B) DIC image. (C) Nuclear distribution. (D) One large and one small subapical actin accumulation. (E) Two subapical actin accumulations different in size. (F) Subapical actin cap. (G) One linear subapical and one small apical actin accumulation. (H) One apical linear actin accumulation. (I) One large apical actin accumulation. (J) One apical actin accumulation. (K) Two actin accumulations, one apical and one slightly displaced subapically. (L) Multiple actin accumulations at a hyphal tip. Triangular arrowheads indicate the hyphal apex. Both bars = 10 um. Bar in image (A) applies to all images except image (L).  Page 145.  Figure 5.4. Colony morphology, hyphal morphology and branching pattern offr and fr; act . 1  (A) A three-day-old colony offr. (B) A n eight-day-old colony offr. (C) Hyphal morphology and branching pattern of a one-day-old colony of fr. (D) A three-day-old colony of fr; act . 1  (E) A n eight-day-old colony of fr; act . (F) Hyphal morphology and branching pattern of a 1  one-day-old-colony offr; act . Bars = 100 fim.  Page 146.  1  Figure 5.5. Actin and nuclear distribution in hyphal tips of fr; act . (A, B, C) Images of the 1  same hyphal tip. (A) DIC image. (B) Nuclear distribution. (C) One subapical and three small xxii  peripheral actin accumulations. (D) Two subapical peripheral actin accumulations, one much smaller than the other. (E) Two large subapical and three small (apical and subapical) actin accumulations. (F) Three subapical actin accumulations. (G) Multiple strongly stained actin accumulations at a hyphal tip. (H) One large linear subapical actin accumulation. (I) A subapical cap-like actin accumulation. (J) Two apical actin accumulations. (K) One apical actin accumulation. (L) One linear subapical, and additional subapical actin accumulations. The triangular arrowheads indicate the apex of the described hypha. Bar =10 pm, and applies to all images. Figure  Page 147.  5.6. Actin and nuclear distribution in a dichotomously branched hypha ofjr. (A) DIC  image. (B) Nuclear distribution. ( C , D) Actin distribution in different planes. (C) Apical actin accumulation is visible in two out of three hyphal tips. Bar = 10 pm. Figure  Page 148.  5.7. Hyphae offir stained with FM4-64. (A-F) Growth observed over several minutes.  The Spitzenkorper is visible in the apical region in most tips. The white triangle in image (C) indicates the hypha that is beginning to branch dichotomously. Bar =10 pm. Figure  Page 149.  5.8. Colony morphology, hyphal morphology and branching pattern of gran and gran  act . (A) A three-day-old colony of gran act . (B) A n eight-day-old colony of gran act . 1  1  1  (C) Hyphal morphology and branching pattern of a one-day-old-colony of gran act . 1  (D) A three-day-old colony of gran. (E) Hyphal morphology and branching pattern of a oneday-old colony of gran. Bars = 100 pm. Figure  Page 150.  5.9. Actin and nuclear distributions in hyphal tips of gran. (A, B, C ) Images of the  same hyphal tip. (A) DIC image. (B) Nuclear distribution. (C) One apical actin accumulation. (D, H) Images of the same hyphal tip. (D) Nuclear distribution. (E, F , G ) Images of the same hyphal tip. (E) DIC image. (F) Nuclear distribution. (G) Absence of actin xxiii  accumulations. (H) Multiple weakly stained actin accumulations. Triangular arrowhead indicates the apex of the described hypha. For all images bar = 10 (im.  Page 151.  Figure 5.10. Actin and nuclear distribution in hyphal tips of act gran. (A) One subapical 1  axial and one small apical actin accumulation. (B) One subapical peripheral actin accumulation. (C) Two subapical actin accumulations. (D) Multiple subapical actin accumulations. (E, F, G) Images of the same hyphal tip. (E) DIC image. (F) Nuclear distribution. (G) Subapical actin cap. (H) Subapical linear, and two small apical actin accumulations. (I) One large apical actin accumulation. (J) Two apical actin accumulations. (K) Multiple weakly stained actin accumulations in a hyphal tip. (L) Absence of actin accumulations in a hyphal tip. For all images bar =10 urn  Page 152.  Figure 5.11. (A-K) Growing hyphae of gran stained with FM4-64 observed over several minutes. Spitzenkdrpers are visible in hyphal tips. (F) Two Spitzenkdrpers are visible before the new apical branches appear. (L) DIC image. Bar = 20 um.  Page 153.  Figure 5.12. Colony morphology, hyphal morphology and branching pattern of col-15 and col-15; act . (A) A three-day-old colony of col-15. (B) A n eight-day-old colony of col-15. 1  (C) Hyphal morphology and branching pattern of a one-day-old colony of col-15. (D) A three-day-old colony of col-15; act . (E) A n eight-day-old colony of col-15; act . (F) 1  1  Hyphal morphology and branching pattern of a one-day-old-colony of col-15; act . Bars = 1  100 um.  Page 154.  Figure 5.13. Actin and nuclear distribution in hyphal tips of col-15; act . (A, B, C) Images 1  of the same hyphal tip. (A) DIC image. (B) Nuclear distribution. (C) One subapical actin accumulation slightly displaced towards the periphery. (D) One subapical peripheral actin accumulation. (E) One subapical actin accumulation and linearly distributed actin that xxiv  connects the subapical actin accumulation with the apex. (F) One subapical and a couple of small apical actin accumulations. (G) Two subapical actin accumulations. (H) One apical actin accumulation. Bars = 10 pm. Bar in image (A) applies to all the images except image (G).  Page 155.  Figure 5.14. Colony morphology, hyphal morphology and branching pattern of col-8 and col-8; act . (A) A three-day-old colony of col-8. ( B ) A n eight-day-old colony of col-8. 1  (C) Hyphal morphology and branching pattern of a one-day-old colony of col-8. ( D ) A threeday-old colony of col-8; act . (E) A n eight-day-old colony of col-8; act . (F) Hyphal 1  1  morphology and branching pattern of a one-day-old-colony of col-8; act . Bars =100 pm. 1  Page 156. Figure 5.15. Actin and nuclear distributions in hyphal tips of col-8. (A, B, C) Images of the same hyphal tip. (A) DIC image. ( B ) Nuclear distribution. (C) One apical actin accumulation. ( D ) Apical actin cloud. (E) Two apical actin accumulations. (F) Multiple actin accumulations. (G) Multiple small and one large subapical actin accumulations. (H) Absence of apical actin accumulations. Arrowheads indicate the apex of the described hyphal tip. Bars = 10 pm. Bar in image (A) applies to images (A-D). Bar in image (E) applies to images (EH).  Page 157.  Figure 5.16. Actin and nuclear distribution at hyphal tips of col-8; act . (A, B, C) Images of 1  the same hyphal tip. (A) DIC image. ( B ) Nuclear distribution. (C) One subapical actin accumulation displaced toward the periphery. ( D ) One large subapical peripheral actin accumulation. (E) One subapical actin accumulation and a small apical actin accumulation. (F) Two tips with one subapical actin accumulation each. (G) One subapical linear actin accumulation. (H) One apical linear actin accumulation and smaller actin accumulations at a xxv  hyphal tip. (I) Multiple strongly stained actin accumulations. (J) Multiple weakly stained actin accumulations. (K) Several weakly stained actin accumulations. (L) One apical actin accumulation. Bar = 10 urn, and applies to all images.  Page 158.  Figure 5.17. Growth of col-8 hyphae stained with FM4-64. (A) DIC image. (B) Spitzenkdrpers in apical regions of hyphae. (C) Same hyphae observed a few minutes later. Bar = 20 |im.  Page 159.  Figure 5.18. Colony morphology, hyphal morphology and branching pattern of spco-4 and spco-4; act . (A) A three-day-old colony of spco-4; act . (B) A n eight-day-old colony of 1  1  spco-4 act . (C) Hyphal morphology and branching pattern of a one-day-old colony of spco1  4; act . (D) A three-day-old colony of spco-4. (E) Hyphal morphology and branching pattern 1  of a one-day-old colony of spco-4. Bars = 100 \im.  Page 160.  Figure 5.19. Actin and nuclear distribution in hyphal tips of spco-4. (A, B, C) Images of the same hyphal tip. (A) DIC image. (B) Nuclear distribution. (C) One apical actin accumulation. (D) One apical actin accumulation and a few small satellite actin accumulations. (E) One star-like apical actin accumulation. (F) One two-part apical actin accumulation. (G) One three-part apical actin accumulation. (H) Multiple weakly stained actin accumulations at the tip. Bar = 10 um, and applies to all images.  Page 161.  Figure 5.20. Actin and nuclear distributions in hyphal tips of act ; spco-4. (A, B, C) Images 1  of the same hyphal tip. (A) DIC image. (B) Nuclear distribution. (C) Multiple weakly stained actin accumulations. (D) Multiple strongly stained actin accumulations. (E) Two subapical actin accumulations. (F) One subapical actin accumulation. (G) One subapical peripheral actin accumulation. (H) One subapical actin cap and a small apical actin accumulation. (I, J) Linear subapical actin accumulations. (K) One apical actin accumulation. (L) Absence of xxvi  actin accumulations in a hyphal tip. Triangular arrowhead indicates the apex of the described hypha. Bar =10 pm, and applies to all other images.  Page 162.  Figure 5.21. Hypha of spco-4 stained with FM4-64. (A-C) Three images collected over a couple of minutes. A Spitzenkorper is visible in the apical region. Bar =10 pm.  Page 163.  Figure 5.22. Colony morphology, hyphal morphology and branching pattern of md and md act . (A) A three-day-old colony of md. (B) A n eight-day-old colony of md. (C) Hyphal 1  morphology and branching pattern of a one-day-old colony of md. (D) A three-day-old colony of md act . (E) A n eight-day-old colony of md act . (F) Hyphal morphology and 1  1  branching pattern of a one-day-old-colony of md act . Bars = 100 pm. 1  Page 164.  Figure 5.23. Actin and nuclear distribution of md. (A, B, C) Images of the same hyphal tip. (A) DIC image. (B) Nuclear distribution. (C) One large apical actin accumulation. (D) One small apical actin accumulation. (E) Apical actin cloud with a stronger stained dot within. (F) Multiple small apical actin accumulations. (G) Multiple weakly stained actin accumulations at a hyphal tip. (H) Absence of apical actin accumulations. Triangular arrowhead indicates the apex of the described hyphal tip. Both bars = 10 pm. Bar in image (E) also applies to image (H). Bar in image (A) applies to the rest of the images.  Page 165.  Figure 5.24. Actin and nuclear distribution in hyphal tips of md act . (A, B, C) Images of the 1  same hyphal tip. (A) DIC image. (B) Nuclear distribution. (C) One subapical actin accumulation and a small apical actin cloud. (D) One subapical actin accumulation. (E) One subapical peripheral actin accumulation. (F) Two subapical actin accumulations. (G) One linear subapical actin accumulation. (H) One small apical actin accumulation. (I) One average-size apical actin accumulation. (J) One slightly peripherally displaced apical actin accumulation. (K) Multiple small actin. accumulations in a hyphal tip. (L) Absence of actin xxvii  accumulations in a hyphal tip. Triangular arrowhead indicates the apex of the described hyphal tip. Both bars =10 pm. Bar in image (D) applies to images (E, H). Bar in image (A) Page 166.  applies to the rest of the images.  Figure 5.25. Hyphae of md stained with FM4-64. (A) DIC image. (B) Spitzenkorper in hyphal tips of multiple apical branches. Bar = 20 pm.  Page 167.  Figure 5.26. (1) One apical actin accumulation. (2) Two apical actin accumulations. (3) One subapical actin accumulation. (4) Two subapical actin accumulations. (5) One or more linear subapical actin accumulations. (6) Subapical actin cap and diffuse staining at hyphal tip. (7) Multiple strongly stained subapical, or subapical and apical actin accumulations. (8) Multiple weakly stained actin accumulations at the hyphal tip. (9) Multiple apical actin accumulations. (10) One linear apical actin accumulation. (11) Absence of actin accumulations at the hyphal tip.  Page 168.  Figure 6.1. Model of hyphal tip growth and branching. Hyphal tip of (A) wild type and (B) act . Circles = vesicles; blue lines = microfilaments; blue stars = actin accumulations; thin 1  black line = expandable cell wall; thick black line = non-expandable cell wall; red colour = Page 193. Figure 6.2. Position of residues substituted in the actin polypeptide of the actin RIP mutants. Numbers indicate distance from the N H end (see Table 6.1.). IA-HB indicate subdomains. 2  Red = category C mutants; Shades of green = category E mutants; Shades of orange = category F mutants; Shades of purple = category G mutants; Shades of blue = category H Page 194.  mutants; Yellow = category L mutants.  xxviii  LIST OF ABBREVIATIONS ADP-Adenosinediphosphate ATP-Adenosinetriphosphate BSA-Bovine Serum Albumin cAMP-Cyclic Adenosinemonophosphate CAP-Calcium Actin Phosphoinositide DIC-Differential Interference Contrast EDTA- Ethylene diaminetetraacetic acid EGTA-Ethylene glycol-0,0 -bis(2-amino-ethyl)-N, N , N \ NMetraacetic acid GAP-GTPase Activating Protein GDI-Guanine Nucleaotide Dissociation Inhibitor GEF-Guanine Nucleotide Exchange Factor GFP-Green Fluorescent Protein GTPase-Guanosinetriphosphatase HEPES-N-[2-hydroxyethyl] piperazine-N -[2-ethanesulphonic acid] HRP-Horseradish Peroxidase MAPK-Mitogen Activated Protein Kinase MIPS-Munich Information Center for Protein Sequences ORF-Open Reading Frame P A K - p i l Activated Kinase PIPES-Piperazine-N, N -bis[2-ethanesulfonic acid] PM-Plating Medium (Sorbose-Containing Medium) PVDF-Polyvinylidene fluoride RIP-Repeat Induced Point Mutations SDS-PAGE- Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis VFG-Vesicle Fusion Gradient VM-Vogel's Minimal Medium VSC-Vesicle Supply Center ,  ,  ,  xxix  ACKNOWLEDGEMENTS  To everyone who contributed to the successful completion of this project: Thank you!  xxx  CHAPTER I INTRODUCTION  Two of the basic characteristics of all cells are shape and motility. Actin plays a key role in the generation of these characteristics. A n actin network contributes to cell morphogenesis and helps create the typical shape of the cell. When associated with the motor protein myosin, actin serves as a track for generating force that leads to movement such as muscle contraction. Movement also occurs in cells where a peripheral actin network contributes to the formation of fdopodia and lamellipodia. These and a multitude of other essential functions render actin one of the most widespread and conserved proteins in cellforming organisms. The present work focuses on the role of actin in tip growth in fungi.  1.1 Participants of the Hyphal Tip Growth Process  Localized polar extension of the cell wall, resulting in a tubular cell shape, is characteristic for fdamentous fungi. During this process new cell wall and plasma membrane components have to be incorporated at the site of apical expansion (Gooday, 1995). Actin takes part in successive steps in the supply route, and is an active participant in organelle positioning, organelle movement, vesicle transport, exocytosis, and localization of plasma membrane-associated proteins (Heath, 1990).  1  1.1.1 Actin  Actins are highly conserved proteins that are present mainly in the cell cytoplasm, but can also be found in the nucleus (reviewed by Sheterline et al., 1998). In species that contain different isoforms of actin, each isoform can have a different position in the cell, and can be differentially expressed in cells of different tissues. Even before translation, 3'-ends of the actin mRNA can bind to a particular region of the cytoskeleton. Unfolded actin proteins are present throughout the cytoplasm. The same complex that folds tubulin, the TCP-1 (toroidal chaperonine protein) complex, is responsible for folding actin into its globular form, G-actin. Globular actin binds to one nucleoside tri- or di-phosphate, usually ATP or A D P , and divalent cations. At physiological conditions, the divalent cations that G-actin binds to are Mg  2 +  ions. Binding a nucleoside and M g  2 +  facilitates the self-assembly of G-actin into  fdamentous F-actin. Polymerization into F-actin is a highly dynamic process (Alberts et al., 1989) that occurs at both ends of the filament, but at different rates. The end that binds monomers more rapidly is referred to as the plus or barbed end, while the slower assembling end is referred to as the minus or pointed end. The terms "barbed" and "pointed" originate from the appearance of the actin fdament after binding to myosin, which resembles the barbed end of an arrow at the plus end, and the pointed end of an arrow at the minus end. The first step in the formation of an actin filament is nucleation. This step, in which three actin monomers assemble to serve as a template for the addition of monomers, is slow, and causes an initial lag in the growth of an F-actin fdament. Once the template is assembled, new monomers add on rapidly. The addition is reversible, but monomers are added much faster than they are removed. As the  2  concentration o f monomers is reduced, it reaches a point at which the numbers o f added and disassembled monomers are equal. The monomer concentration at this point is called the critical concentration. The critical concentration is different at the + and - ends o f the filament, and this difference enables the direction o f fdament growth to change. A t the steady state, the majority o f the monomers are added at the + end, and removed at the - end. A t this point, the length o f the filament is constant, but each monomer within the assembly travels towards the - end, a process called treadmilling. Shortly after a monomer is added to the growing F-actin polymer, the A T P molecule bound to the monomer is hydrolysed. The hydrolysis o f A T P to A D P changes the monomer conformation,.and promotes disassembly. When the monomer concentration is low enough, the whole fdament w i l l dissociate. Besides the free monomer concentration, the rate o f monomer addition depends on several factors, including Pi concentration and p H . Another level of control is binding o f certain proteins to free monomers. This includes profdin, which promotes A D P - A T P exchange, and cofdin that binds to filamentous actin and brings about depolymerization. A complete cycle o f assembly and disassembly o f an actin fdament can be completed in minutes. Besides binding sites for self-assembly, nucleotides and divalent cations, G-actin has binding sites for drugs and toxins, such as cytochalasins. This property makes it possible to investigate actin function in vivo, by inhibitor studies. A c t i n fdaments can also bind to a number o f different proteins. Interactions with myosins, tropomyosin and caldesmon are involved in the formation o f muscle contractile units. Binding to proteins o f the gelsolin and oc-actinin families, as well as a number of other proteins determines the characteristics o f the cytoskeleton or networks at the cell periphery (Sheterline et al., 1998, and references therein). The gelsolin and cc-actinin families cross link actin fdaments, or act as bridges  3  between actin filaments and cellular membranes (see Chapter V I for more details). Integrins, and other plasma membrane and cell wall components can also bind actin, and implicate it in processes related to cellular functions at these sites (Brakebusch and Fassler, 2003). Actincontaining structures then engage in an intricate interplay with other cell structures and organelles ultimately giving the form and dynamic characteristics of the cell. G-actin is present throughout the mycelium of filamentous fungi. On the other hand, F-actin is widespread at sites such as the periphery of hyphae next to the plasma membrane in the form of plaques or actin caps, at the Spitzenkdrper, as components of the cytoskeleton, associated with microtubules, next to septae, next to the nuclear envelope, associated with organelles, surrounding some vesicles and as the fibrillar coating of filasomes (Hoch and Staples, 1983; Roberson, 1992; Srinivasan et al., 1996). The lack of actin mutants in most filamentous fungi necessitated the use of inhibitor studies to deduce the role of actin. In a variety of filamentous fungi disruption of actin filaments with cytochalasins interrupts actin functions and results in changes in actin distribution, formation of cell wall deposits at sites not destined to expand, reduction in enzyme secretion, and ultimately an altered hyphal morphology (Allen et al. 1980; Grove and Sweigard, 1980; El Mougith et al., 1984; Kanbe et al., 1993; Torralba et al., 1998). Consequently, branching frequency and pattern are altered, growth rate reduced, differentiation of the ascomycetous ascogonium hindered and spore germination inhibited (Oliver, 1973; Sabanero, 1994; Liu et al., 1995). However, inhibitors block selected functions of actin, and do not affect other functions. Therefore, these studies did not clarify how the actin network within a hypha is set-up and maintained, the multiple roles of actin, or in which way the functions of the actin network are regulated. Another disadvantage is that although  4  the effect of inhibitors on actin filaments is established, they can have additional effects that are not related to actin function (Thomas et al., 1974; Manavathu and Thomas, 1980). Alterations in the actin gene leading to an altered actin protein can, on the other hand, show changes that are specifically caused by actin. In Saccharomyces cerevisiae there is only one actin gene, ACT 1 (Ng and Abelson, 1980), and a large collection of actin alleles. The first two identified actin alleles were temperature sensitive, and pointed to a function of actin in polarized growth (Shortle et al., 1984; Novick and Botstein, 1985). At restrictive temperatures, the mutants exhibited a different actin distribution, delocalized chitin deposition at the cell surface, partial inhibition of invertase secretion, intracellular accumulation of secretory vesicles, osmotic sensitivity, and cell death after prolonged incubation at restrictive temperatures (Novick and Botstein, 1985). Additional actin mutants were later obtained, and were recessive lethal, conditional lethal, or had a wild type phenotype (Wertman et al., 1992). Other mutants were described with defects in actin organization, actin filament assembly, endocytosis, and sporulation (Belmont et al., 1999; Whitacre et al., 2001). A test for drug resistance identified lantruculin A , phalloidin and tolytoxin binding sites (Ayscough et al., 1997; Belmont, et al., 1999). The changes in the actin protein were at sites shown to interact with the divalent metal ion, nucleotide, other monomers, or at buried sites that may affect proper folding (Wertman et al., 1992). Mutations at the surface of the actin protein were at sites that interacted with actin binding proteins such as myosin and tropomyosin (Wertman et al., 1992, Miller et al., 1996). There are no actin mutants in filamentous fungi so far, with the exception of a recent study designed to isolate mutants with polarity defects in N. crassa, in which one actin mutant was  5  obtained (Seiler and Plamann, 2003). This mutant had multiple branches at lateral branching sites, but besides the phenotype, it was not further characterized.  1.1.2 Plasma Membrane, Cell Wall and the Spitzenkdrper  In any growing hyphal tip, new cell wall and plasma membrane needs to be added so that it can expand. The main constituents of the cell wall are polysaccharides such as Pglucans and chitin (Gooday, 1995). Vesicles containing components of the plasma membrane and cell wall travel from sites where they are generated to sites where they are incorporated (Grove et al., 1970). The terminology that will be used throughout this dissertation to describe position within a hypha is outlined in Figure 1.1. Vesicles are formed at the endoplasmic reticulum in proximal regions of the hyphae. From there they are transported first to the Golgi membranes where additional processing occurs, and then to sites which require incorporation of new membrane material, such as existing or new hyphal tips, or septae. The delivery path of the majority, if not all, of the vesicles destined for growing hyphal tips includes passage through the Spitzenkdrper, as shown in electron micrographs in which vesicles coincide with the position of the Spitzenkdrper in live cells (Grove and Bracker, 1970). Grove and Bracker (1970) summarized the early work on understanding the Spitzenkdrper. The data are as follows: The Spitzenkdrper was considered to be related to apical growth in higher fungi, and to be correlated with the direction of growth. Although the first images of the Spitzenkdrper obtained by light microscopy suggested that it was a distinct organelle, micrographs collected by electron microscopy showed that the Spitzenkdrper was  6  a collection of apical vesicles, microvesicles, and ribosomes. Based on the ultrastructure of various fungi, Grove and Bracker (1970) pointed out that a smaller zone within the apical vesicle cloud corresponded better to the presence and position of the Spitzenkorper. This zone could contain microvesicles, tubules and ribosomes. They also divided fungal tips into three groups with regards to the apical organization in hyphae. One group had apical vesicles distributed more or less evenly throughout the tip of the hypha, and was characteristic for Oomycetes. In the second group, characteristic for Zygomycetes, vesicles had the highest concentration in a crescent-shaped zone at the apex. Septate fungi (including Neurospora crassa) comprised the third group, with a mainly sphaerical apical vesicle cloud, and the presence of a specialized zone with, or in rare occasions without, vesicles that corresponded to the Spitzenkorper. Howard (1981) looked at the same regions of hyphal tips in Gibberella acuminata using light microscopy and electron microscopy. He pointed out that a higher vesicle density leads to a greater shift in refractive index when growing hyphae are observed with a light microscope. Therefore, the vesicle concentration determines the presence and size of the Spitzenkorper and it is not equal to the specialized inner zone of the apical vesicle cloud. Howard (1981) showed the presence of microfdaments in the specialized inner zone of the vesicle cloud, and suggested that it is an organization center of the Spitzenkorper. The microfdaments were present in the form of filasomes, which represented microvesicles surrounded by microfdaments. Actin fdaments also surrounded vesicles indicating the possibility of actin fdament involvement in a mechanism for localized secretion. In addition, Howard (1981) found that microtubules are present at the hyphal tip, often in the Spitzenkorper, and that they are parallel to the direction of growth.  7  Using high resolution video-enhanced light microscopy, Lopez-Franco and Bracker (1996) looked at the dynamics and diversity of 32 septate fungi. They classified these fungal species into nine patterns of apical organization based on the appearance of the Spitzenkdrper. This confirmed the inherent diversity of the Spitzenkdrper, and gave a way to differentiate between common and variable characteristics in different species. In addition, the authors documented that the Spizenkdrper was a highly dynamic complex that changed in size, shape, and position during growth within each fungal species tested. Dynamic changes occurred both inherently, and as a response to stress conditions. The Spitzenkdrper core varied in composition between species but not within a species. The finding that the core persists under stress conditions in Rhizoctonia solani supports the idea proposed by Howard (1981) that the specialized inner region appears to be the organizational center of the Spitzenkdrper. Even though it has been studied for almost eighty years, the structural and functional details of this organized collection of selected cell constituents still need to be refined. Lopez-Franco and Bracker (1996) summarized the current view of the Spitzenkdrper: it is a heterogenous, multicomponent complex without clear boundaries that is dynamic, pleomorphic, variable among different fungi and has a function in hyphal tip growth.  1.1.3 Micro tubules  Microtubules, as a part of the cytoskeleton, are necessary for the transport of vesicles to the hyphal tip. Disruption of microtubules caused the emergence of multiple germination tubes instead of one in N. crassa, but did not affect the outgrowth of branches (Barja et al.,  8  1993). This indicated that microtubules were involved in selecting the germination site, while microfdaments were involved in the assembly of components that build the germination tube. It also indicated that, as with microfdaments, microtubules acted as tracks along which vesicles were delivered to the tip. The disruption of these tracks altered the position at which plasma membrane and cell wall material incorporated. Motors that bind to microtubules, such as dyneins and kinesins, facilitated transport of vesicles and organelles to the tip (Steinberg and Schliwa, 1995; Tinsley et al., 1996; Wu et al., 1998). Disruption of these motor proteins also caused growth defects in hyphal morphogenesis (Riquelme et al., 2000).  1.1.4. Turgor  Early hypotheses on the role of turgor in hyphal growth were influenced by views on cell wall characteristics at sites of cell wall expansion. Based on the observation that hyphal tips burst subapically, not at the apex where the cell wall should be weakest, turgor was not considered to play a role in hyphal expansion (Reinhardt, 1892; Bartnicki-Garcfa et al., 2000). Harold et al. (1996) demonstrated that hyphal tip growth could proceed in Saprolegnia ferax even after reducing turgor pressure close to zero, and concluded that turgor is not required to extend the apex. Because actin distribution appeared normal during these extremely low turgor conditions, microfdaments were proposed as an option for the force generating entity allowing hyphal growth to progress (Kaminskyj et al., 1992). BartnickiGarcia (2002) argued that bursting is a chemical process, not mechanical as implied by Reinhardt (1892), and therefore can be involved in hyphal shape formation. However, he  9  agreed that turgor is not the primary determinant and that only a minimum threshold value is needed to expand the wall (see next segment).  1.1.5 Tip-High Calcium Gradient  Another component present at the hyphal tip is a gradient of [Ca ], with the highest 2+  concentration at the extreme apical region, as in S.ferax (Garrill et al., 1993), or at a small distance from the apical region, as in N. crassa (Levina et al., 1995). The gradient of [Ca ] 2+  disappeared in the absence of tip growth, indicating that it is necessary for growth to proceed (Garrill et al., 1993; Hyde and Heath, 1997). Although evidence showed that stretchactivated channels with a tip-high distribution were involved in the generation and maintenance of the gradient in S.ferax (Garrill et al., 1992; Garrill et al., 1993), this is likely not the case in fdamentous fungi such as N. crassa. In N. crassa, stretch-activated channels were distributed uniformly along the hypha, and inhibitors of calcium channels at the plasma membrane did not disrupt the [Ca ] gradient (Levina et al., 1995). In Achlya bisexualis, 2+  ionophores triggered branching when low C a concentrations were present in hyphal tips, 2+  highlighting the importance of internal calcium gradients (Harold and Harold, 1986). Silverman-Gavrila and Lew (2001) proposed that the [Ca ] gradient observed in N. crassa 2+  was generated by the release of C a simultaneous sequestration of C a  2+  2+  from vesicles located at the hyphal tip, and  in compartments behind the tip (see next section for  details).  10  1.2 Models of Tip Growth and Branching  Selection of a polarization site, initiation of growth at the selected polarized site, and maintenance of polarized growth are the main parts of the hyphal growth process. In the early growth stages, the first hypha that grows out from a spore is often referred to as the germination tube. The formation of a lateral branch consists of the same steps, except that the polarization site is along the trunk of an existing, usually growing, hypha, rather than a spore. How a germination tube, or a lateral branch, is selected and growth initiated, still remain controversial questions. As for hyphal growth itself, there are some characteristics that appear to be common in different fungi, such as the pulsate nature of hyphal growth (LopezFranco et al., 1994). There are several hyphal tip growth models that emphasize the role of different components of the hyphal growth process. Actin is a participant in each one of them, but its position in the hierarchy of the process varies. It cannot be disputed that incorporation of new components of the plasma membrane and cell wall influences the shape of a developing hypha. One group of models stresses this aspect of hyphal tip growth. The endolytic model proposes a balance between lysis and synthesis of the cell wall (Johnson, 1968). In this model glucan chains, one of the main structural components of the cell wall, are lysed by endoglucanases. The weakened cell wall expands, and the disruption is recognized by the cell's repair mechanisms. The breaks in the glucan chains are then rebuilt with the help of appropriate enzymes either by incorporating shorter segments into the break, or by extending the ends of the break until they meet. Both the breakage and the repair occur simultaneously, and require delicate control.  11  The steady state model (Wessels, 1990) invokes enzymes that soften the cell wall to initiate hyphal tip growth. The softened cell wall extends, and new components are incorporated by exocytosis of plasma membrane and cell wall-forming vesicles, and/or vesicles containing enzymes required for cell wall synthesis. Cell wall components need time to establish cross-links between their components, so the softened apex with the smallest number of cross-links has the highest degree of plasticity, while older regions of the cell wall lose their plasticity. Johnson et al. (1996) tested the endolytic and steady state models by looking at the nature and frequency of induced ruptures in cell walls of Schizosaccharomyces pombe. They noticed that ruptures were out-curled both at the extensile tip and non-extensile regions. Outcurl is present if there is a tension gradient that is highest at the outer layer of the cell wall. The out-curled ruptures could be explained by the steady state model in non-extensile region, and by the endolytic model in the extensile regions. The frequency of these ruptures reduced through time, in parallel with the decrease in the mean rate of extension. The endolytic model predicts a reduction of the enzyme gradient in the cell wall with the reduction of the growth rate, which fits the data. To reconcile the contradicting conclusions, Johnson et al. (1996) merged the endolytic and steady-state model into a hybrid model. This model incorporated the exocytosis of carbohydrates and enzymes, the gradient of cross-linking enzymes highest in the outer cell wall layer, and the regulation of cross-linking by stretch receptors from the steady-state model. The endolytic model contributed the continuous endoglucanolytic activity with the highest concentration of lytic enzymes in the inner cell wall layer, and glucan molecule repair. In this model turgor is the internal force-generator that expands the weakened cell wall.  12  Trinci (1978) gave a quantitative description of hyphal growth and branching. He defined the hyphal growth unit, a physiological rather than morphological entity, as the ratio between the total hyphal length and the number of tips. In the finite difference model (Prosser and Trinci, 1979) the authors mathematically defined the flow of plasma membrane and cell wall containing vesicles towards the hyphal tip. They proposed that the duplication cycle regulates the frequency and sites of septation. In this model, a branch is initiated when the rate at which vesicles arrive at the tip exceeds the maximum rate of vesicle incorporation. In subapical segments branching occurs when a critical level of vesicles accumulates. However, the authors did not describe events at the apical extension zone, or the means by which the site of branch initiation would be selected (Trinci, 1978; Prosser and Trinci, 1979). In the Vesicle Supply Center (VSC) model, vesicles carrying new cell wall materials that arrive at the tip first accumulate at a proposed vesicle supply center (Bartnicki-Garcia et al., 1990). From here, they are distributed to their final destination, incorporate into the apical region of the extending hyphal tip, and allow hyphal expansion. This 2D model provides an equation that shows that the dimensions of the hyphal tip depend on the number of vesicles released by the V S C per unit time, and the rate at which the V S C advances. The model is also referred to as the hyphoid model because of the hyphoid shape that the equation defines. The Spitzenkorper is selected as the cellular structure that has the function of the V S C because it is comprised mainly of vesicles, and it coincides with the position of the V S C . The authors used a computer simulator to follow the trajectories of the Spitzenkorper and found that the morphology of the hyphae could be explained if the Spitzenkorper serves as a V S C . Two mechanisms were put forward as options for the tipward displacement of the V S C : a pulling mechanism and a pushing mechanism. In the pulling mechanism actin filaments serve  13  to connect the V S C with the apex. Microfilaments are also a part of the pushing mechanism, in which the cytoskeleton pushes the V S C ahead. The formation of a new branch requires enzymatic action to soften the already rigid cell wall. After this prerequisite is satisfied, growth is maintained in the same way as described for an already existing polarized hyphal tip. In recent years, the authors confirmed that growing tips expand orthogonally, and expand their 2D model to a 3D model (Bartnicki-Garcfa et al., 2000; Gierz and Bartnicki-Garcia, 2001). For a viable mathematical description of 3D hyphal growth, it is necessary to know the pattern of cell surface displacement. Turgor at, or above, a threshold value appears sufficient to generate enough internal force to sustain the direction of cell surface displacement. In conclusion, the authors offer two major factors that maintain growth and generate the shape of a hypha: the cytoskeletal elements that displace the V S C providing a gradient of vesicles that fuse with the cell surface, and turgor providing the force that expands the cell surface. The V S C model can explain the inherent pulsed-growth of a number of filamentous fungi (Lopez-Franco et al., 1994). Satellite Spitzenkdrpers that arise at a short distance from the apex, migrate towards the apex and merge with the main Spitzenkdrper are a part of the rationale (Ldpez-Franco et al., 1995). The pulses of faster growth could arise as a result of the incorporation of a wave of additional vesicles, supplied to the main Spitzenkdrper by its satellite, into the cell surface (Bartnicki-Garcia, 2002). One of the main advantages of this model is that it provides a mechanism for the generation of a vesicle exocytosis gradient through the V S C (Bartnicki-Garcia, 2002). It can be applied to inherent hyphal growth processes, explains a variety of encountered hyphal shapes, and does not require the presence of tags to direct vesicles to their sites of  14  incorporation. Lopez-Franco and Bracker (1996) reported that some fungal tips do not closely approximate the hyphoid shape described by the hyphoid equation. Heath and van Rensburg (1996) tested the V S C model in S.ferax. They argued that the required degree of uniformity in the gradient of exocytosis would be difficult to maintain in living hyphae because of the inherent variability of biological systems. They devised an alternative mathematical description of exocytosis gradient generation that did not require a point source for vesicles, and that fit a wider range of hyphal tip morphologies. In addition, they showed that vesicles did not follow trajectories predicted by the V S C model in S.ferax, that cell components often occupied the predicted site of the V S C without blocking the flow of vesicles to the tip, and that organelles that occasionally wandered into the region between the predicted V S C and tip plasma membrane did not influence the growth rate or hyphal tip shape. The conclusion of this study was that the V S C model does not apply to S,ferax, and probably other fungi. Instead the authors proposed a V F G (vesicle fusion gradient) model that emphasizes the importance of a vesicle fusion gradient, regardless of how it is generated. Although the authors acknowledged that the way in which the gradient is established is unknown, they proposed the involvement of the membrane skeleton partly consisting of actin filaments. It is clear that the consistent presence of actin at sites of polarized hyphal extension compels its incorporation into models of hyphal tip growth. Actin is the focal point of a model developed for fungi by Heath and coworkers (Heath, 1995). The predecessors of this model were models for polarized growth of tubular cells in plants (Picton and Steer, 1982). Heath (1995) developed a model for hyphal tip growth where actin is proposed to regulate the tip extensibility by reinforcing the plasma membrane, or by propelling the tip forward  15  through the rearrangement of the cytoplasmic F-actin system. To reinforce the apical plasma membrane, actin would have to be anchored to the hyphal tip. In S.ferax and N. crassa integrin-like and spectrin-like proteins localize to the hyphal tip, and are proposed to link actin fdaments to the apex (Degousee et al., 2000). After plasmolysis of hyphae, cytoplasmic strands, also referred to as Hechtian strands, remain attached to the plasma membrane and cell wall in S. ferax, Achlya ambisexualis and N. crassa (Bachewich and Heath, 1997). The presence of actin in Hechtian strands at the hyphal tip of S. ferax further supports the proposed actin anchoring to the plasma membrane (Bachewich and Heath, 1997). Beside the established role of actin in polar tip growth maintenance, actin has an obligatory role in the mechanism for initiation of polarized growth. Disruption of F-actin with lantruculin B and cytochalasins prevents the outgrowth of germ tubes from conidia or protoplasts in Aspergillus nidulans, Mucor mucedo, S. ferax, N. crassa, and a number of other fungi (Grove and Sweigard, 1980; El Mougith et al., 1984; Barja et al., 1993; Sabanero, 1994; Heath etal. 2000). The emphasis is on calcium in the next group of models. Reissig and Kinney (1983) showed that an increase in intracellular [Ca ] after treatment with the ionophore A23187 2+  induced branching, and identified C a cytoplasmic C a  2+  2+  as a branching signal in N. crassa. A tip-high  gradient was proposed to be the reason for the absence of dichotomous  branches in N. crassa wild type (Schmid and Harold, 1988). The most recent model of hyphal tip growth (Silverman-Gavrila and Lew, 2001; Silverman-Gavrila and Lew, 2002) explains how the tip high gradient is maintained. In this model, stretch-activated phospholipase C is responsible for the localized production of inositol (1, 4, 5)-triphosphate (IP3) at the hyphal tip. IP3 binds to IP3-receptors on vesicles and facilitates release of calcium  16  contained in the vesicles. This, coupled with subapical C a endoplasmic reticulum, creates a tip-high C a  2+  2+  sequestration into the  gradient. The high concentration of C a  2+  at the  hyphal tip facilitates tip-localized fusion of vesicles. Silverman-Gavrila and Lew (2003) showed that the presence of a [Ca ] difference between the apex and subapical region, not 2+  the steepness of the gradient, is the requirement for growth to occur. The model requires a steady flow of vesicles to the tip, but the authors did not elaborate on how this flow is regulated, or how a switch from lateral to apical branching may occur. None of these models discusses how the branching pattern could switch from lateral to dichotomous branching, although models with the emphasis on calcium give options for one or the other branching pattern. A l l the mentioned models fit together already obtained information deemed relevant for tip growth and branching. This still leaves the possibility that there are other, perhaps novel, components crucial for the tip growth process, whose involvement has not yet been discovered.  1.3 Control Mechanisms in the Growth Process  Up to this point, building blocks that incorporate at sites of polarized cell expansion, and tip growth and branching models were reviewed. The next question that inevitably comes to mind is how distinct events relevant for hyphal morphogenesis are coordinated. Although information on regulatory mechanisms of hyphal growth is accumulating in fungal pathogens and model fdamentous fungi, the largest body of information is gathered from studies of bakers yeast, S. cerevisiae.  17  In unicellular S. cerevisiae cells, budding, shmoo formation, and pseudohyphal growth require polarization events. Selection of a polarization site depends on the cell type, determined by the mating type loci (Chant, 1999). Haploid cells containing either the MATa or MATa locus have an axial budding pattern, while diploid cells containing both MA Ta and MATa loci have a bipolar budding pattern. A transcriptional repressor, A x l l p , regulates the switch between axial and bipolar budding by activating the axial landmark during haploid cell growth, and the bipolar landmarks during diploid growth. Members of the axial landmark include Bud3p, Bud4p and BudlOp. They constitute a ring at the mother-bud neck that marks the site next to which the subsequent bud will be produced. At the bipolar landmark, Bud8p and Bud9p mark sites at opposite poles of the cell. Mutations in any of the landmark proteins cause the budding pattern to switch. The Ras GTPase Budlp (Rsrlp) reads the spatial cues and activates the Rho GTPase Cdc42p that in turn triggers the mitogenactivated protein (MAP) kinase signalling cascade. Budlp activity is regulated by the guanine nucleotide exchange factor (GEF) Bud5p and the GTPase-activating protein (GAP) Bud2p. Once Budlp is localized by the axial or bipolar cues, it is activated by its regulatory proteins, and stimulates the assembly of an apical scaffold, including Bemlp. During mating, an external gradient of pheromone binds and activates a G-protein coupled receptor (Pruyne and Bretscher, 2000). Receptor activation causes the G-protein heterotrimer to disassemble to G a and G(3y subunits. The free G(3y subunit then recruits Farlp, a polarity determinant, and together they set up a scaffold including Bemlp, activating Cdc42p, which in turn triggers the M A P kinase signalling cascade. In conditions of nitrogen starvation, yeast cells are stimulated to enter fdamentous growth (Lengeler et al., 2000).  18  During filamentous growth in diploid cells Ras2p, a small G-protein, sets off downstream signalling cascades through Cdc42p activation (Pruyne and Bretscher, 2000). Regardless of what upstream events recruit it, the Rho-GTPase Cdc42p has a central role in the assembly of a selected bud site and the orientation of the actin cytoskeleton toward this site (Schmidt and Hall, 1998). Several proteins regulate Cdc42p (Fig. 1.2). Cdc42p and Cdc24p are present as a complex at selected polarization sites. The GEF Cdc24p activates Cdc42p and promotes the association of Cdc42 with its effectors. Cdc24p also interacts with Budlp and Bemlp, important for the recognition of the polarization site tag. The GAPs Bem3p and Rgalp control the length of the signal by returning Cdc42 into the inactive form. For proper function, Cdc42p needs to be plasma membrane-bound, in the vicinity of its regulators. The geranyl-geranyl transferase Cdc43p adds prenyl groups necessary for Cdc42 to bind to the plasma membrane. The guanine nucleotide dissociation inhibitor (GDI) Rdilp binds to Cdc42p and translocates it to the cytoplasm, preventing the interaction of its G E F and GAPs. A n apical scaffold, the polarisome, has the function of clustering the Cdc42p-Cdc24p complex at polarization sites. The polarisome contains Spa2p, Pea2p, Bud6p, Bnilp, and Sphlp (Pruyne and Bretscher, 2000). These proteins are required for the assembly of actin, and make the connection between the signalling components and actin filament assembly. Bnilp binds to the Rho-GTPase complex, other polarisome proteins, profilin (Pfylp), and the actin-bundling protein Teflp/Tef2p. Another component of the polarisome, Bud6p (Aip3p), binds to actin filaments. Effectors of Cdc42p are p21-activated kinases (PAKs). In yeast, there are two P A K s that associate with Cdc42: Ste20p and Cla4p (Pruyne and Bretscher, 2000). Ste20p is  19  involved in apical growth, while Cla4p mediates the switch from apical to isotropic growth. Downstream, Ste20p activates a M A P kinase pathway that differs in its components depending on the original stimulus. In response to mating pheromone Ste20p activates the M A P kinase cascade that consists of Stel lp, Ste7p and Fus3p held together by the scaffold protein Ste5p. Fus3p then activates the transcriptional activator Stel2 that interacts with M c m l p to transcribe genes necessary for mating. In response to limited nitrogen availability, Ste20p activates the filament-inducing M A P kinase pathway. Elements of the pathway that differ from the mating type response pathway are the scaffold protein Spa2p instead of Ste5p, the M A P K Ksslp instead of Fus3p, and the association of Stel2 with Teclp instead of Mcmlp. The Stel2p/Teclp complex then activates genes that bring about pseudohyphal differentiation. In parallel with the M A P kinase pathway a cAMP-mediated pathway also results in pseudohyphal growth (Fig. 1.2). This pathway senses fluctuations in glucose, a carbon source, in the environment. Glucose binds to the G protein-coupled receptor Gprlp, and triggers the release of Gpa2p, the Got subunit of G-protein. Gpa2p then activates adenyly cyclase that produces cAMP. A t increased concentrations, c A M P binds to the regulatory unit of cAMP-dependent protein kinase (PKA), Bcylp, and releases Tpklp/Tpk3p, and Tpk2p, the active catalytic units of PKA.Tpk2p induces the production of Flol l p , a cell surface protein necessary for pseudohyphal growth, through the transcription factor Flo8p, and by inhibiting the transcriptional repressor Sfllp. Cross talk between the cAMP-mediated and M A P kinase pathways includes an activating role for Ras2p in both pathways, regulation of Flol l p expression by both pathways and possible sharing of Stel2p by both pathways. Other signalling pathways may 20  also be involved in regulation of pseudohyphal growth. Phospholipase C, Plcplp, bound to the Gprlp receptor appears to be necessary for the association of the Get subunit Gpa2p with the G-coupled receptor Gprlp. The exact role of Plclp in pseudohyphal growth is not clear. In fungi, most of the C a  2+  is sequestered in vacuoles, and phosphoinositide signalling is  involved in regulation of C a  2+  release from these stores (Davis, 2000).  Although some aspects of yeast unicellular growth are shared with filamentous cell growth, there are differences as well (Lengeler et al., 2000). The cAMP-mediated and the M A P kinase signalling pathway components are present and involved in filamentous or pseudofilamentous growth in S. cerevisiae, Schizosaccharomyces pombe, Candida albicans, Cryptococcus neoformans, Ustilago maydis, Magnaporthe grisea, Cryptonectria parasitica, Aspergillus nidulans and N. crassa. The way that components within these pathways are triggered and connected may vary depending on the environmental conditions and stage of the cell cycle, but the components themselves are conserved. Some of the genes and gene products involved in signalling pathways are presented in Tab. 1.1. Another signalling pathway, the Ca /calmodulin/calcineurin pathway could be 7-4-  important in tip growth through the regulation of the [Ca ] gradient. In N. crassa external calcium does not restore the disturbance in the [Ca ] gradient in hyphal of mutants that 2+  lacked the catalytic subunit of the calcineurin heterotrimer (Prokisch et al., 1997). Finally, regulation of a large number of actin binding proteins, e.g. proteins from the spectrin family (Hartwig, 1994), by phosphoinositides implicates the phosphoinositide signalling in regulation of actin function during hyphal development.  21  1.4 The Objective of This Study  The presence of actin at developmental hot spots in fdamentous fungi, such as sites of polarized growth, has long been established. To deduce the exact function of actin at sites of hyphal tip growth in fdamentous fungi, the main strategy was to disturb actin function by the use of inhibitors. This yielded information that implicated actin in processes such as cell wall deposition, secretion, hyphal morphogenesis, and polarization events. However, the way in which actin is involved, the components that actin interacts with and the regulation of the actin roles still remains unclear. N. crassa is a fdamentous fungus. It is an established model organism with a sequenced genome and a large collection of mutant strains, and has great potential to address issues related to hyphal developmental. The position of actin in the hierarchy of hyphal tip growth in N. crassa is not determined. Its role could be very different depending on whether it acts as an initiator or simply an effector. The involvement of actin in determining the branching pattern is also not clear. The mechanism regulating branching site selection is not known, and although it is possible to predict the formation of a lateral branch along the trunk of a hypha, the exact place where a lateral branch will be initiated, cannot be foretold. By generating actin mutants, I intend to contribute to the resolution of the role of actin in hyphal tip growth and branching. I expect changes introduced into the actin gene to disrupt the structure and function of the actin protein and result in a non-wild type phenotype. Mutants of this kind have a strong potential in identifying other components in hyphal morphogenesis, such as actin-binding proteins.  22  N. crassa has only one actin gene (Tinsley et al., 1998) yet produces three isoforms of the actin protein (Barja et al., 1991). In a previous study the importance of actin in hyphal morphogenesis led us to seek actin mutants by selecting for cytochalasin A resistance (Virag, 1999). Cytochalasins are compounds of fungal origin that cap the barbed ends of F-actin fdaments, and prevent the addition of G-actin monomers (Brown and Spudich, 1981; Cooper, 1987). Wild type strains of N. crassa react to the addition of cytochalasin A by changing the branching pattern and frequency creating a "starburst" phenotype at low concentrations, and by forming abnormal hyphae with swollen tips at high concentrations (Allen et al., 1980). In vitro experiments showed that cytochalasin binds to the barbed ends of actin filaments (Flanagan and Lin, 1980; Brown and Spudich, 1981), suggesting that this is the mode of cytochalasin action in vivo. Cytochalasin A-resistance of colonies derived from a mutagenized wild type strain could indicate a disruption in actin function or the function of an actin related protein. Using this genetic approach, I isolated the first actin mutant in N. crassa from such a screen, named it ccr-100, and characterized it. Besides being cytochalasin A-resistant, the ccr-100 mutant strain had a different colony morphology, branching pattern and frequency, as well as hyphal morphology (Virag, 1999). It was cold sensitive and female sterile. Crosses with mapping strains revealed that the mutated gene was a member of linkage group V . The actin gene maps to the same region of linkage group V , suggesting that the ccr-100 may be an allele of the actin gene. A more detailed overview of the characteristics of ccr-100 is presented in Chapter III. In this study, I determined by functional complementation that the gene mutated in the ccr-100 strain is the actin gene, so I renamed the mutant act .1 further analysed act at 1  1  the molecular and cellular level. I generated more actin mutants by Repeat Induced Point  23  mutations (RIP) and analysed them as well. Finally, I looked at the interactions between selected genes, and analysed the double mutants. As a result, I propose a model of hyphal tip growth and branching that assigns multiple roles for actin, and at the same time incorporates concepts from existing models. This model has the capacity to rationalize the various phenotypes encountered in the generated actin mutants, as well as phenotypes of mutants with mutations in genes other than actin.  24  Name of gene NC-ray smco-7/NC-ras2 gna-1 gna-2 cr-1 mcb nrc-1 nrc-2 cna cnb  Proposed function of gene product ras homolog ras homolog G protein a subunit G protein a subunit adenylyl cyclase protein kinase A regulatory subunit MAPKKK Ser/Thr protein kinase calcineurin A , catalytic subunit calcineurin B , regulatory subunit  Table 1.1. Some members of signaling pathways in N. crassa.  25  Distal region  Proximal region  —  *~  Hyphal tip region  r-S  Hypha  Apex  Subapical region Apical region  Retrograde movement  Anterograde movement  Figure 1.1. Terminology used to describe position and movement of components within a hypha. The hyphal tip region is the region in which the cell wall is still expanding. The part of this region closest to the apex (tip) is the apical region, while the region immediately behind, but still in the hyphal tip region, is the subapical region. Distal refers to a position closer to the tip. Proximal refers to a position further away from the apex. Retrograde movement is in the direction away from the apex. Anterograde movement is in the direction towards the apex. Also see Fig. 6.1.  26  Internal or external cues Ras >r ^ cAMP/PKA  . ^ G protein Sivpy subunits a subunit \ Rho-GTPases (clustering) Polarisome  cascade  \ p21 activated kinases MAP kinase cascade  /  Transcriptional regulators  I  Filamentous growth  Figure 1.2. Signal transduction cascades regulating polarized growth in fungi, with upstream and downstream components.  27  CHAPTER II  MATERIALS AND METHODS  2.1 Strains and Their Maintenance N. crassa wild type strains 74-OR23-1A (FGSC# 987), 74-OR8-la (FGSC# 988), mat a; gran (FGSC# 793), and the auxotrophic strain arg-3 mat A (FGSC# 1068) were obtained from the Fungal Genetics Stock Center (FGSC, University of Kansas Medical Center, Kansas City, K S ; Tab. 2.1).The mutant strain ccr-100 (Virag, 1999) was renamed act after 1  establishing that it is an allele of the actin gene. Strain 2-17-124 ad-3A mat a (FGSC# 4987) from Dr. Griffiths' lab collection was used to construct the double mutant ad-3A; act for 1  forced heterokaryons. Standard media and methods were used for growing and maintaining N. crassa strains (Brockman and de Serres, 1963; Davis and de Serres, 1970). The strain N2283 mat a his-3 ::Pccg-l hHl GFP , +  +  +  kindly provided by Dr. Michael  Freitag, was crossed with strain mat A; act , and the double mutant mat A his-3+::Pccg-l 1  hHl GFP ; act with an act phenotype and GFP-tagged nuclei was obtained. +  +  1  1  Strains used for constructing the double mutants were obtained from the FGSC, and are presented in Table 2.1.These strains were crossed with the act strain of the opposite 1  mating type. A heterokaryon containing act and a 1  ml  ad-3B cyh-1 nuclei was made before  crossing^- and act . Octads were picked and scored for the presence of tetratypes. The 1  double mutants isolated from these tetratypes were backcrossed with wild type to ensure the presence of both mutations. The double mutants were then analysed.  28  2.2 Cloning and Subcloning  A N. crassa chromosome V-specific genomic D N A library and the Orbach/Sachs cosmid library of 7Y. crassa genomic D N A sequences were obtained from the FGSC (Orbach, 1994; Kelkar et al., 2001). Transformations of act conidia with cosmids from the genomic 1  D N A libraries were conducted according to a modified electroporation protocol (Margolin et al., 1997). The conidia of act were not stored on ice because of the cold-sensitivity of the 1  strain. Fragments from partial restriction enzyme digests of cosmid G17B12 with Nhe I, Eco RVmd Sma I were re-ligated and transformed into act . A 2.1 kb fragment containing the 1  wild type actin gene was cut out from a pCR®II Invitrogen vector (a gift from Dr. Patrick Shiu, Stanford University, Stanford, CA) using the restriction enzyme Eco RI and ligated into vector pCB1004 (Carroll et el., 1994) at the multiple cloning site. Subcloning was done using standard techniques (Sambrook et a l , 1989). In some cases the DNeasy Plant Mini kit (Qiagen, Mississauga, ON) was used for genomic D N A isolation, and the QIAprep Spin Miniprep and QIAfilter Plasmid Midi kit (Qiagen, Mississauga, ON) were used for cosmid and plasmid isolations. The Expand High Fidelity PCR Kit (Roche Diagnostics, Laval, QC) was used for PCR amplifications. The primers used for amplification of the act and wild 1  type actin gene were A C T - F (5' A G C C A A A G C A T C A T C G C T C C 3') and A C T - R (5' T G C A G A C A T G C T A C T T G A C G C 3'). The PCR-amplified actin fragments were cloned into the pCR®2.1-TOPO vector using the Invitrogen TOPO T A Cloning Kit (Invitrogen, Burlington, ON). D N A was sequenced at the Nucleic Acid Protein Service Unit at the University of British Columbia using an Applied Biosystems PRISM 377 automated sequencer and the Applied Biosystems B i g D y e ™ v3.1 Terminator Chemistry. Sequences  29  were compared to the genomic D N A sequences from the N. crassa Genome Project at the Munich Information Center for Protein Sequences (MIPS, http://www.mips.biochem.mpg.de/proj/neurospora/).  2.3. Determining the Presence of Actin in the act Strain 1  The cytoplasmic protein fraction was extracted from act and wild type mycelium 1  using a modified protocol (Miles et al., 2002). Mycelium was grown on Vogel's minimal medium (VM) agar plates on cellophane sheets, and harvested five days after inoculation. For both wild type and act 500mg of mycelium was ground with sand under liquid nitrogen, 1  and suspended in the extraction buffer (1 mL extraction buffer per 1 g wet weight). The extraction buffer contained 50 m M HEPES, 1 Complete Mini EDTA-free Protease Inhibitor Cocktail Tablet (Roche, Diagnostics, Laval, QC) and 3.5 p L of P-mercaptoethanol per 10 mL of distilled deionized H2O. The solution was gently mixed for 1 h at 4°C and then centrifuged in a table-top centrifuge at maximum speed for 1.5 h. The pellet was discarded, and the total protein concentration in the supernatant was measured using the Bradford method (Sambrook et al., 1989). Proteins from the cytoplasmic fraction were separated on a SDS-PAGE gel and transferred to a PVDF membrane (Sambrook et al., 1989; Samuel, 2000). For both wild type and act the amounts of loaded proteins per lane were: 70 pg, 50 pg, 30 pg, and 10 pg. Full1  range rainbow molecular weight markers were used to determine the size of the separated proteins (Amersham Biosciences Corp., Piscataway, NJ). Actin was immuno-labeled with a 1:3000 dilution of 1 mg/mL purified mouse monoclonal anti-actin primary antibody 30  (Cedarlane, Hornby, ON). A 1:7500 dilution of goat anti-mouse horseradish peroxidase (HPvP)-conjugated immunoglobulin ( D A K O A/S, Denmark) was used as the labeled secondary antibody. The presence of HRP was detected using E C L Western blotting detection reagents (Amersham Biosciences Corp., Piscataway, NJ), and the chemiluminescence was recorded on a BioMax M R fdm (Fisher Scientific, Nepean, ON). The intensity of the detected actin bands was measured using the Multiimage Light Cabinet with the Alpha Imager 1220 ver.4.0 Documentation and Analysis System (Alpha Innotech Corporation, San Leandro, CA).  2.4 RIP (Repeat Induced Point Mutations) of act  Conidia of wild type were transformed by electroporation with the construct containing the 2.1 kb wild type actin fragment in vector pCB1004 (Orbach, 1994; Kelkar et al., 2001). Selection for hygromycin B resistance yielded several transformants. The transformants were crossed with the wild type strain of the same background but opposite mating type. Growth of heat-shocked ascospores from these crosses was observed on V M or on sorbose-containing agar medium (PM). Colonies that had a phenotype that differed from wild type were selected and characterized at the colony, hyphal morphology, cytological and molecular level. These colonies were then backcrossed again with the wild type strain with the same background but opposite mating type to assess the progeny phenotype, phenotype ratios and the presence of hygromycin B resistance. The actin gene from selected progeny was PCR amplified and cloned into a pCR®II Invitrogen vector and sequenced in the same manner as the act and wild type actin genes (see section 2.2). Sequences of the actin alleles 1  31  were compared to the genomic D N A sequences from the Neurospora crassa Genome Project at MIPS.  2.5 Hyphal Staining  For actin immuno-labeling act and wild type hyphae were grown on cover slips 1  dipped in warm V M containing molten 1.25% agar and placed on a wet fdter paper in a Petri dish. Conidia were inoculated on the cover slips and grown overnight or until the hyphae covered half of the area. Actin immuno-labeling was based on the method developed by Heath et al. (2000). The method I used differed from the original method in the concentrations of the cell wall-digesting enzymes, incubation times, and the absence of glutaraldehyde, phenylmethylsulfonyl fluoride and m-maleimidobenzoil N hydroxysucccinimide ester in the solutions. Glutaraldehyde was omitted to prevent autofluorescence. Healthy growing hyphae were fixed and permeabilized with 180 U.L of freshly made 3.7% paraformaldehyde or formaldehyde and 0.05% Triton X-100 in P E M buffer (60 m M PIPES buffer, pH 7.0 10 m M M g S 0 and 10 m M EGTA) for 20 min. The 4  hyphal wall was then digested with digestion enzyme solution for 45 s. The digestion enzyme solution contained 1 mg/mL Driselase and 1.6 mg/mL P-D-glucanase (InterSpex Products, Foster City, CA). This gave a satisfactory degree of cell wall digestion resulting in a higher resolution of actin distribution compared to previous reports. In the original method the cell wall was digested for 6-8 min with 9 mg/mL Driselase and 1 mg/mL Novozyme (Heath et al., 2000). This was followed by incubation with a 1:500 dilution of the primary antibody (mouse anti-actin monoclonal antibody, C4 clone from Cedarlane, Hornby, ON) in a humid  32  chamber at 37°C for 1 h. Before treating the sample with the secondary antibody, nonspecific protein binding was blocked by incubating the samples with 4% B S A in P E M buffer for 20 min. A 1:600 dilution of the secondary antibody conjugate (tetramethylrhodamine goat antimouse IgG from Cedarlane, Hornby, ON) was added next and incubated in a humid chamber at 37°C for 1 h. Nuclei were stained with 10 u.g/mL Hoescht 33258 in P E M buffer for 30 min followed by three rinses in P E M . Dilutions were made from a stock solution of 1 mg/mL (Raju, 1982). Staining with the vital fluorescent dye FM4-64 was by the protocol of Hickey et al. (2002).  2.6 Image Capturing  For photography colonies were grown on V M at 25°C in 8.5 cm-diameter Petri dishes. Images were captured with a Nikon Coolpix 5000 digital camera. Hyphae were observed on a Zeiss Axioplan 2 Fluorescence microscope and a BioRad Radiance 200 Confocal Microscope using appropriate filter sets. Images were collected from the fluorescence microscope with an Empix Video Image Capture system including a D V C Digital Mono C C D Camera or a Mono cooled 12B Digital (Retiga 1300) camera (Qlmaging, Burnaby, BC) and Northern Eclipse v.6.0 image capturing software (Empix Imaging Inc., Mississauga, ON). Images were organized and false-coloured in Adobe Photoshop 5.0. The three-dimensional model of actin was retrieved from Swiss-Model, a protein structure homology-server, and the program DeepView-spdv 3.7 (Guex and Peitsch, 1997; Schwede et al., 2003).  33  FGSC#  Genotype  988 987 1068 4987 4564 1068 1401 3848 3847 3462 3461 1372 2233 102 103 1175 793 794 1296  74-OR8-la 74-OR23-1A arg-3 A ad-3A a a ad-3B cyh-1 a arg-3 A col-8; a col-15; a col-15; A col-16; a col-16; A spco-4; a spco-4; A fra frA spa gran; a gran; A md; a ml  Table 2.1. List of strains from the FGSC used in this study, including morphological mutants used to generate double mutants with act . 1  34  CHAPTER III CLONING OF act AND SOME CHARACTERISTICS OF THE act MUTANT 1  1  STRAIN  3.1 Introduction  In a previous study, selection for cytochalasin A resistance in UV-mutated conidia of  N. crassa yielded a total of 28 cytochalasin A-resistant colonies (Virag, 1999). These colonies grew more vigorously on cytochalasin A-containing medium and/or had a different branching pattern compared to the control colonies from non-irradiated conidia. I focused on one particularly promising mutant strain that I named ccr-100 (cytochalasin A-resistant, isolate number 100), and subsequently re-named act . To determine whether mutation 1  occurred at one or more loci, the original act mutant strain was backcrossed with the wild 1  type strain of the opposite mating type. The mutant and wild type progeny segregated in a 1:1 ratio in each of 24 asci tested, indicating that there was only one mutated locus. The act  1  strain was crossed with the mapping strain alcoy, and subsequently with the mutant strains aur mat A; cot-1; inl, mat a; gran, and mat a; pk. The segregation of the progeny showed that act was a part of linkage group V , and was linked 7.8% to inl, 6.4% to gran and 7.7% to pk. 1  Tinsley et al. (1998) showed by RFLP analysis that the actin gene is also a member of linkage group V and is located in the same region, close to inl, revealing the possibility that act is an allele of the actin gene. 1  Besides being cytochalasin A resistant, the phenotype of act differed from the wild 1  type by having different colony morphology, branching pattern, branching frequency, and  35  hyphal morphology (Fig. 3.1). I divided the growth of act into four stages. In the initial stage 1  of act growth, germinating conidia and young hyphae just beginning to branch could not be 1  distinguished from wild type. The distance between branching points corresponded to the same distances in wild type, but the branching pattern was mainly dichotomous (Fig. 3.ID). The branching pattern remained predominantly dichotomous throughout all the stages of growth. Very few aerial hyphae developed in this stage, and the colony appeared flat on the medium surface. In the second, intermediate, stage the intervals between branching sites decreased in length (Fig. 3.IE). Towards the end of this period, development of aerial hyphae intensified. The colony still appeared flat, but thicker. The conidiating stage was characterized by mass development of aerial hyphae, accompanied by the abundant production of conidia. At this stage, the colony resembled the shape of a doughnut (Fig. 3.IB). The distance between new branching sites continued to decrease (Fig. 3.IF). Next was the constant stage, which lasted until the colony reached the limits of the medium. New branches formed closer to each other compared to the previous three stages, and continued growth did not result in a further decrease in the distance between branches (Fig. 3.1G). However this seemingly continuous growth included some cycling in the frequency of branching: periodically there was an escape of less frequently branched hyphae. These hyphae then continued decreasing the distance between branches until a new wave of less frequently branched hyphae overtook them. Aerial hyphae and conidia continued to develop, although they were less abundant than in the previous stage (Fig. 3.1C). Hyphae of act had 1  what looked like small aborted branches, giving them a less linear look than wild type hyphae (Fig. 3.ID). Their tips were not as tapered as in wild type hyphae.  36  The average of lengths between branching sites at various stages of act growth with 1  and without cytochalasin A in the medium can be seen in Fig. 3.11. The average length between branching sites in wild type on V M was 172.07 (±4.00, N=1323). Dichotomous branching occurred in 85% of the total number of branching events in act , considerably 1  more than the 1% of dichotomous branches observed in wild type. On cover slips covered with solid V M act had a slower growth rate than the wild type. The average growth rate of 1  individual act hyphae in the initial stage was 2.66 pm/min (±0.26, N=42), which was less 1  than the 12.37 pm/min (±1.64, N=28) recorded in wild type. Average colony growth rate, increase in wet weight, increase in dry weight and increase in biomass in act followed the 1  same trend through time and relative to wild. On medium containing cytochalasin A , the growth rate of wild type was slower than the growth rate of act . 1  Dicker and Turian (1990) showed that the frost and spray mutants can be converted into a wild type phenocopy by adding C a  2 +  to the medium, suggesting a calcium-dependent  function for the altered proteins. To test if C a  2+  added exogenously can revert act to wild 1  type, I grew act on solid V M containing 5 mM, 50 mM, and 100 mM CaCl -2H20. Neither 1  2  act nor wild type showed any differences on these concentrations of calcium. V M 1  containing 300 mM CaCl2-2H20 slightly slowed down the growth of the wild type strain, but did not affect act . There were no noticeable changes in the morphology in any cases. These 1  results showed that even if the internal calcium concentrations in act are modified, they do 1  not affect growth. Actin filaments exposed to low temperatures undergo de-polymerization in vitro (Hayashi, 1967). Microfilaments also undergo changes after a shift to lower temperatures in vivo, e.g. in platelets (Winokur and Hartwig, 1995). These results prompted us to test the  37  effect of low temperatures on the growth of act . N. crassa wild type strains show a cold1  shock effect after sudden reductions in temperature (Watters et al., 1999). The cold-shock effect includes changes in the branching pattern and frequency. When transferred from 25°C to 6°C, growth of act stopped completely showing that act is cold sensitive. A subsequent 1  1  shift from 6°C to 25°C restored growth, showing that the cessation of growth was reversible. The wild type control strain continued growing after the temperature shift, albeit at a slower growth rate, and showed changes in branching pattern and frequency characteristic for the cold-shock effect. Crosses in which the mutant act strain was used as the female strain produced 1  perithecia that did not form beaks, and did not contain viable ascospores, indicating that act  1  strains are female sterile. However they are fertile as paternal parents.  3.2 Results  3.2.1 Cloning and sequencing of act  1  As a prerequisite for sib-selection, I needed to ensure that the mutation was recessive. I assessed the phenotype of forced heterokaryons consisting of nuclei of ad-3A mat A; act  1  and arg-3 mat A. The phenotype of heterokaryotic colonies was almost entirely wild type, except for small regions of the colonies that showed a higher incidence of dichotomous branching, probably a result of an imbalance in the nuclear ratios. The wild type phenotype of the heterokaryons indicated that the mutated allele was recessive.  38  The wild type act allele was cloned by sib-selection, using hygromycin B resistance 1  as an indicator of vector entry. Clone G17B12 from the Orbach/Sachs cosmid library of N. crassa genomic D N A sequences rescued the mutant phenotype (Orbach, 1994; Kelkar et al., 2001). At the time, the cosmids of the library were not ordered, so I end-sequenced cosmid G17B12. The end-sequences were compared to the genomic D N A sequences from the N. crassa genome project at MIPS. The comparison showed that cosmid G17B12 was a part of contig c7al6 on linkage group V . The cosmid contained 6 open reading frames (ORFs), so I subcloned fragments of the cosmid insert to pinpoint the region in which the act mutation 1  occurred (Fig. 3.2). The fragment containing the actin gene complemented the mutant phenotype. Because there was more than one open reading frame in the complementing fragment, I inserted the wild type actin gene PCR amplified from a clone generously donated by Dr. Patrick Shiu, Stanford University, Stanford, C A , into vector pCB1004 (Carroll et al., 1994) and transformed the act mutant strain with this construct. This actin clone complemented the 1  mutant phenotype, strongly suggesting that the mutation had occurred in the actin gene. I amplified, cloned and sequenced the act mutant and wild type alleles using selected primers. 1  The wild type sequence was identical to the actin sequence made available by the Neurospora Genome Project (MIPS), but only partially identical to the sequence originally published by Tinsley et al. (1998), which is missing a 64 base pair sequence at intron V. The mutant actin allele act has a G C to T A transversion at position 1667 (exon VI) of the 1673 base pair 1  open reading frame. To confirm this change and to check its segregation in relation to act ,1 1  sequenced the actin gene of each member of an octad from the cross between wild type and act . The single consistent change in all four act progeny was the point mutation at position 1  1  39  1667. This change was absent in all four of the wild type progeny. The possibility of this cosegregation occurring by chance is 4/8 x 3/7 x 2/6 x 1/5=1.4%, supporting the hypothesis that the mutation is the basis for the act phenotype. Additional point mutations that were present 1  in the individual progeny due to errors in the PCR or sequencing reactions indicated that there was one error per every 3.35 x 10 base pairs, i.e. 1 base pair mistake per two sequenced actin genes. The ORF of both wild type and act translate to a polypeptide 375 amino acids long. 1  The mutation results in a substitution of the penultimate residue (cysteine) at position 374 in actin by phenylalanine (Fig. 3.3). The position of the amino acid substitution was in domain IA of the folded protein. This domain is exposed at the barbed end of F-actin fdaments, and interacts with actin binding proteins in other organisms (Sheterline et al., 1998).  3.2.2.Cytology of act  1  Ultimately, the colony and hyphal morphologies as well as the branching characteristics of act are the result of the mutation in the actin gene. Knowing that there is a 1  change in the amino acid sequence of the actin protein, I hypothesized that if this change affects actin function, there might be an altered actin distribution. Therefore, as the first step to determine how the change in actin can result in this phenotype, I looked at actin distribution in hyphae of act by immuno-labelling. Previously published images of actin in 1  wild type hyphae showed a cloud of fluorescence in the hyphal tip, and at the very apex actin was proposed to be concentrated at the position of the Spitzenkdrper (Heath et al., 2000). However, fine details of the actin distribution were not known. I therefore modified the  40  immuno-labelling protocol to reduce the amount of fluorescence in the hyphal tip to give better resolution of actin distribution. To make sure that I was not observing non-specific binding, I immuno-labelled wild type and act cytoplasmic actin on Western blots using the 1  same primary antibody and observed only one strong band after signal detection in both cases (Fig. 3.4). This confirmed that the primary antibody binds specifically to actin. To determine if the defects in act could be partially due to the amount of produced actin, I assessed the 1  amounts of actin in the wild type and act strains. I measured the intensity of the detected 1  bands, and compared them using Student's paired t-test. The band intensities (Fig. 3.4) are presented in Table 3.1. The t value was 2.71, n=3, and the probability of this result assuming the null hypothesis was 0.073. This shows that the amount of actin, although somewhat higher in wild type than in act , is not significantly different. 1  In a growing wild type hypha, I found, as expected, that actin typically formed a cloud adjacent to the extreme apex (85.71% of cases, N=35), and a number of peripheral plaques subapically (Figs. 3.5A, B and D). The number of plaques diminished further away from the tip. The range of shapes of the apical actin accumulation included spherical (with or without a less intensely stained center), half-spherical, ovoid, horseshoe-shaped, triangular, and irregular. The most frequently encountered shape was an intermediate between spherical and half-spherical. Small actin plaques co-localized with some, but not all nuclei (Figs. 3.6AC). I also observed small spherical structures that bind actin strongly but which I could not identify (Fig. 3.6D). These structures were usually absent from the tip, and were positioned either individually or a few in the same hyphal compartment. The most striking feature of the actin distribution in the act mutant was the mainly 1  subapical position of the actin accumulation at the hyphal tip. Subapical actin had either the  41  shape of a cap (12.87% of cases, N=101), a large round accumulation (31.68%), two accumulations beside each other (8.91%), or multiple small accumulations (19.80%, Figs. 3.5E-H, Fig. 3.7). Actin accumulations were mainly present in the central region of the hypha, but occasionally could be observed in the peripheral subapical region (Figs. 3.5J and K). In some hyphae (2.97%) one actin apical accumulation, smaller and less intensely stained, was present at the extreme apex together with an intricate subapical peripheral network (Fig. 3.51). Sometimes combinations of different distributions were present. Some hyphae did not have any actin accumulation at the tip, and I suspect that they were not growing at the time of fixation (23.76%). The Differential Interference Contrast (DIC) microscopy images and presence of actin staining distal from the position of the subapical actin accumulations show that the changes did not occur as the result of plasmolysis. As in wild type, some actin co-localized with nuclei. Nuclei were found closer to the hyphal tip in act , and were more abundant in the subapical region compared to the wild type hyphae in 1  fixed cells (Figs. 3.5C and L). The presence of actin at sites of tip growth in N. crassa has been well established (Degousee et al., 2000; Heath et al., 2000). However, there is no information about the distribution of actin at the hyphal tip during a branching event. The fact that the dichotomously branching phenotype of act is a result of a mutation in the actin gene makes 1  this mutant particularly interesting for this kind of investigation. M y preliminary observations show that in the act mutant before a branch occurs no significant actin 1  accumulations are present. Sometimes there would be multiple weakly stained subapical actin dots or accumulations that had a thin filamentous appearance. However, in hyphal tips that have committed to a dichotomous branching event, strongly stained actin accumulations  42  became visible when the new branches could be distinguished. In some hyphae at various stages of a dichotomous branching event one large subapical accumulation was present at the basis of the branch. The dynamic changes in the size, shape and position of the Spitzenkdrper during hyphal growth have been demonstrated in several fungi, including N. crassa, by phasecontrast microscopy and by vital staining with the dye FM4-64 (Lopez-Franco and Bracker; 1996, Fischer-Parton et al., 2000). Fisher-Parton et al. (2000) showed that the shape of the Spitzenkdrper core region stained with FM4-64 ranged from circular to horseshoe-shaped, usually having an area of reduced fluorescence within it. In the present work, the range of shapes of the actin accumulation at the hyphal tip in the wild type strain corresponds to the range of shapes of the Spitzenkdrper core region. Growing hyphal tips of act treated with 1  FM4-64 have an apical structure that corresponds to the Spitzenkdrper (Fig. 3.8, Fig. 3.9). The size of the Spitzenkdrper is smaller in act than in wild type, probably a result of the 1  slower growth rate of act (Fig. 3.8). Subapically, a less stained circular or cap-like region is 1  usually visible. The position of this region corresponds to the position of subapical actin accumulations (compare Figs. 3.5 and 3.8). During growth, on some occasions the hyphal tip starts an apical branching sequence, but one of the two branches aborts, while the other continues growing, giving the typical "aborted branch" appearance. Staining with FM4-64 revealed that a Spitzenkdrper is present at each branch and that it disappears in the aborted branch when growth stops. During dichotomous branching in act two Spitzenkdrpers appear 1  at the apex prior to the emergence of two new branches (Fig. 3.9). Some observations of the growth of wild type hyphae provided information about the behaviour of the Spitzenkdrper during lateral and apical branching. Hyphae stained with 4-8  43  p M FM4-64 continued growing with a wild type hyphal morphology and branching pattern after the addition of the vital stain. During growth, the Spitzenkorper was constantly changing its shape, but remained in the apical region at all times. Growth proceeded at a constant growth rate, but preceding the emergence of two lateral branches behind the hyphal tip region, the growth rate slowed down, and the Spitzenkorper disappeared. As soon as the two new branches emerged, the hyphal tip resumed growth, and the Spitzenkorper became visible again (Fig. 3.10). Shortly after the lateral branches started growing, a Spitzenkorper became visible in the apical region of each branch. After the addition of 32 p M FM4-64, hyphal tips stopped growing. Growth proceeded a few minutes later with a few rounds of atypical apical or dichotomous branching events, followed by a return to the lateral branching pattern. The growth of a tip undergoing an apical branching event was recorded (Fig. 3.11). A single Spitzenkorper was visible before branching. Simultaneously with the emergence of two new apical branches, two additional Spitzenkdrpers became visible at their apices. During this time, the initial Spitzenkorper did not disappear, and maintained its position at the site that became the third branch. Growth continued with one Spitzenkorper in the apical region of each of the three branches. The dynamics of the Spitzenkorper during lateral and apical branching was different. Wild type and act strains with GFP-labelled nuclei were also stained with FM4-64, 1  and their growth was followed. The presence of FM4-64 at the apex of hyphae showed that the hyphae had a Spitzenkorper, an indicator of healthy, growing hyphae. Leading wild type hyphae had abundant nuclei throughout the hyphal diameter except in the hyphal tip region (Figs. 3.12A-D). Primary and secondary lateral branches had a smaller diameter, and fewer nuclei per same length of hypha (Fig. 3.12E). Only long lateral branches had nuclei (Fig. 44  3.12B), while short, young lateral branches lacked them (Fig. 3.12A). Nuclei close to the hyphal tip were often elongated both in wild type and act strains (Figs. 3.12C, D, F, and I), 1  indicating that they may be dividing. In both strains the nuclei migrated towards the tip as the hypha grew, but maintained a distance from the apex. FM4-64-stained membranes showed anterograde movement as well. In the act strain, nuclei were present closer to the hyphal tip 1  than in wild type (Fig. 3.12J-L). A n unstained vacuole-like compartment was often present at the tip of act hyphae, behind the Spitzenkdrper (arrows in Figs. 3.12F-I). This compartment 1  was present regardless of whether the hyphae were in the process of apical (Figs. 3.12G and I) or lateral branching (Fig. 3.12H). The FM4-64-free compartment at the tip did not colocalize with a nucleus, as was the case with many subapical compartments that were not stained with the vital dye.  3.2.3 Loss of polarity in act at threshold temperature 1  The act strain was cold sensitive and stopped growing after its colony was incubated 1  at 6°C. I shifted the temperature from 25°C to various lower temperatures, and established that in act a unique effect occurred at 7°C. This effect does not occur in other newly induced 1  actin mutants (see Chapter IV). When act colonies were transferred from 25°C to 7°C they 1  initially stopped growing (Figs. 3.13A and 3.13B). There were no prominent actin accumulations at the hyphal tip at this stage. A few hours after the temperature downshift the hyphal tips went through a stage of isotropic growth, indicating a loss of polarity (Figs. 3.13C and 3.13D). During this stage, I could still not observe prominent actin accumulations at the hyphal tip. Polarity was subsequently resumed at seemingly random sites. Hyphal 45  growth continued at a significantly slower rate, and with an enlarged hyphal diameter (Figs. 3.13E and 3.13F). A large subapical actin accumulation could be seen in hyphal tips at this stage, mainly in the axial region of the hypha. Eventually the hyphae directed their growth towards the colony periphery, retaining the subapical position of the actin accumulation in each. Wild type control hyphae showed the expected lag, starburst, and recovery phases after the temperature downshift, as observed by Watters et al. (2000). In wild type at 7°C an apical actin accumulation was present during linear growth, as well as in dichotomous branches (Figs. 3.13G-3.13J).  3.3 Summary  The following is a summary of results related to act , with some additional details. 1  The incorporation of these results into a new hyphal tip growth and branching model is discussed in Chapter VI. In designing this study, I hypothesized that a selection for cytochalasin A-resistance will yield mutants in the actin gene or genes of actin-related proteins. The hypothesis was confirmed, and I succeeded in isolating the first actin mutant in N. crassa. In this study, sequencing of the mutant actin allele identified the mutation in the D N A sequence as a transversion that translates into a single change in the amino-acid sequence. The properties of the act mutant were: resistance to cytochalasin A , an abnormal colony and hyphal 1  morphology, an abnormal branching pattern, cold sensitivity and loss of polarity, an abnormal actin distribution, aberrant nuclear distribution and small Spitzenkorper size. The  46  amount of actin in wild type appeared to be slightly higher than in act , but the difference 1  was not significant. Characteristics of the act phenotype give some insight in the relationship between the 1  Spitzenkorper and actin present at the hyphal tip. Actin showed a spherical accumulation with a subapical position in most hyphae of act . At the same time, this change did not 1  prevent hyphal elongation, and the apex still contained a Spitzenkorper, although smaller in size than in wild type. This indicated that most of the actin at the hyphal tip is not a functional part of the Spitzenkorper. However, since some tips of act have a second small 1  apical actin accumulation at the extreme apex, a part of the apical actin accumulation may still function within the Spitzenkorper. The smaller size of the act Spitzenkorper may result 1  from a slower hyphal growth rate, independent of the actin distribution at the tip. Alternatively, if the apical actin within the Spitzenkorper is functioning as a Spitzenkorperorganizing centre, then the smaller size of the Spitzenkorper in act may reflect the presence 1  of a reduced amount of actin, and a consequent decreased organizing capacity. This would also mean that all the growing tips should have a small apical actin accumulation, which does not correspond to the data unless the amounts of apical actin are too low to be detected, or i f the actin antibody-binding domains are occupied at the Spitzenkorper. The positions of the FM4-64-free compartments behind the act hyphal apices correspond to the positions of 1  subapical actin accumulations in fixed immuno-labelled hyphae, and corroborate the actin distribution data obtained in fixed cells. Actin and the Spitzenkorper may also functionally cooperate in determining the branching pattern. The act strain had dichotomous branching, as opposed to the lateral 1  branching pattern in wild type. The switch from lateral to dichotomous branching may reflect  47  a change in the hyphal tip polarity maintenance apparatus. A t early stages of a dichotomous branching event, before the two new branches emerged, apical actin accumulations were absent, with a large subapical accumulation present in some cases. During the same process the Spitzenkdrper disappeared immediately before the two new branches emerged, and two Spitzenkdrpers appeared after emergence of the two new branches. The absence of the Spitzenkdrper at this stage is in agreement with the disappearance of the Spitzenkdrper during early stages of dichotomous branching in the ramosa-1 mutant of Aspergillus niger, suggesting that the same mechanism of branch pattern regulation is in action in both fungi (Reynaga-Pena and Bartnicki-Garcia, 1997). A further discussion on the possible interaction between the actin accumulations and the Spitzenkdrper is presented in the context of the new model for hyphal tip growth and branching (see Chapter VI). The Spitzenkdrper behaviour during lateral and apical branching in wild type gives some insight in the branching mechanism. The main Spitzenkdrper feature important for branching is its origin. Previous studies in fungi proposed de novo formation (Reynaga-Pena and Bartnicki-Garcia, 1997). The slower growth rate and the disappearance of the Spitzenkdrper prior to the emergence of two lateral branches indicated the dependence of the formation of the two new Spitzenkdrpers on the disassembly of the existing one. This could mean that at least some vesicles of the existing Spitzenkdrper were incorporated into the two new Spitzenkdrpers, and that the new Spitzenkdrpers were formed with the vesicle contribution of the previously existing one. Alternatively, the disassembly of the existing Spitzenkdrper may be necessary to allow the formation of more than one new Spitzenkdrpers without the incorporation of the disassembled vesicles into the new Spitzenkdrpers. In contrast, during apical branching, the persistence of the one existing Spitzenkdrper during the  48  formation of two new ones indicated independence in their formation. It is possible that the same branching mechanism is underlying both formation of a Spitzenkdrper from an existing one, and de novo formation. The mode that will be switched on would then depend on the conditions at the hyphal tip. In this case, one option is that the apical dominance of C a , 2+  proposed to inhibit additional branches at the tip (Schmid and Harold, 1988), is creating conditions that would favour a certain mode of Spitzenkdrper formation. If Spitzenkdrpers are formed de novo in all conditions, then events during lateral branching can be explained by competition for the vesicle pool destined for incorporation at growth sites. The two lateral branches may simply be draining away the vesicles from the leading hyphal tip causing the temporary dissipation of the existing Spitzenkdrper. The different nuclear distribution in act is most likely not significant for hyphal tip 1  growth or branching, but is instead an additional effect that the actin mutation confers, emphasizing the pleiotropic nature of changes in the actin gene. The positioning of nuclei closer to hyphal tips in act both in fixed and live cells could be related to a wider diameter 1  of hyphae and a slower growth rate in act , or to a disruption in the mechanism by which the 1  nuclei are maintained at a constant distance from the hyphal apex. A downshift of act colonies to a threshold low temperature accentuates the existing 1  polarity defect to the extent of complete polarity loss following the shift. However, the loss of polarity is transient, and a random site is selected for continued growth. Although adaptation to cold is a complex process, this loss of polarity additionally implicates actin in the process of polarization site selection.  49  Strain Lane on film Amount of total protein [p,g] Area of peak  wild type B A 50 70 2805 2150  act  1  C 30 1478  D 10 658  E 70 1858  F 50 2000  G 30 942  H 10 382  Table 3.1. Amount of actin in bands from Fig. 3.4 expressed through the peak area measured using the Alphalmagerl220 ver.4.0 system. The lanes correspond to the lanes in Fig. 3.4. The amount of total protein is the amount of protein in the cytoplasmic fraction that is loaded in the appropriate lane.  50  Figure 3.1. (A) Three-day-old wild type colony. (B) Three-day-old act colony. (C) Six-day-old act colony. Hyphal morphology of (D, E , F, G) act and (H) wild type. (D) Initial stage. (E) Intermediate stage (F) Conidiating stage. (G) Constant stage. AU bars = 100 Jim. (I) The median of lengths between branching sites in strain act on solid V M containing 10 jxg/ml cytochalasin A (VM+10), and without cytochalasin A (VM). The bars represent the standard error. White columns show the resultsfromthe initial stage, light gray columns show results from the intermediate stage, and dark grey columns show the results from the constant stage. 1  1  1  1  51  CD O O  r^. CL -Q CO CO  a  M-  3  CO  CL  -Q  <  Q . -Q  0  Z  2=  CD CL  C £  -9  O  CN CN ID Q .  S  O  -9 -° °y  /CvNi " LO <N ID ^ 0  CL  CD co\<u o •— i- < D  CL -Q  CD C L + 5 h~ CL _ O -Q CM i CL CD o C O U 3 CO C D C N CU CM £ E  ca  CO  h-  CM CO CL  Cl i  Q. d  Q - O S> C L  CO ^ CD CD  o  CO  o  CD C M  CL  O  CL •== - Q CD  I1 s "  3  CD CO  •4-*  O CO  CL'fl)  I  ° CD CM  c CD  2  -*-'  CO  >  u CJ  •B  I <+-!  5 o  CL  "TO O CCL"m CD  0  CD  £ ?  CD  ck ro 0 t; t; o a o 0 Q . B IS Q . < O f * > D 4) > 1 Q. L t I V- T-cMco-sriDCor^ CD • • • • • • • CM  1  to  1 C  •a  O  -9  ro  5  o  o  £ T3 CD CD  So  co  .£ o  1  G)  CO  co  0  r^.  o g  D-T-  co  _  o  o 1  CN CL JD CM C O i  Q.CO CL O CD CN . O ^ co o_ x O j Q CO h~ ^ID CL uT CL C L CO  S8 -Q  2?  i CN CO CO CO Q_  o  .2  CM CD  CN  o  CO CO  +=  52  c c  Figure 3.3. (A) Three-dimensional model of actin. Arrow and red colour in ribbon indicate the position of C 3 7 4 at the carboxyl terminal. IA-IIB indicate the subdomains on the actin molecule. The model was retrieved from Swiss-Model, a protein structure homology-server, and the program DeepView-spdv 3.7. (B) Position of actin domains in an F-actin filament.  53  A B C D  E F G H  50kD — 35kD —  F i g u r e 3.4. Actin presence in total protein extracts of w i l d type and act . Lanes ( A - D ) show w i l d type actin. Lanes (E-G) show act actin. Amount o f total protein loaded into the wells o f the S D S - P A G E gel were 70 u g (lanes A and E), 50 u g (lanes B and F), 30 p:g (lanes C and G), and 10 fig (lanes D and H). The arrows indicate the positions o f recombinant proteins from the full-range molecular weight markers (see Chapter II). 1  1  54  B  D  C  #  E  T  F  '  G  H  '  *  T *-»  K  1  *  L  ' gjj  •  Figure 3.5. (A) Brightfield image, (B) apical actin distribution, and (C) nuclear distribution of a wild type hyphal tip. (D) Head-on view of the apical actin distribution in a wild type hyphal tip. Actin distribution in act : (E) subapical actin cap, (F) large spherical subapical actin accumulation, (G) two subapical actin accumulations, (H) multiple small subapical actin accumulations, and (I) small apical actin accumulation. (J) Brightfield image, (K) peripherally displaced subapical actin accumulation, and (L) nuclear distribution of an act hyphal tip. Arrowheads indicate the position of the extreme apex. A l l bars = 10 fim. Bar in image (A) applies to images (B, C). Bar in image (E) applies to the rest of the images without bars. 1  1  55  Figure 3.6. Co-localization of actin with some nuclei in wild type. (A) Merged image of actin distribution and nuclear distribution (B) Actin distribution. ( C ) Nuclear distribution (D) A different hyphal segment of wild type with ring-like actin structures. Bar = 10 pm. Colour was added using the program Photoshop 5.0.  56  A  B  C  D  E  F  •  |  1  H  G  1  -:  *  L  K  J 1  Figure 3.7. Several act hyphal tips. (A-L) Successive images of longitudinal planes 0.45 pm apart. Subapical and apical actin accumulations are present in hyphal tips. Bar= 10 pm. 1  57  Figure 3.8. FM4-64 staining of (A) wild type and (B) act strains The farrighthypha in image (B) is branching dichotomously. Arrowheads indicate less stained areas that correspond to the position of subapical actin accumulations in act . Note the smaller size of the Spitzenkdrper in act . Bar = 10 jam. 1  1  1  58  A  —  c  B ft  JK  E  D i" •1  '•'•A  :  .  | G  F  v. ' "  J  1  H  •  ;  L  K  .  ',;  *  M 1,1  :'; *t  N  0  A  M  M  • •  P  Q  (  1  •  s  R  T  1  r  U  V  W  x  •  ffl  ©•  Figure 3.9. (A-Y) Growth of two act-' hyphae stained with FM4-64 viewed at 15 s intervals. (F) Two Spitzenkdrpers are visible at the apex before the emergence of two new branches in the hypha on the left. ( O ) Two Spitzenkdrpers are visible at the apex before the emergence of two new branches in the hypha on the right. Bar = 19 |xm.  59  A  B  C  F  G  H  L  M  D  1  E  J  **  K  N  0 -*  P  Q  R  S  T  U  V  W  X  Y  Figure 3.10. Wild type hyphae stained with 8 pM FM4-64. (A-Y) Successive images collected 15 s apart. (L) The Spitzenkorper disappears. (O) The triangular arrowhead indicates the emergence of thefirstlateral branch. (P) The triangluar arrowhead indicates the emergence of the second lateral branch. (V) The Spitzenkorper reappears. (L-V) The growth rate is slower. Bar - 20 pm.  60  A  C  B jib  D  E  n  G  H  J  K  1  L  Figure 3.11. Growth o f w i l d type hyphae stained with 32 p M FM4-64. ( A - L ) Images taken 5 s apart. (F) The two triangular arrowheads indicate the emergence o f two new apical branches with Spitzenkdrpers. Bar = 20 pm.  61  Figure 3.12. FM4-64 stained (A-E) wild type and (F-L) act strains with GFP-tagged nuclei. (A) A leading hypha with abundant nuclei and two lateral branches without nuclei. Each tip has a Spitzenkorper. (B) A leading hypha with three branches. The two longer branches have nuclei, and all tips have a Spitzenkorper. (C, D) Leading hyphae with round and elongated nuclei. (E) Primary lateral branch with a smaller diameter and less nuclei. (F) Leading hypha, (G) small dichotomous branch, (H) small lateral branch, and (I) an unbranched and branched hypha. Arrows indicate the region that often has a compartment not stained with FM4-64. (J) Hyphal tips with less nuclei per diameter than wild type. (K) An early dichotomous branch. (L) Hyphal tips with nuclei closer to the tip than in wild type. Bar in image (A) = 20 pm and applies to images (A, B, J , K , L). Bar in image (C) = 10 pm, and applies to the rest of the images. 1  62  Figure 3.13. (A, C , E, G , I) DIC images and (B, D, F, H, I) actin distribution in (A-F) act and (G-J) wild type after temperature downshift. (A, B) Initial absence of actin accumulation at hyphal tip. (C, D) Loss of polarity and tip swelling. (E, F) Regained polarity with an increase in hyphal diameter and a subapically displaced enlarged actin accumulation. ( G , H) Absence of actin accumulations in early stages of apical branching. (I, J) Presence of apical actin accumulations in later stages of apical branching. Bars = 10 pm. Bar in image (A) applies to images (C-F). Bar in image (G) applies to images (i-J). 1  63  C H A P T E R IV RIP-GENERATED ACTIN MUTANTS  4.1 Introduction  In N. crassa hyphae, the actin protein participates in a multitude of processes (see Chapters I and III). A n efficient way of determining which processes actin is involved in is through a genetic approach. It is based on altering or disrupting a gene, and observing the range of deficiencies that the alteration or disruption causes. Each deficiency points to a process in which a specific region of the gene, or the gene as a whole, is involved. This genetic approach yielded the act allele of the N. crassa actin gene. Although the act allele 1  1  allowed the deduction of some roles of actin (see Chapter III), I suspected that there were other aspects of actin function that were not affected by the act mutation. It was, therefore, 1  necessary to generate additional actin alleles to address this issue. To create more actin mutants, I took advantage of the N. crassa RIP (repeat induced point mutations) machinery. The RIP machinery recognizes duplicated D N A sequences (Selker and Garrett, 1988), and introduces point mutations into the duplicated and original sequences during the sexual cycle (Irelan and Selker, 1996). The multiple point mutations are typically G C to A T transitions that occur after fertilization but prior to meiosis (Selker et al., 1987). The frequency of point mutations varies. It depends on how many rounds of pre-meiotic divisions occurred after fertilization, and how close the copies are to each other (Singer et al., 1995; Irelan and Selker, 1996). If there are fewer divisions prior to meiosis, the D N A sequence is likely to have fewer point mutations (Singer et al., 1995). Also, unlinked copies tend to have  64  fewer changes than linked copies (Cambareri et al., 1989). In subsequent crosses, the duplicated sequences are still susceptible to RIP, but this susceptibility is a function of the similarity of the duplicated sequences and genetic linkage (Cambareri et al., 1991). The RIP mechanism acts on pairs of sequence copies (Selker, 1990). This does not prevent an uneven number of copies from getting mutated because RIP occurs multiple times during the premeiotic nuclear divisions (Irelan and Selker, 1996). It is not clear how the duplicated sequence triggers the point mutations, but the action of an enzyme was proposed to catalyse the process, based on the high density and single type of mutation (Irelan and Selker, 1996). The trigger may consist of two steps: methylation of cytosine residues by a D N A methyltransferase, and induction of RIP by the methylated residues (Irelan and Selker, 1996). The presence of methylated cytosine residues in the duplicated sequences also offers an explanation for the conversion of G-C to A-T. A 5-methylcytosine from the G C base pair could be converted to thymine through deamination. Alternatively, deamination of a cytosine could produce a uracil residue, and a T-A base pair in the next round of D N A replication (Selker, 1990). RIP was the choice for mutagenesis of the N. crassa actin gene because it introduces single base pair mutations, with various frequencies, throughout the targeted gene in a random manner. The N. crassa actin gene was subjected to RIP in a previous study, but no actin mutants were retrieved (Tinsley et al., 1998). The authors attributed the absence of actin mutants to gene silencing in vegetative tissues, a process termed quelling (Irelan and Selker, 1996). It is also possible that point mutations did occur, but were at sites in actin that are involved in processes essential for the survival of the cell, and therefore resulted in unviable ascospores.  65  4.2 Results  After transforming wild type conidia with circular or linearized constructs (see Chapter II), I identified transformants by selecting for the most vigorously growing hygromycin B-resistant colonies. Hygromycin B-resistance predicted the integration of the construct into the genome, and the presence of at least one extra copy of the actin gene in the genome. Control transformations without the presence of transforming D N A did not yield hygromycin B-resistant colonies, while control transformations with only the vector yielded hygromycin B-resistance colonies, supporting this prediction. To introduce point mutations in the actin genes by the RIP machinery, transformants were crossed with the wild type strain of the opposite mating type. Two transformants were not able to produce normal perithecia and/or ascospores. Out of the ones that were capable of producing ascospores, I selected nine for further analysis. From each of these nine crosses 50 random ascospores were picked and their colony growth assessed (Tab.4.1). Only ascospores that were dark brown and elliptical were picked because the light-coloured ascospores do not usually germinate. The percentages of nonviable ascospores in Tab. 4.1 represent the percentages of dark brown ascospores that did not grow. There was also a high incidence of white ascospores and ascospores of an unusual shape and size in most of the crosses, indicating problems with ascospore maturation. The cross between wild type and transformant (Tr) #4 C (C = transformed with a linearized plasmid) yielded a particularly high number of aberrant elongated banana-shaped ascospores  66  that probably correspond to ascospores from the same ascus that failed to separate from each other. A second screen was set up on sorbose-containing agar medium that restricts colony growth and allows growth of a large number of colonies in a relatively small area. Abnormal looking colonies from ascospores from the RIP-inducing crosses were picked, and a total of 7503 germinated ascospores were assessed. Mutant strains with a variety of phenotypes were isolated from these screens. Some progeny derived from different transformants had the same mutant phenotype, and at the same time progeny derived from the same transformant gave different mutant phenotypes. To make sure that there was only one copy of the mutated actin gene, I backcrossed the actin RIP progeny with the wild type strain and looked at the ratio of wild type and mutant phenotype(s) in individual asci (Tab. 4.2). I correlated this with the presence or absence of hygromycin B-resistance to determine if the ectopic actin gene was eliminated during segregation (Tab. 4.2). The properties that were assessed included germination ability, hyphal morphology, branching pattern, branching frequency, growth direction, abundance of aerial hyphae, presence of conidia and growth rate. A l l mutants were additionally assessed for actin distribution in hyphal tips. During the assessment of the actin distribution at hyphal tips, I noticed that a certain number of tips in each mutant, as well as in wild type, did not have prominent actin accumulations at the hyphal tip. However, even without prominent actin accumulations, most tips had a peripheral actin network that stained the brightest in the subapical peripheral region.  67  I sequenced the actin gene from some of the actin RIP mutants to determine the regions in which the point mutations occurred, and to see i f there is a correlation between the phenotype of the mutant and mutated regions. In general, strains that were sequenced within a couple of months of isolation from the screen had a higher incidence of G-C to A-T mutations (88%) than strains that were sequenced at a later time (65%). I eliminated the possibility that the variations in the sequence are due to the background of the strains by selecting strains with the same background for transformation and crosses. Based on their phenotype, the mutant strains were grouped into 12 categories labeled A - L (Tab. 4.3): apolar growth (A), germination tube defects (B), delayed germination (C), early growth abortion (D), severe polarity defects (E), bursting hyphae (F), colonial phenotype (G), higher branching frequency (H), reduced branching frequency (I), one-sided curl (J), wavy hyphae (K), and aberrant growth at low temperatures (L).  4.2.1 Category A Apolar Growth Mutants, and Category B Mutants With Germination Tube Defects  Having in mind that actin is most likely an essential gene in N. crassa, it is not surprising that some of the ascospores did not form hyphae, or aborted soon after germination. A n examination of a set of these ascospores provided interesting insights in the importance of actin at early stages of polarization. Two category A ascospores from the screens formed a few bulbous outgrowths before aborting growth altogether (Fig. 4.1). Because of the presence of several "bulbs", it is likely that the initial polarization site was selected before isotropic growth ensued. After a period of  68  time, isotropic growth was switched off, one or more polarization sites were selected along the bulbous outgrowth, and isotropic growth continued at these sites. This infers that the defect in actin function is related to initiation and maintenance of polarized growth during germination tube differentiation. Several ascospores germinated without forming branches. The germination tubes were long, usually fairly straight, and aborted growth. The absence of branching indicates that these mutants may not have the ability to initiate branching. However, the initial selection of a polarization site within the ascospore must have occurred when the germination tube was initiated. This suggests that selection of a polarized site at germination may be a different process than the selection of a lateral branch site, although both processes are expected to share most, if not all, of the components involved. In one mutant, the nonbranched germination tube burst at the hyphal tip and released the cytoplasmic material before growth ceased, indicating the loss of cell wall integrity.  4.2.2 Category C Mutants With Delayed Germination  A large group of mutants was selected from sorbose-containing medium due to the smaller size of their colonies compared to wild type. A total of 10 colonies were isolated (Tab.4.3). Vegetative hyphae, aerial hyphae and conidiospores were produced later in these mutant strains than in wild type, so the colonies were smaller, with sparse mycelium compared to the wild type colony of the same age (Fig. 4.2A). The mutants had delayed germination from ascospores and conidiospores, and a slower growth rate than wild type (Fig 4.3). Once the ascospore germinated, the germination  69  tube formed new branches more frequently, and the growth rate was reduced compared to wild type. The branching pattern was lateral with approximately the same branching frequency as in wild type (Figs. 4.2C and 4.2D). Hyphae had a high occurrence of short lateral aborted branches (Fig. 4.2B). One mutant from this group, act (3.4UC), was selected for sequence analysis. There 16  were two point mutations in the actin gene (Tab. 4.4). One silent transition (G to A) was present at position 1702 of the sequence that follows the coding region. A second point mutation was a transition at position 1227 of the D N A sequence. This mutation was in exon V , and translates to a T249A switch. In most hyphal tips, there was one prominent apical actin accumulation. A t rare occasions, two apical actin accumulations, multiple actin accumulations, or one subapical actin accumulation were visible (Fig. 4.4, Tab. 4.5). Staining of already polarized hyphal tips of the actin mutant 1.4UC with the vital stain FM4-64 showed that their apex contained a spherical Spitzenkdrper that resembled the wild type Spitzenkdrper (Figs. 4.5A-4.5D). Although consistently present in growing hyphae, the Spitzenkdrper was more or less compact in different hyphae (compare Figs. 4.5E, 4.5F and 4.5G, 4.5H), and disappeared when growth ceased. The stain bleached out in a short time interval at higher magnifications, so a time sequence of growth could not be obtained (Figs. 4.5B, 4.5C, and 4.5D).  70  4.2.3 Category D Early Growth Abortion Mutants  Another group of mutants formed only a few branches and then aborted growth. The branching pattern was lateral, or a combination of lateral and dichotomous. A l l the mutants had in common an exceptionally slow growth rate. Hyphae had problems maintaining direction in some mutants, in others hyphae burst. The phenotypes suggested defects in polarity establishment and maintenance, cell wall integrity, and hyphal growth maintenance. Photographs of this group of mutants are not shown.  4.2.4 Category E Mutants With Directional Growth Defects  Four mutants showed extensive defects in directionality and polarity accompanied with growth termination. The colonies of these mutants produced very few, i f any, aerial hypha, did not produce conidia, and had a severely reduced growth rate compared to the wild type. Mutant act (37.9UC) had hyphae with a range of different morphologies. Some 15  hyphae did not have branches near the tip (Fig. 4.6A). Some of these branches lost their polarity and widened their diameter (Fig. 4.6B, 4.6C, 4.6D). Rarely, regions of the sparse colony had a less pronounced polarity maintenance defect, and had a lateral branching pattern with frequently occurring short aborted lateral branches (Fig. 4.6E). Loss of polarity and short aborted branches also occurred in regions distant from tips (Fig. 4.6F). Hyphae of mutant act (36.2C) often had irregular shapes with an enlarged diameter throughout their 14  length (Fig. 4.7). On some occasions the tip had a pronounced taper, but an enlarged tip was  71  more frequent. In the latter case constrictions behind the enlarged region formed septae, indicating they may represent arthrospores (Davis, 2000; Fig. 4.8). The presence of a large number of nuclei in these segments supports this interpretation. In regions where the polarity defect is not as pronounced, the branching pattern is inconsistent, and is a combination of lateral, dichotomous and multiple apical branching (Fig. 4.9). The terms for position along a hypha are outlined in Fig. 1.1. Mutant act (31.9UC) lacked branches in the distal region of 13  hyphae. It lost hyphal polarity along the length of the hypha, and hyphae additionally could not maintain well the direction of growth. This resulted in the presence of beaded or enlarged regions at the tip and along the length of meandering hyphae (Fig. 4.10). In mutant 33.9UC hyphae were enlarged throughout their length. Nuclei within hyphae were clumped in groups, most likely announcing imminent hyphal death (Fig. 4.11). In all four mutants, hyphae that lost polarity stopped growing. The actin gene of three of these mutants was sequenced (Tab. 4.4). Mutant act  15  (37.9UC) had two mutations, one in intron II, and another in exon V . The point mutation in the exon translated to a change from valine to alanine at residue 159. The actin gene of act  14  had two nonsilent mutations, H101R in exon V , and D310N in exon VI, as well as another silent mutation in exon V . Only one silent mutation in intron IV is present in mutant act  13  (31.9UC). One apical actin accumulation was present in hyphal tips of act (37.9UC), act 15  14  (36.2C), act (31.9UC), and 33.9UC, in instances in those cases in which hyphae progressed 13  without loss of polarity. However, when polarity was disturbed, there was diffuse actin, multiple small actin plaques at hyphal tips, or no visible actin (Figs. 4.12 and 4.13). Apical  72  enlargements at hyphal tips generally did not have actin prominent accumulations, with rare exceptions (Fig. 4.8). A subcategory of mutants expressed only the inability to maintain a consistent growth direction (Fig. 4.14). The colonies of these mutants were sparse, and did not produce abundant aerial hyphae or conidiospores, as was the case with the previous mutants. Hyphae had a slow growth rate, and often changed the direction of growth. In contrast to the other mutants, these mutants did not enlarge the diameter of their hyphae.  4.2.5 Category F Mutants With Bursting Hyphae  Two mutants, act (1.2C) and act (2.5UC), with hyphae that burst frequently were 11  5  isolated (Tab. 4.3). Common properties of these two strains were that their colonies had a slower growth rate, and produced a reduced number of aerial hyphae (Fig. 4.15A) as well as fewer conidiospores. Although in young colonies the hyphal bursting defect was not obvious, the surface of older colonies was covered with droplets of orange cytoplasm that flowed out from bursting points (Fig. 4.15D). Particularly large droplets were visible in colonies of mutant act (1.2C). Having in mind that aerial hyphae were more numerous at proximal sites 11  in colonies, the abundance of cytoplasmic droplets in proximal regions of the colony may indicate that aerial hyphae are more prone to bursting than hyphae that grow on the surface of the medium. The colonies in general had a more dense appearance than a wild type colony (Fig. 4.15C). The two strains also had unique properties, as follows. Hyphae had a lateral branching pattern in mutant act (1.2C), and short lateral 11  branches were frequently seen close to the hyphal tip (Fig. 4.15B). The actin gene of act  11  73  (1.2C) had six C to T transitions (Tab. 4.4). One of them was at position 464 of the noncoding region, in intron II, while the rest were in the coding region, in exon VI. In the coding region, mutations at positions 1458,1497,1533, and 1647 of the D N A sequence that encode amino acids 304, 317, 329, and 367 in the polypeptide sequence, were silent. The only nonsilent mutation was a point mutation at position 1466 of the D N A sequence that encodes amino acid at position 307 of the polypeptide sequence. The P307L at this site could have a significant effect on the actin protein structure because introduction of proline into the polypeptide chain can produce kinks. At hyphal tips, actin was mainly present as one apical accumulation, together with small actin plaques at the subapical periphery (Fig. 4.16). A smaller percent of hyphal tips had multiple small subapical actin accumulations (Tab. 4.5). In hyphae of mutant act (2.5UC) the multiple lateral branches were longer (Fig. 5  4.17) than in mutant act (1.2C). Some hyphae had tips with a more pronounced taper than 11  others. Mutant act (2.5UC) had three base pair changes, a silent change in intron I, and two 5  non-silent changes in exon III (Tab. 4.4). The base pair changes in the exon resulted in the G23S and D24N substitutions in the polypeptide chain.  4.2.6 Category G Mutants With a Colonial Phenotype  Colonial growth was described in N. crassa as continuous restricted growth of a colony in which the colony has a dense appearance, hyphae are frequently branched, and growth rates are slower than in wild type (Garnjobst and Tatum, 1967). A total of 10 mutants with a colonial phenotype were isolated from both screens. Colonies had uneven edges, and aerial hyphae formed cloud-like clumps that developed conidia abundantly, giving the colony  74  a bright orange colour (Fig. 4.18 A). There appeared to be a cycle of growth in which less frequently branched hyphae alternated with very frequently branched hyphae, as in act . 1  However, the colonies of the colonial mutants did not have the appearance of more or less concentric rings of different density, due to the uneven colony edge (Fig. 4.18D). The mutants had a mainly dichotomous branching pattern (Fig. 4.18B). Branching events were more frequent than in wild type, and the branching frequency increased through time (Fig. 4.18C). Hyphae were not linear and appeared to meander slightly during growth (Fig. 4.18B). Aerial hyphae were abundant and produced conidia. The actin gene of three of the colonial mutants was amplified, cloned and sequenced (Tab. 4.4). Mutant act (1.5UC) had one point mutation (G to A ) at base pair position 1030 of 3  the coding region in exon V , and translated to a R183H non-silent change. Mutant act  4  (5.5UC) had one transversion at position 592 (A to C) in intron r v . This mutation was in the intron IV internal Lariat regulatory sequence (Bruchez et al., 1993), and most likely affects splicing of this intron during pre-mRNA processing. A mistake in the excision, or religation of the surrounding exons could cause a changed codon sequence, and subsequent incorrect amino acid incorporation and/or termination during translation, resulting in an altered actin protein. Mutant act  17  (7.4UC) had one transition event in the coding region: the A to G base  pair change at position 864 of exon V resulted in the non-silent N128D substitution in the polypeptide chain. At the hyphal tips actin could be seen mainly in one apical actin accumulation, although occasionally two apical actin accumulations were observed (Figs. 4.19 and 4.20). One stronger subapical actin accumulation plus multiple actin accumulations were also encountered, but less frequently than the apical actin distribution (Tab. 4.5). In all cases there  75  was a peripheral network of small actin plaques most pronounced in the subapical region. As in wild type, in proximal regions of the hypha the position of some small actin accumulations coincided with the positions of some of the nuclei. During the course of a dichotomous branching event, two apical actin accumulations became visible before the two apical branches emerged (Fig. 4.21). This indicated that actin is engaged in the early stages of the formation of a dichotomous branch. Staining with the vital dye FM4-64 revealed that a spherical Spitzenkdrper was absent in the apical region. Instead there were multiple small subapical stained particles or diffuse staining in the apical region of the hyphal tip (Figs. 4.22 and 4.23). During observation, some hyphae increased their diameter in the tip region, and temporarily stopped elongating (Fig. 4.23). This was most likely a reaction to the heat released as a result of high light intensity, because it occurred less frequently when the light intensity was reduced.  4.2.7 Category H Mutants With Frequently Branched Hyphae  Some of the isolated actin RIP mutants showed a higher incidence of branching events. One mutant from this group, act (4.2C), was particularly interesting because its 2  colony was similar to the colony of act (Fig. 4.24). It had the four stages of growth 1  characteristic for act with the typical doughnut shape of the colony in the conidiating stage. 1  There were twelve mutations in the act (4.2C) actin gene (Tab. 4.4), seven in the non-coding 2  sequence immediately after the stop codon, and five in exon VI. Only one of the mutations was non-silent, and translated to a M355I change. Therefore, the amino acid change in act  2  (4.2C) was at a different position than the one in act . Most hyphae of act (4.2C) had one 1  76  2  apical actin accumulation at the hyphal tip (Fig. 4.25, and Tab. 4.5), a smaller number had multiple small actin accumulations, and only a few had two apical actin accumulations. I followed the position, shape and changes of the Spitzenkdrper during growth of act (4.2C) 2  hyphae stained with FM4-64 (Fig. 4.26). Images were recorded in 30s intervals. Although splitting of the existing Spitzenkdrper was not recorded, two Spitzenkdrpers were visible before the two branches of a dichotomous branching event emerged. Colonies of other mutants from this group produced aerial hyphae with fewer conidia (Figs. 4.27, 4.28, and 4.29). In mutant 8.2C, abundant aerial hyphae were produced close to the edge of the colony, contributing to the dense appearance of the colony. The growth rate of mutants from this diverse group was faster than in the colonial mutants, but slower than in the wild type. Hyphae of all the mutants initiated branches more frequently than the wild type. Branching patterns were a combination of lateral and dichotomous. Very rarely, an apical branch formed more than two branches. The actin gene from three mutants in this group was sequenced (Tab.4.4). Mutant act  6  (2.8C) had two silent base pair changes in introns I and IV, and three base pair changes in the coding region of exons IV, V , and VI. Changes in the coding region resulted in the changes at three positions in the polypeptide: G42R, M190I, and G308S. Mutant act (52.9UC) had 18  one silent base pair change in intron V , and three more non-silent changes in exons V and VI resulting in the amino acid residue substitutions S235P, L320P and V339I. In act (6.2C) 19  there were three silent base pair changes in introns IV and V , and five non-silent changes in exons V and VI that translated into the following amino-acid replacements: N l 15S, M299I, M305I, A319T,and A321T.  77  When stained with FM4-64, the hyphal tips had a wild type-like Spitzenkdrper position and behaviour (Figs. 4.30, and 4.31) that was maintained as long as the hypha was growing. The peripheral network of actin plaques in the subapical area was present as well.  4.2.8 Category I Mutants With a Reduction in the Frequency of Branching, Category J Mutants With a One-Sided Curl and Category K Mutants With Wavy Hyphal Growth  Three actin RIP mutants, 18.4UC, 38.4UC, and 1.6C, had colonies with a sparse mycelium, few aerial hyphae and few conidiospores. Hyphae formed branches and had a lateral branching pattern, but the frequency of branching events was lower than in wild type hyphae (Figs. 4.32,4.33 and 4.34). This was most prominent in early stages of growth. As the colony aged, the frequency of branching increased. Growth rates were reduced compared to the wild type strain at all stages of growth. Two mutants showed a tendency of hyphae to grow in a counterclockwise wide curl. The extent of the curl and number of hyphae that grew in this manner varied within the strain, and between strains. One additional mutant with less frequent branching showed a tendency of hyphae to curl as well, but in contrast to the other two mutants, the curl was clockwise (Fig. 4.35). The growth rates of these strains were greatly reduced compared to the wild type strain. After several rounds of subculturing, the phenotype of one of the two mutants changed to wild type, probably as a result of reversion of the original point mutation. Hyphae of the second mutant stopped growing, and could not be revived.  78  One mutant, 2.9UC, had wavy hyphae (Fig. 4.36). The colony of this mutant was sparse, and growth rates reduced, as in the previous two groups of mutants. There were very few aerial hyphae, and no conidia.  4.2.9 Category L Mutants With Aberrant Growth at Low Temperatures  Mutant act (20.2C) could not be distinguished from wild type at the usual growth 20  temperature (25°C). However, it showed a different hyphal morphology at low temperatures. This mutant went through the lag and hyper-branching phase, but could not proceed to the recovery phase and continued to form branches. Eight progeny from a backcross were observed after a shift from 25°C to 7°C (Fig. 4.37). Four progeny had the branching defect after the temperature shift, and four recovered to the wild type branching pattern. The act actin allele was sequenced, and showed a total of seven base pair changes 20  (Tab. 4.4). Two point mutations occurred in intron V , and another five in exons V and VI. A l l of the mutations were silent except two that translated into changes in the amino acid residues 257 (alanine to glycine) and 331 (alanine to valine).  Three mutants, 2.4UC, 5.9UC, and 21.9UC, with aberrant phenotypes did not have any mutations in the actin gene. A couple of mutants, 2.4C (act ), and 1.9UC (act ) with an 9  7  almost wild type phenotype were sequenced as well. Mutant 2.4C (act ) had one mutation 9  that translated into a Q246L change in the polypeptide chain. The 1.9UC (act ) mutant had a 7  mutation that changed K18R in the polypeptide chain. In both cases after several rounds of  79  sub-culturing the phenotype of the strains changed to wild type, indicating that a reversion may have occurred, and I did not further look at the properties of these strains.  4.3 Summary  A collection of actin mutants was generated by RIP, and isolated from two screens. The RIP mutants were assessed for their characteristics at the colony, hyphal and cellular level. Based on these characteristics, all the actin mutants were divided into 12 categories of mutants (Tab. 4.3). Knowledge about the role of actin allowed predictions of phenotypes that actin mutants may display. Mutants with some of the predicted phenotypes were isolated from our screens, while others were not. A summary of predicted and observed phenotypes is presented in Fig. 4.38 and Tab. 4.6. To confirm that the mutants were a result of mutations in the actin gene, and to further reveal the possible ways in which changes in actin lead to phenotypic changes, the actin gene of some of the mutants were sequenced. The sequences showed the type and extent of the mutations in the actin gene. One of the concerns with regards to the function of the mutated actin allele was that RIP is usually followed by extensive methylation (Irelan and Selker, 1996) that can result in gene silencing. The signal for methylation comes from mutations introduced by the RIP machinery. However, if there are few mutations, it appears that the signal is not strong enough to trigger the methylation process (Selker et al., 2002). The sequenced actin RIP mutants have few point mutations, and have therefore most likely not been methylated. The positions of point mutations introduced into the actin gene of the actin RIP mutants are presented in Fig. 4.39. Mutations generated in the RIP process are thought to be  80  always G-C to A-T (Irelan and Selker, 1996). Not all the point mutations in the sequenced actin genes are of this category. Having in mind that each type of nucleotide can theoretically be substituted with three other nucleotides, there are 4 x 3 = 12 possible nucleotide changes. Only two of out of these twelve possibilities are G-C to A-T, giving a 16% chance that this mutation will occur by chance if all types of nucleotides are represented equally. The G-C to A-T content of the actin ORF is 56.93% : 43.07%, demonstrating appropriate equality. Out of a total of 59 mutations, 44 were G-C to A-T, which is 75%, much higher than the percent expected if mutations were random even when the slight difference in the G-C to A-T content is taken into account. The presence of point mutations not typical for RIP could be due to changes that occurred independently of the RIP machinery, or they could be atypical creations of the RIP machinery. Reports on the RIP mechanism are mainly based on examples of sequences that underwent extensive RIP-induced changes. It is possible that in such cases an overwhelming majority of mutations are G-C to A-T, and that other rare mutations, although they did occur, were attributed to amplification mistakes. However, there is no proof that this could be occurring. In the present study, the polymerase product specifications claimed a polymerase error rate of 8.5 x 10" . A more accurate estimate might 6  be the one obtained in act , i.e. one error per 3.35 x 10 base pairs. This error rate is expected 1  3  to generate 10 mistakes along 33.5 kb of D N A sequence, which is the length of twenty actin genes. The total number of point mutations in the twenty actin genes represented by the twenty actin RIP mutants tested was 61, suggesting that one out of six changes may be due to PCR and sequencing errors. The actin distribution pattern was quantified in some of the actin RIP mutants, and was different than in the wild type strain. Although the most frequent actin pattern in all the  81  mutants was the one typical for w i l d type, the range o f different actin patterns present in each o f the mutants was wider than i n w i l d type. In various fdamentous fungi, absence o f growth correlates with the absence of the Spitzenkorper in the apical region of the hyphal tip (see Chapter I). This correlation is closely related to the function of the Spitzenkorper in the hyphal growth process. Considering the importance of actin for the hyphal growth process and the presence o f actin accumulations in most tips of both the actin RIP mutants and the w i l d type, the absence o f actin may be an indication that active growth has ceased at the hyphal tip at the point that the hyphae were fixed. A further discussion of the results can be found in Chapter V I .  82  (C)  Tr4 (Q  Tr6 (Q  Tr8 (Q  50  50  50  50  42 1  42 5  42 6  37 6  42 4  48 2  43 7  47 3  48 8  43 7  46 4  96% 4%  86% 14%  94% 6%  86% 14%  86% 14%  92% 8%  Cross between wild type and:  Tr2 (UC)  Tr4 (UC)  Tr5 (UC)  Tr8 (UC)  Tr9 (UC)  Tr2  Total number of picked ascospores Wild type Other  50  50  50  50  50  43 2  38 6  45 4  40 8  Germinated Not germinated  45 5  44 6  49 1  Viable Non-viable  90% 10%  88% 12%  98% 2%  Table 4.1. Phenotype of germinated ascospores, number of germinated ascospores and viability percentage of ascospores from crosses between wild type and nine transformants. The transformants contain at least one extra copy of the wild type actin gene. Tr = transformant. U C = transforming plasmid was circular (uncut). C = transforming plasmid was linear (cut).  83  Backcrossed actin RIP mutant including mating type 4.2 C 1.6 C 2.8 C 1.5 UC A 2.5 UC 5.5 UC a 2.8 C a 35.4 UC a 3.4 UC a 29.4 UC A 28.4 UC A 34.4 UC a 2.4 UC 1 A 23.2 UC a 43.9 UC a 1.9 UC 1 A 38.4 UC a 21. 9 UC a 5.4 UC 1 a 4.4 UC 1 A 21.4 UC 1 A 5A.4 UC 1 A 6.4 C 1 a 18.4 UC a 17.4 UC a 1.4 UC 1 a 52.9 U C A 23.2 UC a 1.2 C 1 a 44.2 C A 1.2 C 1 7.4 UC 1 11H.9 UC 1 111.9 UC 1 22.4 UC 1 3.4 C 1 A 15.4 UC 1 a 24.4 UC 1 A 23.4 UC 1 A 10.2 C a 6.2 C 1 A 2.9 UC 1 a 2.4 UC 1 A 14.4 UC 1 A 40.9 UC 1 a 65.2 C 1 A 32.2 C I A  Hygromycin B resistance of actin RIP mutant  Number of screened octads  Resistant Sensitive Sensitive ?  8  Resistant Sensitive Sensitive Sensitive Sensitive Sensitive Sensitive Sensitive Sensitive Sensitive Sensitive Sensitive Sensitive Sensitive Sensitive Sensitive Sensitive Sensitive Sensitive Sensitive Sensitive Sensitive Sensitive Sensitive Resistant Resistant Resistant Sensitive Sensitive Sensitive Sensitive Sensitive Sensitive Sensitive Sensitive Resistant Resistant Sensitive Sensitive Sensitive Sensitive Resistant Resistant  5 22 25 12 7 21 24 17 20 20 20 23 24 24 24 24 24 24 24 24 24 7 29 22 21 23 24 24 24 24 24 24 24 24 24 22 24 24 24 24 24 24 24 24 24  Continued on next page. 84  1:1 ratio of wild type: mutant phenotypes in octads 3 3 15  Other ratio of phenotypes in octads  10 3 19 21 14 16 20 16 7 ? ? 21 21 22 21 21 20 21 3 23 20 21 7 (+12) 19 12 17 22 ? ?  2 4 4 3 3 4 0 4 16  21 ?  3 ?  18 14 21 ? 22 12 23 ? ? ? ?  6 8 3 ? 2 10 1 ?  5 2 7  ?  ? 3 3 2 3 3 4 3 4 6 2 0 4 (+20) 5 12 7 2 ? ?  Numbe rof alleles (1 or more) other more (?) more (?) more 1 more (?) 1 1 1 1 1 1 more 1 1 1 1 1 1 1 more (?) 1 1 1 1 1 more 1 ? ?  1 ? 1 more 1 ?  1 more 1 ?  9  ?  ? ?  ? ?  20.2 C 1 a 8.2C 1 a 4.2 C 1 a  Resistant Resistant Resistant  ? -  24 0 8  -  ? -  Table 4.2. Mating type, hygromycin resistance and number of actin copies in actin RIP mutants. Actin RIP mutant names: e.g. 4.2C, 4=serial number of mutant progeny from the RIP-generating cross, 2=number of transformant used as one of the parents in the RIP-generating cross, C=in generating the transformant that was later used as one of the parents in the RIP-generating cross, a linear plasmid was used. UC=in generating the transformant that was later used as one of the parents in the RIP-generating cross, a circular plasmid was used. ?=indicates that progeny could not be scored with confidence (+#)=indicates number of octads for which the phenotype could not be clearly scored).  85  Category Mutant phenotype A B  C D E F G  H I J K  L  Apolar growth Germination tube defects Delayed germination Early growth abortion Directional growth defects Bursting hyphae Colonial  High branching frequency Low branching frequency One-sided curl Wavy hyphae Aberrant growth at low temperature  Mutants in this category 2 unnamed few unnamed 1.4UC, 2.4UC, 3.4UC (act ), 4.4UC, 5.4UC, 5A.4UC, 6.4UC, 21.4UC, 23.4UC, 24.4UC few unnamed 37.9UC (act% 36.2C (act ), 33.9UC, 31.9UC 16  14  (act ) 13  2.5UC (act ), 1.2C (act ) 1.5UC (act ), 5.5UC (act ), 7.4UC (act"), 15.4UC, 17.4UC, 22.4UC, 28.4UC, 29.4UC, 34.4UC, 35.4UC 4.2C (act ), 2.8C (act ), 8.2C, 6.2C (act ), 52.9UC 3  11  3  2  4  6  19  (act ) 18  1.6C, 18.4UC,38.4UC 2 unnamed, 1.6C 2.9UC 20.2C (act ") 1  Table 4.3. Actin RIP mutants grouped in categories based on their phenotypes. Mutant name abbreviations: e.g. 1.4UC, 1 = serial number of mutant progeny from the RIPgenerating cross, 4 = number of transformant used as one of the parents in the RIPgenerating cross, U C (uncut plasmid) = in generating the transformant that was later used as one of the parents in the RIP-generating cross, a circular plasmid was used. C (cut plasmid) = in generating the transformant that was later used as one of the parents in the RIP-generating cross, a linear plasmid was used. Name in brackets indicates that the actin gene of the mutant has been sequenced and named.  86  1o co to  u w  OL CL  CC  co  CO  87 cc  LO  *~  o co <  03 CO CO c o CO  1458 1466 1497 1533 1647 i  QL  CC  1  o  CM  O (0  o  Tt  S Tt  CO c o CO LU UJ  T— T—  o O O z  CO  :>  o o  co co  w  0. Q.  CC CC  ACC to ACT CCT to CTT ATC to ATT ATC to ATT CCC to CCT  T (Thr) to T (Thr) | P (Pro) to L (Leu) | I (lieu) to I (lieu) | I (lieu) to I (lieu) | P (Pro) to P (Pro) |  GGT to AGT | K (Lys) to R (Arg) | CAG to CTG Q (Gin) to L (Leu) |  'r  o  CD  AtoG  i  CM  E6 E6 E6  GGT to AGT ] G (Gly) to S (Ser) | GAT to AAT | D (Asp) to N (Asn) |  | | | | | |  GGG to AGG | G (Gly) to R (Arg) | ATG to ATA | M (Met) to 1 (lieu) | GGT to AGT | G (Gly) to S (Ser) | 1  1219  | | | | | |  | C (Cys) to F (Phe) |  K (Lys) to K (Lys) Q (Gin) to Q (Gin) Q (Gin) to Q (Gin) M (Met) to I (lieu) V (Val) to V (Val) R (Arg) to H (His)  i  CtoT AtoG AtoT CtoT CtoT CtoT CtoT CtoT CtoT  |  TGC to TTC  Amino acid change  Codon change  AAG to AAA CAG to CAA CAG to CAA ATG to ATA GTG to GTA CGT to CAT  CM CO o c o c o CO CO CO  CO  Other (S)  E3 E5  1052 1468  E4 LO CO LU LU  T—  RIP (S)  1  1  | E3 CO c o c o CM LO LO  co  1  5.9UC | 31.9UC  O O O z z 55" z  o co CM O) o CO  i  2.4C 1.2C  CM CO LU  1  i  Other (NS) Other (NS) RIP (S) RIP (S) RIP (NS)  T— T—  RIP IS) RIP (NS) RIP (NS) RIP.(NS)  CO LU  RIP (NS)  1  E6 | | E5 | CO CO LO l O CO CO  1  NC (1)  CC  RIP (S) RIP (NS) Other (NS) RIP (S)  Q.  CO CM CM  C(3)  CC  w z CM O) LO  1  acf  O Ct  I E6  co co LU UJ  RIP (S)  I  | | | | | | | | | | | | I | j  Amino acid number  1  NC (2)  RIP (S) RIP (S) RIP (S) RIP (S) RIP (S) RIP (S) RIP (S)  PS I PS I PS I PS I PS I PS I PS  CtoT | GtoT I GtoA | GtoA | GtoA I GtoA I GtoA J GtoA | GtoA I GtoA I GtoA | GtoA | GtoA | GtoA | GtoA AtoC GtoA GtoA GtoA CtoT GtoA GtoA I GtoA GtoA  Nucleotide change  i o o CO CO CO CO CO LO i o CO CO CO c o CO  1  1  co 1  C(2)  UV (S) UV (NS) CC 1 1  1  CM Q. 1  1  1 2.4UC 1.9UC  o z O w 1 1  1  2.8C  I  •r—  NC (7)  Type of mutagenesis and effect  1667 I 1682 I 1688 J 1695 I 1699 I 1737 I 1741 J 1791 1554 1605 1608 1611 1656 | 1030  Intron/Exon number  CD CM Ol  1 |  Number of nucleotide  CM 1 1  t  1  4.2C  Position and number of changes in regions •* 1  1  1.5UC 1 5.5UC 2.5UC  acf  ccr-100  Number of DNA changes  CO 1 1  1  Renamed  i 1  (S)O  \s  Mutant  i  i  a>  o>  cn LO  O z  o  T—  M  1  u co  fl  PQ U o (3 Z,  a.  < Q "to  I X  < < o a  01  ro  <  >  co Q  ro •' co S i £ j >; >  0)  Id  co co  2  </>  3|£ >  •S CD  i  s  15 '  <  ^  00 J3  o  8  a g r g3  o  S3 O  a a  o  C3  o  a  H  II  U  o <  Io  u <J  o H  aQ CO  C3  in  £2  C3  xi o  e  o  «>  JO o  CM i  •1*1 O a)  I.a a a°| CO ^ a °  C  11  &£  a  s  2  o o o co " o O PH  S g (D 00  II  D O CO  c a g *  a ft° v» CO  co  co.  co co  1 ^  -co  D  o CO  o o  CT O 1  •a  S §  J3  W 0) O  13 * =3 CO.  m  O  10  co, O  ccj  o  O  11  |.a  J  00 (3  1 s .aB 2  lO  d  O  a> cs  a 2 co  00 O D ^ 00 J3 T3  9 S •2 § • ^i II° •a <a (3  I  co  O  CM CD CO  O  O  8 O CM CD  c3  O 3 o  O  CM O CM  88  •i § 00-2  ral cu  o  H  g  co oo « cu .«  •2 ">  00 (3  ©  u C  3  u  <u S  5 <~ ** .& O S  o  ^  ccj  C  •ass CS  o  ft  iI f  H  Kg  «  _  o  .  S  I CCJ  I ^ 5 o ® h «u «a e  T  O D bO  e ft «-S -  cj cj cs  < u  2  S | £1  H  I  2  a ft  o is a o o  ft: i2s  ft  «  a. te  B  OH  2  ,* 3 I f s ca  —  o ccj  M U  o  ft .  - s a* .a S « 3  c>  .2  .a s a  =  «  £ « —  .S <+3 < u ° 43 cd bO  s *"  o ^  | Z "S  sI  ^ • •2 § in ti  e  ft —  * 2 2  t  % s  . V  -4->  S3 cd  "° t2  * 3 89  3 O  •a  u  o  T3  CJ 3  Q 1  o  4  o  P  to  "ft  s  o >  Ed  •a  =1  11  3  X!  e fc -  a o  4  =  ul  .4*1  o —  o  CJ  x>  3  IX) 4>  a o  a  o e  3  1=8 3 B  If & XJ  XI  *j a  s aa3d  oo  o xs g tSo 9M  —-  U  fc,  ea  5>  5 P 00=u H-  1 £ fc  XI  V5  <o «J  II  gj  B  « a •CJ  —  ac  2£ §  *as a g£  |fs  J £ §s 2a £ oo ¥>  r —  o  t  o «  00  ©  ra  b  ex o  | T3  ft  a  a o  .H o  I  CJ  N  •c _ c 3  O ft |(  s  a o 4)  |  ca  o .a  h I?  I  ro  f- S _  £ <H xi 09  '€ s  o 3  •2io -a. 13j3 2 p & ca§ 8S a15 I  33-  °a .fc = _  M  ft | a  2  X)  u a e C>>a — ca et Ti *s» o x>  w B  xi B9  DO  g  2 2 e. ft  X)  C£  Ji  «J  2 x>  T3 3  43 < • > — u s 2 3  3  ob °f>  o -a  a  £ c >• o a 5a  ^  ob °o  "O 3  *  j= s x> & 1 2 5 fx ft  g  ox:  3  as 6 do °P  g fj  o  E xs xi  I  w  5  ? 1ca -  T3 9  o P B  c5  3  q  s i  sr. So^T  u XJ  d  d o  90  XJ ?  2 x:  P o -5  CJ  Figure 4.1. A category A isotropic germination mutant ascospore unable to polarize growth. Bar = 30 jim.  91  92  Figure 4.3. ( A ) Germination of the wild type strain after one day. (B) Germination o f the category C delayed germination actin R I P mutant 1.4UC after one day, (C) after two days, (D, E) after three days, (F, G) after four days. A l l bars = 100 |im. B a r in image (D) applies to images (E, F, G).  93  A  B  E  C  D  G  H  Figure 4.4. Actin and nuclear distributions in hyphal tips of the category C actin mutant act (3.4UC) with delayed germination. (A, B) Images of the same hyphal tip. (A) One apical actin accumulation in median plane. (B) Peripheral actin accumulations in peripheral plane. (C) Three actin accumulations. (D) Four apical actin accumulations. (E) Multiple actin accumulations at a hyphal tip. (F, G , H) Images of the same hyphal tip. (F) DIC image. (G) Nuclear distribution. (H) Absence of actin accumulations. Subapical co-localization of actin with some nuclei. Bar = 10 Lim and applies to all images. 16  94  B  C  D  /  \  H  F  Figure 4.5. FM4-64-stained hyphal tips of the category C actin mutant 1.4UC with delayed germination. (A, E , G) DIC images. (B, C , D ) Time series of images of an FM4-64 stained hyphal tip illustrating bleaching of the vital stain. (B, C) The Spitzenkdrper is visible at the tip. (F) A diffuse Spitzenkdrper. (H) A compact Spitzenkdrper. Bar = 10 Lim, and applies to all images.  95  Figure 4.6. Hyphal morphology of the category E actin RIP mutant 37.9UC (act ) with a directional growth defect. (A) A long unbranched hypha. (B) An unbranched hypha with enlarged segments at the tip, indicating loss of polarity. (C) A hyphal tip that temporarily lost polarity, and resumed polarized growth at more than one point. (D) Hyphae with disrupted polarity resulting in a meandering appearance, and dichotomous branching. (E) Hyphae with a less pronounced polarity problem that retain the lateral branching pattern. Bar = 100 Lim, and applies to all images. 15  96  C  B  Figure 4.7. Hyphal tips of the category E actin mutant 36.2C (act ) with a directional growth defect, (A, B , C) Images of the same hyphal tip. (A) DIC image. (B) Nuclear distribution. (C) One apical actin accumulation ( D , E , F) Images of the same hyphal tip. (D) DIC image. (E) Nuclear distribution. (F) Almost no visible actin staining. (G, H , I) Images of the same irregularly swollen hyphal tip. (G) DIC image. (H) Nuclear distribution. (I) Diffuse actin staining. Bar = 10 pun, and applies to all images. 14  97  B  Figure 4.8. Hyphal tips of the category E actin mutant 36.2C (act ) with a directional growth defect. (A, B , C) Images of the same swollen hyphal tip (A) DIC image. (B) Nuclear distribution. (C) One small apical actin accumulation. (D, E, F) Images of the same contorted hyphal tip. (D) DIC image. (E) Nuclear distribution. (F) Several peripheral actin plaques. (G, H , I) Images of the same swollen sections of a hypha. (G) DIC image. (H) Nuclear distribution. (I) Actin accumulations at the septae. Bar = 10 pm and applies to all images. 14  08  Figure 4.9. Eight-day-old hyphae of act (36.2C), a category E actin RIP mutant with a directional growth defect. (A) A dichotomous branch and two unbranched hyphae that cannot maintain direction well. (B) A combination of dichotomous and lateral branching. Bar = 100 pm, and applies to both images. 14  99  Figure 4.10. Eight-day-old hyphae of the category E mutant  act  13  (31.9UC)  with a directional growth defect (A) Long unbranched hypha. (B) Hypha with regions close to the tip that lost polarity. (C) Wavy growth of hypha and loss of polarity at the tip. (D) A lateral branch that lost polarity. (E) Wavy growth, with bead-like regions that lost polarity. Bar = 100 pm, and applies to all images.  100  Figure 4.11. Hyphae of the category E actin mutant 33.9UC with a directional growth defect. (A, C, E; and B, D, F) Images of the same group of hyphae, respectively. (A, B) DIC images. (C, D) Nuclear distributions. (E, F) Mainly diffuse actin distribution distributed around groups of nuclei. Bar - 30 (im in image (A) applies to images (C, D). Bar = 10 Lim in image (B) and applies to images (D, F). 101  Figure 4.12. Actin and nuclear distributions in hyphal tips o f the category E actin mutant 3 7 . 9 U C (act ) with a directional growth defect. (A, B) Images of the same hyphal tip. (A) One apical actin accumulation. (B) Nuclear distribution. (C, F) Images of the same hyphal rip. (C) D I C image. (D, E) Images o f the same hyphal tip. (D) Two apical actin accumulations at the tip of a leading hypha and one apical actin accumulation in the lateral branch. (E) Nuclear distribution. (F) N o prominent actin accumulations. (G, H, I) Images o f the same hyphal tip. (G) D I C image. (H) Nuclear distribution. (I.) Peripheral actin patches at a bulbous enlargement. Bar = 10 pm and applies to all images. 15  102  Figure 4.13. Hyphal tips of the category E actin mutant 33.9UC with a directional growth defect. (A, B , C; D , E, F; and G, H , I) Three sets of images of three different hyphae. (A, D, G) DIC images. (B, E, H) Nuclear distributions. (C) Small apical actin accumulation. (F) Apical actin cap of peripheral plaques. (I) Two small subapical actin accumulations. Bar = 10 mm, and applies to all images. 103  A  B  • 1  •  /  'I  *  Figure 4 . 1 4 . (A, B ) Hyphae o f a subcategory o f mutants with severe polarity defects (category E), unable to maintain growth direction. Magnification = 100X.  104  105  Figure 4.16. Actin and nuclear distribution in hyphal tips of the category F actin mutant act (1.2C) with bursting hyphae. (A, B , C) Images of the same hyphal tip. (A) DIC image. (B) Nuclear distribution . (C) One apical actin accumulation. (D) One large apical actin accumulation. (E) Multiple small actin accumulations. (F) Three apical actin accumulations. (G) Few apical actin accumulations. (H) Hyphal tip without apical actin accumulations. All bars = 10 Lim. Bar in image (E) applies to images (G) and (H). Bar in image (A) applies to the rest of the images. 11  106  107  108  Figure 4.19. Actin and nuclear distribution in hyphal tips of the category G actin mutant act (7.4UC) with colonial growth. (A, B , C ) Images of the same hyphal tip. (A) DIC image. (B) Nuclear distribution. (C) One apical actin accumulation. (D) Two apical actin accumulations. (E) Three actin accumulations at apex. (F) One subapical actin accumulation. (F) Multiple weakly stained actin accumulations. (H) Absence of actin accumulations. All bars = 10 (am. Bar in image (D) applies to images (D) and (E). Bar in image (A) applies to the rest of the images. 17  109  Figure 4.20. Actin and nuclear distribution in hyphal tips of the category G actin mutant acP (1.5UC) with colonial growth. (A, B, C) Images of the same hyphal tip. (A) DIC image (B) Nuclear distribution. (C) One apical actin accumulation. (D, E) Two apical actin accumulations. (F) Multiple weakly stained actin accumulations. (G) One subapical actin accumulation. (H) Hyphal tips with diffuse actin caps. Bar = 20 pm.. Bar in image (A) = 10 Lim, and applies to all images except image (H).  110  Figure 4.21. Actin distribution in hyphal tips of actin RIP mutant 5.5UC (act ). (A) One apical actin accumulation in a hyphal tip that is not branching. ( B ) Dichotomous branching before the emergence of two new tips. There are two apical actin accumulations, one for each new branch. (C) Dichotomous branching after the emergence of two new tips. There are two apical actin accumulations at the tip of each new branch. (D) A dichotomous branch with longer branches. The apical actin accumulation persists at each branch tip. Bar = 10 pm, and applies to all images. 4  Ill  Figure 4.22. FM4-64-stained hyphae of category G actin RIP mutants (A, B) 17.4UC and (C-H) act (5.5UC) with colonial growth. Images are presented in pairs, with the DIC image first, followed by the fluorescence image. (A, B) Multiple small accumulations of membranous material replacing the Spitzenkdrper at the hyphal tip. (C, D) Hypha without prominent staining. (E, F) Multiple small membranous accumulations. ( G , H) Multiple small membranous accumulations at dichotomous branch. Bar in image (C) = 20 Jim and applies to image (D). Bar in image (A) = 10 (am, and applies to the rest of the images. 4  112  A  E  B  C  G i %  \  I  \  act  3  Figure 4.23. FM4-64-stained hyphal tip of category G actin RIP mutant (1.5UC) with colonial growth. (A-C) Time sequence of the growth of a hyphal tip. A diffuse stronger stained area is visible in the apical region instead of a Spitzenkorper. (D) DIC image of the same hyphal tip. (E, G) Multiple small stained accumulations in the apical region. (F, H) DIC images of the appropriate hyphae. Bars = 10 pm. Bar in image (A) applies to images (B-D). Bar in image (E) applies to the rest of the images.  113  114  E  B  C  F  G  D  H  Figure 4.25. Actin and nuclear distribution in the category H actin RIP mutant act (4.2C) with high branching frequency. (A, B , C) Images of the same hyphal tip. (A) DIC image. (B) Nuclear distribution. (C) One apical actin accumulation. (D) Peripheral actin network. (E) Multiple weakly stained actin accumulations at hyphal tip. (F) Two apical actin accumulations. (G) Multiple strongly stained actin accumulations at a hyphal tip. (H) Absence of actin accumulations. All bars = 10 pm. Bar in image (D) applies to image (G). Bar in image (A) applies to the rest of the images. 2  115  Figure 4.26. Growth of an FM4-64-stained hyphal tip of the category H mutant act (4.2C) with a high branching frequency. (A-K) Sequence of images following the growth and dichotomous branching event. (A-D) Before branching the tip has one Spitzenkdrper. (E) Before the two new branches emerge, the Spitzenkdrper is divided, and each of the two Spitzenkdrpers is assigned to a new branch. The emergence of a lateral branch is also visible, but at a different focal plane. (L) DIC image. Bar = 10 Lim, and applies to all images. 116 2  117  Figure 4.28. The category H actin RIP mutant act (2.8C) with a high branching frequency. (A) A three-day-old colony. ( B ) Hyphal morphology of a one-day-old colony. Bar = 200 pm. 6  118  119  A  B  D  E  C  )  r X  • '  \  1  Figure 4.30. FM4-64 stained hyphal tip of the category H actin RIP mutant act (2.8C) with high branching frequency. (A-E) Time series of images of a growing hyphal tip. A Spitzenkdrper is visible at the apex of the leading hypha, but not at the apex of the new lateral branch. (F) DIC image of the same hyphal tip. Growth of a second lateral branch subapically in the z-plane is visible. Bar in image (A) = 10 Jim, and applies to all images. 6  120  Figure 4.31. Growth and Spitzenkorper position in an FM4-64-stained hyphal tip m of the category H actin RIP mutant a&t (52.9UC) with a high branching frequency. (A-D) Time sequence of hyphal tip growth. The Spitzenkorper is at the apex during growth. In image (D), the hypha growed out of the initial plane. Images ( E , F) are DIC images of different planes of the same hypha. Bar = 10 pm and applies to all the images.  121  A  B  Figure 4.32. The category 1 actin RIP mutant 18.4UC with low branching frequency. (A) Branching pattern of a one-day-old colony. Bar = 1 mm. (B) Hyphal morphology of a one-day-old hyphae. Bar = 200 pm. (C) Branching pattern of a two-day-old colony. Bar from image (A) applies to this image. (D) Hyphal morphology of two-day-old hyphae. Bar= 100 pm.  122  123  Figure 4.34. The category I actin RIP mutant 1 6C with a low branching frequency. (A) A three-day-old colony. (B) Branching pattern of a three-day-old colony with an anticlockwise turn. Bar = 1mm. (C) Anticlockwise turn of a two-day-old hypha. Bar = 200 Ltm. (D) A one-day-old hypha with a mild direction maintenance problem. Bar = 100 Lim. (E) Hyphae that lost poslarity in some regions Bar = 100 pirn. (F) A long unbranched hypha. Bar = 100 Lim. 124  Figure 4.35. A category J actin RIP mutant with curling hyphae. (A) Branching pattern of colony. Bar = 1 mm. (B) Hyphal morphology and branching of colony. Bar = 200 pm.  125  c  mmmm  Figure 4.36. The category K mutant 2.9UC with wavy hyphal growth, (A) Wavy hyphal morphology of a one-day-old colony. Bar = 100 pm. (B) Hyphae of a two-day-old colony. Bar = 200 pm. (C) Hyphal of a four-day-old colony. Bar = 200 pm. (D) Branching pattern of a four-day-old colony. Bar ~ 1mm.  126  Figure 4.37. The category L actin RIP mutant 20.2C (act ) with aberrant growth at low temperatures. ( A ) Branching pattern of colony at 7°C. Bar - 1mm, and also applies to image (C). (B) Hyphal morphology at 7°C. Bar = 200 Lim, and also applies to image (D) Wild type control. ( C ) Branching pattern of colony at 7°C. (D) Hyphal morphology of wild type. All strains were grown at 25°C for one day, then shifted to 7°C forfivedays. 20  127  Delayed conidiospore germination Isotropic germination Absence of conidiation Non-viable ascospores Isotropic germination Delayed ascospore germination Germination tube defects /  y~. macroconidium  Abnormal ascospores Absence of ascospores  /f  x  ascospore  Absence of asci Abnormal asci  branched multinucleate mycelium 'Defects in polarized growth "Defects in polarized growth  mature ascus  Abnormal perithecia  perithecium  microconidium macroconkJhirrt or mycelium Absence o f perithecia Female/Male sterility  protoperithecium  Figure 4.38. Steps in the life-cycle of N. crassa where defects due to actin modification might occur. Green = observed defects Red = predicted defects that were not observed. * = for more details about defects in polarized growth, see Table 4.6. Drawings of the life cycle are taken from Perkins et al. (2001).  128  — CO CO CO LO CO  Q. LL S  55  CD  CO CO  co  ST CM  m CO  CO CO LL. ifl CM CO,  K CM  ID  CM CO  L L . g r -  UJ  t  J  U.  CO  CO  «0 o  LL. a  o CO  ,  z  2 3  *o co  5  N  CM  0  £  ffl  §| £  1  lo  a ~  ^2  CD  • — ' a>  CM  r-  CO  T M  00  m r  T-  l i l CO O  •  CD  C cd  £ cn2  .is  o <*> - 9  X  C . oX  C •—  c  O  3  CD  X "t  *• is 00  . S  •a £ co CD J  m TU J O r t oo J O  o c  u  I  o <o M  CM  1)  g  H ^ 13 ~o o*  CD  c3  CD  cn  , oo o> LU m to  w  CN  OK  £J .£  0  CO  2CD ? |  3  r  -rI £ °2  d  >S  oiS £ 2CL.  ™  °  V c g oo  co  I•  CO  CO  o  v 5  2 co  T -  D  O  j£  X  _ 3  53  cn c: CD CD  CS  «  u a w. c CD cn CD O  —  1-  S 1 53  -o CS  g  c o  CS  | —cc U  c= CD J_  | m oS £ •r= 3 £ £ £ CS X  LU g to  (!)  ° CD CO  '  s  00  oo  LU to  x83 J- tti  LL. CO  £  o  CN 0 5  O -  CD c t • £ cn C 2 ca X  C_ V3 CD  O  1H;| 3  5  +-•  cn  £  « l T3 ca  £  CD  c .1 |  53 *£x-  c g 2  "55  § £  S£  11  o £  x  oo co  r  $ s cn CCJ  = 1 Q. 1X3  § I CcaQ CD CD  1 TJ  O  cn CD  f-  c _  B g — CD  129  ' ca 5O  c o CD C -a •-  &1  •C  CD  % p a. U C  cn  CB CD  gO  > , 60 •CJ- T3CD  O  CQ  i  CD  3  2 £  _i  CD o  CO  <  c3 CD X  ...  ° S 00  O Z  i_ »  c  £ »  CO  c "5 "8 £ o o. ~  00 C CD  CD  S  s  s  CHAPTER V GENE INTERACTIONS  5.1 Introduction  The characteristics of the N. crassa actin mutants suggest that actin is involved in a multitude of cellular processes. It is likely that many of these processes require an interaction of actin with one or more actin binding proteins. These putative interactions could be a part of a process necessary for normal development, such as the assembly of the cytoskeleton, or they could be required for the reaction to an external signal, such as the assembly of networks at the periphery of the cell. In the latter case, the changes can be transient or permanent. The observation of epistasis or antagonism in double mutants can provide evidence for genetic interactions between actin and other genes. Based on colony and hyphal morphology Gavric and Griffiths (2003) divided interactions in N. crassa into four categories: full epistasis, partial epistasis, costasis and novel. If both colony and hyphal morphologies of the double mutant are identical to one of the parents, then the mutated gene in that parent is fully epistatic to the one in the other parent. In partial epistasis there is epistasis in hyphal morphology, but not in colony morphology. The third category is costasis, in which interaction between the mutated genes is not present. In the novel category double mutants show phenotypes not found in the single mutants. The interaction of mutations affecting tip growth and branching in N. crassa was investigated (Gavric and Griffiths, 2003). Analysis of double mutants yielded a Y shaped  130  chart of epistatic interactions, and suggested the positions of gene products in pathways involved in hyphal morphogenesis. In the Y shaped chart two parallel paths merge into one. The backbones of the two parallel paths consist of col-16, fr, col-4, spco-10, and gran on one side, and pk, smco-8, col-15, col-8, and smco-6 on the other side. After merging, the path consists of spco-1, spco-4, smco-7, mcb and col-17. To investigate the possible position of the actin gene in this proposed network, and to search for other interactions, I looked at the interaction between act and selected mutants 1  from this network. The selection of the mutants included their ability to serve as female parents, because act is female fertile. Only one of the mutants, frost, was sequenced (Sone 1  and Griffiths, 1999). The fr gene encodes a membrane protein with a function in M n  2 +  homeostasis, but the role it has in polarized growth is not clear.  5.2 Results  A l l the parents and all the double mutants were assessed for their colony morphology, hyphal morphology and actin distribution in hyphal tips. These characteristics of act have 1  already been described (see Chapter III, Fig. 3.1 and Fig. 3.5) and will not be repeated here. The sequence in which data will be presented is the following: colony and hyphal morphology of the non-act parental strain, colony and hyphal morphology of the double 1  mutant strain, actin distribution in the non-act parental strain and double mutant strain, and 1  in some cases, Spitzenkorper characteristics.  131  5.2.1 The col-16; act Double Mutant 1  The colony of mutant col-16 had restricted growth with abundant aerial hyphae, and produced conidia that detached readily, producing satellite colonies on the medium (Garnjobst and Tatum, 1967; Perkins et al., 2001; Fig. 5.1A and 5.IB). Hyphae branched mainly dichotomously, and had an "aborted branch" appearance (Fig. 5.1C) that is more pronounced in early stages of growth than in the act mutant. 1  Overall, the colony of the the col-16; act double mutant resembled the col-16 parent 1  more than it did the act parent, while the hyphae resembled hyphae of act . The colony had 1  1  the restricted growth typical for col-16, but a smooth colony edge, and a miniature doughnutshaped stage, as in act (Fig. 5.ID and 5. IE). Hyphae of col-16; act hyphae had the same 1  1  branching pattern and hyphal morphology as act (Fig. 5.IF). 1  In most col-16 hyphae actin was distributed in one apical actin accumulation combined with the typical subapical peripheral plaques (Fig. 5.2 and Tab. 5.1). Less frequently actin was present in the form of multiple small actin accumulations at the hyphal tip, and rarely in the form of two apical actin accumulations. Hyphae of col-16; act had a 1  bigger variety of actin distributions. Most hyphae had one subapical, usually large, actin accumulation most reminiscent of act (Fig. 5.3 and Tab 5.1). 1  132  5.2.2 Thefr;act Double Mutant 1  A coral-like colony phenotype (Figs. 5.4A and 5.4B) and hyphae that had an almost exclusively dichotomous branching pattern (Fig. 5.4C) were typical for the fr mutant (Garnjobst and Tatum, 1967; Dicker and Turian, 1990; Perkins et al., 2001). The fr; act double mutant had a colony morphology that was more similar to fr than 1  act , but the hyphal morphology and branching pattern resembled the fr parent. Colony 1  growth of the fr; act double mutant was more restricted than either of the parents, but 1  resembled a fr colony (Figs. 5.4D and 5.4E). At the same time it had more aerial hyphae than a fr colony, and the mycelium appeared thicker. In hyphae offr; act the frequency of 1  branching events was higher than in act (Fig. 5.4F). 1  Hyphal tips of the fr; act double mutant predominantly had actin in one subapical 1  actin accumulation. Actin was present in a variety of other distributions less frequently (Fig. 5.5 and Tab. 5.1). Most of the observed hyphal tips offr did not have any actin accumulations at the hyphal tip. Out of the small number that did, most had multiple actin accumulations at the tip, and few had one apical actin accumulation (Fig. 5.6). The number of hyphal tips with these distributions was not counted because of the possibility that most of the hyphae had ceased growing at the time of fixation. Growing hyphae offr that were stained with FM4-64 had an apical Spitzenkdrper. During dichotomous branching, the Spitzenkdrper was not visible immediately before two new branches emerged. One Spitzenkdrper became visible in each branch after the two new braches emerged (Fig. 5.7).  133  5.2.3 The gran act Double Mutant 1  The colony of gran had granular conidiation, and sparsely branched hyphae (Perkins et al., 2001; Fig. 5.8D). Hyphae had numerous lateral aborted branches. Occasionally, multiple branches formed at the tip of a leading hypha (Fig. 5.8E). The gran act double mutant had unique colony morphology, and hyphae had the 1  combined characteristics of the parental strains. The double mutant colony resembled the colony of act . It had the doughnut-shape stage with an abundance of aerial hyphae, and 1  •continued to grow in a manner similar to act . However, the colony differed from both gran 1  and act by having a slower growth rate, by having less aerial hyphae and conidiospores, and 1  by producing a dark exudate in older parts of the colonies (Figs. 5.8A and 5.8B). Hyphae of the double mutant had the prominent meandering and aborted lateral branches typical for gran, but they also had dichotomous branching, and an increase in the branching frequency (Fig. 5.8C). Actin was observed in one apical accumulation at the hyphal tip of most gran hyphae (Tab. 5.1). At tips of hyphae that were contorted, or going through an apical branching event, there were multiple small apical actin accumulations, or actin was not visible (Fig. 5.9). In gran act most hyphae had one large subapical actin accumulation, but a variety of other 1  actin distributions were also present (Fig. 5.10). Growing hyphal tips of gran stained with FM4-64 showed single Spitzenkdrpers in the apical region. However, before an apical branching event, two (or more) Spitzenkdrpers appeared before the new branches were visible (Fig. 5.11).  134  5.2.4 The col-15; act Double Mutant 1  Mutant col-15 grew flat, but later the colony became elevated (Perkins et al., 2001). Sometimes the colony formed aerial hyphae with conidia (Figs. 5.12A and 5.12B). Hyphae had a mainly dichotomous branching pattern, and a high frequency of branching events (Fig. 5.12C). The colony of the double mutant col-15; act had more similarities with col-15 than 1  act , and the hyphae resembled col-15 as well. The colony resembled the colony of col-15 1  (Fig. 5.12D and 5.12E), except that the mycelium appeared slightly denser. In addition, microscopic droplets of a dark exudate were sometimes visible, as observed in gran act . 1  Hyphae of col-15, act had the same morphology as col-15, with dichotomous branching 1  dominating, and a high frequency of branching (Fig. 5.12F). Tips of col-15 frequently burst during sample processing, and consequently data for actin distribution was not collected. The most frequent actin distribution in col-15; act was 1  one subapical actin accumulation (Fig. 5.13 and Tab. 5.1). Actin in one apical accumulation, two apical accumulations or multiple actin accumulation at the hyphal tip occurred less frequently.  5.2.5 The col-8; act Double Mutant 1  The mutant col-8 had colonial growth and produced aerial hyphae (Garajobst and Tatum, 1967; Perkins et al., 2001; Figs. 5.14A and 5.14B). Hyphae branched mainly dichotomously, with a high branching frequency (Fig. 5.14C).  135  The double mutant col-8; act had a colony phenotype that was intermediate, while 1  the hyphae resembled col-8. Colony morphology of col-8; act was intermediate (Figs. 5.14D 1  and 5.14E), with growth rates slower than act , but faster than col-8. The hyphal morphology 1  and branching of the double mutant was the same as in col-8. In col-8, actin was mainly distributed in one apical actin accumulation, but many of the hyphae had multiple small actin accumulations at the hyphal tip (Fig. 5.15 and Tab. 5.1). The actin distributions were different in col-8, act , where the distribution with one subapical 1  actin accumulation at a hyphal tip was most frequent (Fig. 5.16), followed by hyphae with multiple small actin accumulations at the hyphal tip. Growing hyphae of col-8 stained with FM4-64 showed a continuous presence of Spitzenkdrpers in the apical regions. The Spitzenkorpers disappeared as soon as growth ceased (Fig. 5.17).  5.2.6 The act ; spco-4 Double Mutant 1  The colony of spco-4 started with colonial growth, but then continued spreading on the medium (Garnjobst and Tatum, 1967; Perkins, 2001). The colony did not produce conidiospores on V M (Fig. 5.18A and 5.18B). Hyphae branched more frequently than wild type, and had a lateral branching pattern (Fig. 5.18C). The double mutant act ; spco-4 had a colony phenotype that was more similar to act , 1  1  but hyphae resembled spco-4. Growth was more restricted than either of the parents (Fig. 5.18D And 5.18E). The colony had a compact appearance with a less pronounced doughnut-  136  shape stage than in its act parent. The hyphae of this double mutant had mainly lateral 1  branching, as in spco-4 hyphae (Fig. 5.18F). One apical actin accumulation was present in the majority of spco-4 hyphal tips (Fig. 5.19). Two apical actin accumulations and few small actin accumulations at the hyphal tip occurred rarely (Tab. 5.1). In contrast, most hyphae of act ; spco-4 had multiple small actin 1  accumulations at the hyphal tip, and a significant number of tips had one subapical actin accumulation (Fig. 5.20, and Tab 5.1). Other distributions, such as a subapical actin cap, and multiple prominent actin accumulations at the hyphal tip, were present in a smaller number of hyphae. A Spitzenkdrper was present in hyphae of spco-4 during growth (Fig. 5.21), and had a similar appearance as in wild type.  5.2.7 The md act Double Mutant 1  The mutant md was identified as a gene modifying the band size of the clock gene (Durkee et al., 1966; Perkins, 2001). Growth was described as spreading (Figs. 5.22A and 5.22B), with a branching pattern that is characterized by the occasional occurrence of outbursts of mainly apical branches (Figs. 5.22C). The double mutant had unique colony and hyphal morphologies. The colony of md act was more restricted than either of the parents (Figs. 5.22D, and 5.22E). Growth occurred 1  at a faster growth rate in regions where flares of hyphae with less frequent branching events escaped the compact colony (Fig. 5.22F). As a result, the colony had a highly irregular shape,  137  with an erratic distribution of puffs of aerial hyphae (Fig. 5.22 E). Hyphae branched more frequently than either of the parents. In the majority of hyphal tips of md that had a lateral branching pattern, actin was present in one apical accumulation (Fig 5.23 and Tab. 5.1). Hyphae that started an apical, dichotomous or multiple, branching event lacked the apical actin accumulation, but instead had multiple small actin accumulations in the apical region, or throughout the branching hyphal tip. The md act double mutant had a range of different actin distributions at hyphal 1  tips (Fig 5.24). One subapical actin accumulation was the most frequently occurring distribution. In md hyphae stained with FM4-64 A Spitzenkdrper was visible at tips before branching, as well as in tips of the apical branches (Fig. 5.25).  5.3 Summary  It is important to note that the act allele of the actin gene is not a null mutant. Based 1  on the results presented in Chapter III, it is likely that this mutation affects only some of the multitude of functions that actin has, many of which are essential for hyphal morphogenesis. The second component in the double mutant may also not be null. Therefore, the absence of an epistatic interaction between the act allele and a mutant allele of another gene does not 1  exclude that an interaction is possible. Characteristics of the double mutant strains and their parental strains were assessed for colony morphology, hyphal morphology and actin distribution (Tab.5.2). Complete epistasis of the colony morphology of one parental strain was not encountered in any of the  138  seven double mutants. However, partial epistasis was present. Three double mutants, col-16; act ,fr; act , and col-8; act , had a colony morphology similar to their non- act parent. In 1  1  1  1  contrast, the colony morphology of act ; spco-4 was similar to its act parent. Three of the 1  1  double mutant strains, gran act , col-15; act , and md act , had additional characteristics that 1  1  1  were not present in either of the parental strains. Mutants fr; act , col-15; act , col-8; act , and act ; spco-4 had hyphal morphologies 1  1  1  1  of their non- act parent. Mutant col-16; act , on the other hand, had the hyphal morphology 1  1  of act . A hyphal morphology that appeared to be a combination of the parental hyphal 1  morphologies was present in gran act . Mutant md act had a novel hyphal morphology 1  1  distinct from both parents. There are 11 categories of encountered actin distributions (Tab. 5.1). In each category there are variations of that particular actin distribution, and these are schematically presented in Fig. 5.26. Category 11 consists of tips in which the absence of actin accumulations may simply be the result of the absence of growth in these tips. Therefore, this group will not be considered in the following comparisons. Wild type had a monotypic actin distribution, and only the category 1 pattern with one apical actin accumulation was observed. In contrast, all of the parental and double mutant strains were multitypic, showing from two categories of actin distribution in gran, to ten categories in fr. Out of these, the double mutant strains always showed a bigger range of distribution patterns (four to ten) than their non-act parents 1  (two to four), and the range was radically shifted away from the monotypic wild type distribution. Although none of the strains showed the same set of actin distribution categories, the variability in the double mutant strains was bigger than in the non-act  1  parental strains, and resembled the variability of their act parent. In six double mutants the 1  139  majority of hyphal tips had a category 3 actin distribution, with one large subapical actin accumulation at the hyphal tip, as found in the act parent. Only act ; spco-4 had most hyphal 1  1  tips with a category 8 distribution, with multiple prominent actin accumulations at the hyphal tips. All the seven double mutants differed from their non-act parents, in which the category 1  1 distribution, with one apical actin accumulation, was most common. These comparisons, and the difference between the variability of the double mutants and their parents, suggest that gene interaction occurs in each one of the seven tested combinations. None of the tested double mutants of act showed full epistasis, or costasis. Four 1  double mutants, col-16; act',fr; act , col-8; act , and act ; spco-4, showed partial epistasis. 1  1  1  However, col-16; act and act ; spco-4 are exceptional, because while their hyphae look like 1  1  hyphae of one of the parental mutants, their colony resembles that of the other parental mutant. Three mutants, gran act , col-15; act , and md act , are in the novel category. The 1  1  1  novel characteristics include dark exudate and different hyphal morphology in gran act and 1  col-15; act , and a different hyphal and colony morphology in md act . 1  1  In conclusion, the double mutants can be assigned to two out of four categories of epistatic interactions defined in N. crassa (Gavric and Griffiths, 2003): partial epistasis and novel. The presence of epistatic interactions suggests that actin can interact with different gene products in diverse ways. This can be expected for a protein that has a multitude of roles in different cellular processes, and supports the heterogeneity of the phenotypes of the actin RIP mutants.  140  3 ^ „  8  Sa g |  * I f 8*1 33 1 rt 2 a, " l ft-l  ^ 1 §^ nj i—i  00  l i s  I  <3\  ltr>  i  AH  s o  M  5  W  5=1  £ o  o .a JS &  h  af  o 3  a ©§  M  a I."~ C+H-  o  -a £>  C/3 OH  \8  03  .fl .g  9  fl O  13  '£  53 1^  C/3  flO  to  g  VH  3 « a sS &  73  S «  l/l 1)  o fl •£ « '5b  3  J  C3 >i § , V fe < * >  ^ a fl §  cu  a  2  B 2 3  <U  gi O .ID fl  °  ft  CO  £ S  s I rSfl *.2 g tu  2 8  cd .fl  c/5 «  S.31 +3  "3 -fl 53 23  o >rt >  -rt fl cu  fl ^  O  §5 3  u  o  fl  .2 3 Sow  cu  -  fl.ro  O  (50 CU  S ^ fe o o fl g ta cd o  c  cu g *  .g  rg  g  •s § 1 = o ts .s | fl ^ in 3 1 | l .a 8 aAH  5  3  Ill  1 141  S  §  cd H  O  PHT3  H  O  2  rv  '  X>  2  <3  (col-15) col-8 + act act  Hyphal morphology and branching pattern act fr act + gran col-15 col-8 spco-4  new  new  Double mutant  Colony phenotype  col-16;  (col-16) (fr)  fr;  act  act  gran  _a col-15;  1  act  act  spco-4; md  1  act_  col-8; _ J  1  _  act  1  1  act  new  1  Actin distribution new new new new  (act ) (act ) (act ) (act ) new (act) new (act) new (act) 1  1  1  1  Table 5.2. Colony, hyphal morphology, branching pattern and actin distribution in the double mutants. Brackets indicate partial similarity; two phenotypes connected with a "+" indicate and intermediate phenotype; "new" indicates the presence of novel characteristics.  142  143  Figure 5.2. Actin and nuclear distribution in hyphal tips oi col-16. (A, B , C) Images of the same hyphal tip. (A) DIC image (B) Nuclear distribution. (C) One apical actin accumulation. (D, E) Two apical actin accumulations. (F) Three stronger stained dots in an apical actin accumulation. (G) Multiple small actin accumulations in a hyphal tip. (H) Absence of actin accumulations in a hyphal tip. All bars = 10 pm. Bar in image (G) applies to image (H). Bar in image (A) applies to the rest of the images. White triangles indicate the position of the hyphal apex.  144  A  D  C  B  0  E  F  G  H  J  K  L  T  1  T  Figure 5.3. Actin and nuclear distribution in hyphal tips of col-16; act , (A, B , C) Images of the same hyphal tip.(A) One large subapical actin accumulation. (B) DIC image. (C) Nuclear distribution. (D) One large and one small subapical actin accumulation. (E) Two subapical actin accumulations different in size. (F) Subapical actin cap. (G) One linear subapical and one small apical actin accumulation. (H) One apical linear actin accumulation. (I) One large apical actin accumulation. (J) One apical actin accumulation. (K) Two actin accumulations, one apical and one slightly displaced subapically. (L) Multiple actin accumulations at a hyphal tip. Triangular arrowheads indicate the hyphal apex. Both bars = 10 pm. Bar in image (A) applies to all images except image (L). 1  145  146  Figure 5.5. Actin and nuclear distribution in hyphal tips of fr; act . (A, B, C) Images of the same hyphal tip. (A) DIC image. (B) Nuclear distribution. (C) One subapical and three small peripheral actin accumulations. (D) Two subapical peripheral actin accumulations, one much smaller than the other. (E) Two large subapical and three small (apical and subapical) actin accumulations. (F) Three subapical actin accumulations. (G) Multiple strongly stained actin accumulations at a hyphal tip. (H) One large linear subapical actin accumulation. (I) A subapical cap-like actin accumulation. (J) Two apical actin accumulations. (K) One apical actin accumulation.(L) One linear subapical, and additional subapical actin accumulations The triangular arrowheads indicate the apex of the described hypha. Bar = 10 Lim, and applies to all images. 1  147  B  c  D  Figure 5.6. Actin and nuclear distribution in a dichotomously branched hypha of fr. (A) DIC image. (B) Nuclear distribution. (C, D) Actin distribution in different planes. (C) Apical actin accumulation is visible in two out of three hyphal tips. Bar = 10 Lun.  148  Figure 5.7. Hyphae of fr stained with FM4-64. (A-F) Growth observed over several minutes. The Spitzenkdrper is visible in the apical region in most tips. The white triangle in image (C) indcates the hypha that is beginning to branch dichotomousiy. Bar = 10 Lim.  149  150  Figure 5.9. Actin and nuclear distributions in hyphal tips of gran. (A, B , C) Images of the same hyphal tip. (A) DIC image. (B) Nuclear distribution. (C) One apical actin accumulation. (D, H) Images of the same hyphal tip. (D) Nuclear distribution. (E, F, G) Images of the same hyphal tip. (E) DIC image. (F) Nuclear distribution. (G) Absence of actin accumulations. (H) Multiple weakly stained actin accumulations. Triangular arrowhead indicates the apex of the described hypha. For ail images bar = 10 (im.  151  A  1  B  C  D  p  G  H  J  K  L  T  Figure 5.10. Actin and nuclear distribution in hyphal tips o f act gran. (A) One subapical axial and one small apical actin accumulation. (B) One subapical peripheral actin accumulation. (C) Two subapical actin accumulations. (D) Multiple subapical actin accumulations. ( E , F, G ) Images o f the same hyphal tip. (E) D I C image. (F) Nuclear distribution. ( G ) Subapical actin cap. ( H ) Subapical linear, and two small apical actin accumulations. (I) One large apical actin accumulation. (J) Two apical actin accumulations. ( K ) Multiple weakly stained actin accumulations in a hyphal tip. ( L ) Absence of actin accumulations in a hyphal tip. For all images bar = 10 (im. 1  152  A  B  C  D  E  F  G  H  J  K  1  Figure 5.11. (A-K) Growing hyphae of gran stained with FM4-64 observed over several minutes. Spitzenkdrpers are visible in hyphal tips. (F) Two Spitzenkdrpers are visible before the new apical branches appear. (L) DIC image. Bar = 20 Jim.  153  154  B  Figure 5.13. Actin and nuclear distribution in hyphal tips of col-15; act . (A, B, C ) Images of the same hyphal tip. (A) D I C image. (B) Nuclear distribution. ( C ) One subapical actin accumulation slightly displaced towards the periphery. (D) One subapical peripheral actin accumulation. ( E ) One subapical actin accumulation and linearly distributed actin that connects the subapical actin accumulation with the apex. (F) One subapical and a couple of small apical actin accumulations. (G) Two subapical actin accumulations. ( H ) One apical actin accumulation. Bars = 10 p m . Bar in image (A) applies to all the images except image (G). 1  155  156  E  B  C  D  F  G  H  y  f  Figure 5.15. Actin and nuclear distributions in hyphal tips of col-8. (A, B , C ) Images of the same hyphal tip. (A) DIC image. (B) Nuclear distribution. (C) One apical actin accumulation. (D) Apical actin cloud. (E) Two apical actin accumulations. (F) Multiple actin accumulations. (G) Multiple small and one large subapical actin accumulations. (H) Absence of apical actin accumulations. Arrowheads indicate the apex of the described hyphal tip. Bars = 10 pm. Bar in image (A) applies to images (A-D). Bar in image (E) applies to images (E-H).  157  ....  E  B  C  D  F  G  H  K  L  ••.[.:>:.  *  1  J  Figure 5.16. Actin and nuclear distribution at hyphal tips of col-8;  act . (A, B, C) Images of 1  the same hyphal tip. (A) DIC image. (B) Nuclear distribution. (C) One subapical actin accumulation displaced toward the periphery (D) One large subapical peripheral actin accumulation. (E) One subapical actin accumulation and a small apical actin accumulation. (F) Two tips with one subapical actin accumulation each. (G) One subapical linear actin accumulation. (H) One apical linear actin accumulation and smaller actin accumulations at a hyphal tip. (I) Multiple strongly stained actin accumulations. (J) Multiple weakly stained actin accumulations. (K) Several weakly stained actin accumulations. (L) One apical actin accumulation. Bar = 10 |im, and applies to all images.  158  Figure 5.17. Growth of col-8 hyphae stained with FM4-64. (A) DIC image. (B) Spitzenkdrpers in apical regions of hyphae. (C) Same hyphae observed a few minutes later. Bar = 20 pm.  159  U *• > = w  Cj  oe  «  2 s « PL •  «  Cj  3  CO  CQ  -  ?L £ .  V •!  C+- C*«, C+a  fa o o o 160  Figure 5.19. Actin and nuclear distribution in hyphal tips of spco-4. (A, B, C) Images of the same hyphal tip. (A) DIC image. (B) Nuclear distribution. (C) One apical actin accumulation. (D) One apical actin accumulation and a few small satellite actin accumulations. (E) One star-like apical actin accumulation. (F) One two-part apical actin accumulation. (G) One three-part apical actin accumulation. (H) Multiple weakly stained actin accumulations at the tip. Bar = 10 pm, and applies to all images.  161  B  c  D  F  G  H  K  L  1  E  /  SB  1  J  •  T  Figure 5.20. Actin and nuclear distributions in hyphal tips of act', spco-4. (A, B, C) Images of the same hyphal tip. (A) DIC image. (B) Nuclear distribution. (C) Multiple weakly stained actin accumulations. (D) Multiple strongly stained actin accumulations. (E) Two subapical actin accumulations. (F) One subapical actin accumulation. (G) One subapical peripheral actin accumulation. (H) One subapical actin cap and a small apical actin accumulation. (I, J) Linear subapical actin accumulations. (K) One apical actin accumulation. (L) Absence of actin accumulations in a hyphal tip Triangular arrowhead indicates the apex of the described hypha. Bar = 10 pirn, and applies to all other images.  162  Figure 5.21. Hypha of spco-4 stained with FM4-64, (A-C) Three images collected over a couple o f minutes. A Spitzenkdrper is visible in the apical region. B a r = 10 Lim.  163  164  B  C  D  G  H  'w $%  ?  ;  E  "  F  T  Figure 5.23. Actin and nuclear distribution of md. (A, B , C) Images of the same hyphal tip. (A) DIC image. (B) Nuclear distribution. (C) One large apical actin accumulation. (D) One small apical actin accumulation. (E) Apical actin cloud with a stronger stained dot within. (F) Multiple small apical actin accumulations. (G) Multiple weakly stained actin accumulations at a hyphal tip. (H) Absence of apical actin accumulations. Triangular arrowhead indicates the apex of the described hyphal tip. Both bars = 10 pm. Bar in image (E) also applies to image (H). Bar in image (A) applies to the rest of the images.  165  B  D  C  *  F  G  H  J  K  L  •  1 *  Figure 5.24. Actin and nuclear distribution in hyphal tips of md act . (A, B, C) Images of the same hyphal tip. (A) DIC image. (B) Nuclear distribution. (C) One subapical actin accumulation and a small apical actin cloud. (D) One subapical actin accumulation. (E) One subapical peripheral actin accumulation. (F) Two subapical actin accumulations. (G) One linear subapical actin accumulation. (H) One small apical actin accumulation. (I) One average-size apical actin accumulation. (J) One slightly peripherally displaced apical actin accumulation. (K) Multiple small actin accumulations in a hyphal tip. (L) Absence of actin accumulations in a hyphal tip. Triangular arrowhead indicates the apex of the described hyphal tip. Both bars = 10 pm. Bar in image (D) applies to images (E, H). Bar in image (A) applies to the rest of the images. 1  166  Figure 5.25. Hyphae of md stained with FM4-64. (A) DIC image. (B) Spitzenkdrp in hyphal tips of multiple apical branches. Bar = 20 pm.  167  'r\r\ ' ftp] r\f\ r\r\  10  2  Of)  r\r\  11  Figure 5.26. (1) One apical actin accumulation. (2) Two apical actin accumulations. (3) One subapical actin accumulation. (4) Two subapical actin accumulations. (5) One or more linear subapical actin accumulations. (6) Subapical actin cap and diffuse staining at hyphal tip. (7) Multiple strongly stained subapical, or subapical and apical actin accumulations. (8) Multiple weakly stained actin accumulations at the hyphal tip. (9) Multiple apical actin accumulations. (10) One linear apical actin accumulation. (11) Absence of actin accumulations at the hyphal tip.  168  CHAPTER V I DISCUSSION  6.1 Conclusions Based on act Characteristics 1  The screen for cytochalasin A resistance resulted in the isolation of act , the first actin 1  mutant identified in Neurospora crassa. The act mutant strain has a point mutation in the 1  actin gene and generates an actin protein that has a C374F substitution. The cysteine at position 374 is the penultimate amino acid at the carboxyl end of actin, located in subdomain IA (Fig. 3.3). After polymerizing into F-actin, this subdomain is exposed at the barbed end of F-actin (Sheterline etal, 1998). Cysteine at this position is proposed to be involved in binding to myosin, tropomyosin, profilin, cofilin, proteins of the spectrin family, troponin-1, calponin, caldesmon, and proteins of the gelsolin family (Crosbie et al., 1991; Graceffa and Jancso, 1991; Sheterline et al, 1998 and references therein) based on a series of in vitro studies. Impaired binding ability to one of these actin-binding proteins may lead to the act  1  phenotype. In vivo studies are sparse, and in Saccharomyces cerevisiae a C374A substitution did not result in any change in the morphology although the deletion of the two terminal amino acids caused temperature sensitivity (Johannes and Gallwitz, 1991). In another in vivo study in S. cerevisiae a C374S substitution prevented the mutant actin from binding to profilin, and reduced the efficiency of polymerization (Aspenstrom et al, 1993). In N . crassa the cytochalasin A resistance of act suggests that the residue 374 in N. 1  crassa actin is involved in binding cytochalasin A. Ohmori et al. (1992) showed that in a mutant strain of mammalian cells two simultaneous amino acid substitutions (V139M and  169  A295N) in the actin protein confer cytochalasin resistance. The residues that interact with cytochalasin have to be exposed at the barbed end of F-actin in order to fulfil their function. Both the residues 139 and 295 reported by Ohmori et al. (1992) as well as the residue 374 in N. crassa satisfy this requirement. The external position of the cysteine residue at position 374 additionally supports our proposal that it is responsible, at least in part, for the capping of F-actin fdaments with cytochalasin A.  6.1.1 The Calcium-Actin-Phosphoinositide (CAP) model  Reissig and Kinney (1983) showed that an increase in intracellular [Ca ] after 2+  treatment with the ionophore A23187 induced branching, and identified C a signal in N. crassa. A tip-high cytoplasmic C a  2+  2+  as a branching  gradient was proposed to be the reason for  the absence of dichotomous branches in N. crassa wild type (Schmid and Harold, 1988). The most recent model of hyphal tip growth (Silverman-Gavrila and Lew, 2001; SilvermanGavrila and Lew, 2002) explains how the tip high gradient is maintained (see Chapter I). In this model, stretch-activated phospholipase C is responsible for the localized production of inositol (1, 4, 5)-triphosphate (IP3) at the hyphal tip. IP binds to UVreceptors on vesicles 3  and facilitates release of calcium contained in the vesicles. This, coupled with subapical C a  2+  sequestration into the endoplasmic reticulum, creates a tip-high Ca gradient. The high concentration of C a  2 +  at the hyphal tip facilitates tip-localized fusion of vesicles. Silverman-  Gavrila and Lew (2003) showed that the presence of a [Ca ] difference between the apex 2+  and subapical region, not the steepness of the gradient, is the requirement for growth to occur. The model requires a steady flow of vesicles to the tip, but the authors did not  170  elaborate on how this flow is regulated, or how a switch from lateral to apical branching may occur. I propose a calcium-actin-phosphoinositide (CAP) model in which actin regulates the rate of vesicle flow from proximal to distal regions of a hypha. By doing this, actin controls the tip high gradient of cytoplasmic calcium. Hence, according to the C A P model actin controls the calcium gradient at the hyphal tip, which is both necessary for maintenance of tip growth and the dominance of one polarized site at the hyphal tip. Actin can regulate the transfer from lateral to apical branching by regulating the concentration and gradient of calcium at the hyphal tip. The CAP model (Fig. 6.1) can explain the multiple changes in act at the molecular, 1  cellular, and phenotypic level. I propose that the altered actin protein results in a changed property of cytoskeletal actin that affects the vesicle supply route and reduces the rate at which incoming vesicles arrive at the hyphal tip. This change has a multiplicative effect. Providing that the IPvmediated release of C a  2 +  from vesicles is proportional to the number of  vesicles delivered to the hyphal tip, the smaller number of vesicles at the tip results in a smaller amount of released C a . The effect is a shallower gradient of [Ca ] along the hyphal 2+  2+  tip but whose peak is still at the apex. This is the most significant consequence of the mutation in act in this context. The reduced number of vesicles at the hyphal tip, coupled 1  with the reduction of the [Ca ], results in a slower incorporation of plasma membrane and 2+  cell wall material at the hyphal tip, and a slower growth rate. The slower expansion of the cell wall and plasma membrane could also result in a reduced activity of stretch-activated phospholipase C and a subsequent alteration of phosphoinositide concentrations. Both  171  calcium and phosphoinositides are involved in the implementation of the actin-related functions in a variety of organisms (Janmey, 1994; Yin and Janmey, 2003). The reduction of the amount of cytoplasmic calcium and/or phosphoinositides at the hyphal tip could affect the binding ability of one or more actin-binding proteins involved in anchoring actin to the apical plasma membrane and cell wall, either by direct binding or by activating a regulatory protein. A role for proteins like spectrins and integrins in anchoring actin to the apex has already been proposed for N. crassa (Degousee et al., 2000). The external position of the residue substituted in the mutant actin makes it available for interactions of this kind. Mutant act F-actin fdaments could themselves have a modified 1  binding affinity to cell components that are involved in its anchoring, resulting in the actin usually distributed at the apex not being able to closely follow the expansion of the plasma membrane and cell wall, and acquiring a subapical position. This implies that actin accumulation in the extreme apical region may not be an absolute requirement for maintaining tip growth. The presence of a Spitzenkorper at the apex of act hyphae indicates that vesicles still 1  arrive, and have their highest concentration, at the apex. However, the number of arriving vesicles must be smaller, because of the smaller Spitzenkorper size. In my model for hyphal tip growth and branching, actin from act disrupts vesicle transport to the hyphal tip and 1  consequently reduces the apical cytosolic [Ca ] due to less C a 2+  2+  being released from  vesicles. The actin-regulated cytosolic [Ca ] at the apex, although lower than in wild type, is 2+  still high enough to attract more vesicles than surrounding regions, allowing the formation of a smaller Spitzenkorper and hyphal growth at a slower growth rate. The shift of the apical actin cap to a subapical position without preventing the formation of the Spitzenkorper shows  172  at least partial independence of the Spitzenkorper formation and actin located at the apex. This does not exclude the possibility that a small amount of actin is present as part of the Spitzenkorper because the region distal to the subapical actin accumulation is not entirely devoid of actin. Positioning of this subset of actin would have to be different than the bulk of apical actin, and could be through an alternative anchoring to the hyphal apex, or through associations with the Spitzenkorper. The CAP model accomodates the idea of apical dominance. The model proposes a tip-high calcium concentration that imposes apical dominance, preventing the formation of branches at the hyphal tip, and allowing the formation of lateral branches at a distance form the tip. The high calcium concentration at the apex prompts fusion of vesicles with the apical plasma membrane and cell wall, and prevents fusion in adjacent regions with lower calcium concentrations. At the same time, the V S C is positioned to direct the vesicle supply to the apical region of the hyphal tip. It is not clear what trigger would release calcium from its stores to create the gradient necessary for polarized growth at a lateral site. The CAP model offers an explanation of how a change in actin can cause a shift from a lateral branching pattern to dichotomous branching. In N. crassa wild type branching is lateral, with new hyphal tips emerging subapically at non-regular intervals (Davis, 2000). Disruptions in diverse genes can result in a switch to a dichotomously branching phenotype (Sone and Griffiths, 1999; Bok et al., 2001; Perkins et al., 2001), indicating the involvement of a plethora of gene products. The fact that a change in the actin protein can exclusively trigger dichotomous branching indicates that the wild type actin can control the shift from lateral to apical branching. Based on our present study, it is doubtful whether actin located at the apex is directly responsible for the branching pattern. According to our model, the main  173  function of actin in branching is through regulating the rate of vesicle flow to the hyphal tip and, consequently, the tip-high C a because the C a  2 +  2 +  gradient. However, in act apical dominance is disrupted 1  gradient at the tip is altered. A shallower gradient results in vesicles being  more evenly incorporated throughout the expandable region of the hyphal tip. This creates conditions for additional vesicle accumulation and incorporation that eventually result in a dichotomous branch. A satellite Spitzenkdrper formed during directional mass transport of vesicles towards the hyphal tip could be the focus of further vesicle accumulation that produces a dichotomous branch (Lopez-Franco et al., 1995). The "aborted branch" appearance of act is consistent with this branching model. A n aborted branch starts off as a 1  dichotomous branch, but one of the two hyphal primordia stops growing at an early stage. Growth most likely ceases because the amount of vesicles reaching the tip is not sufficient to maintain the locally increased calcium concentration that drives polarized growth. So far, it would seem that all slower growing hyphae are bound to have a dichotomous branching pattern, but this is not the case. The crucial point may be the C a concentration of C a  2+  2 +  gradient. Even if the  is lower at the hyphal tip in different mutants, the subapical [Ca ] may 2+  not be the same in all of them, resulting in different gradients of C a . Only in some cases 2+  would the gradient be shallow enough for apical branching to occur. An alternative to the mechanism underlying the onset of a dichotomous branching event is given by reaction-diffusion theory. Reaction-diffusion theory proposes that patterns in development can be generated from random concentration fluctuations of chemical substances (Harrison et al., 2 0 0 1 ) . Crucial for the process is the appearance of at least two chemical substances termed morphogens, with the properties of nonuniform distribution in space, nonlinear dynamics of their interactions, and transport by diffusion. Interactions  174  between the two morphogens ultimately result in the formation of developmental patterns such as a radial branching pattern, as seen in the whorls of the unicellular seaweed Acetabularia acetabulum (Dumais and Harrison, 2000), or a dichotomous pattern, as seen in algae of the genus Micrasterias (Holloway and Harrison, 1999). An excellent candidate for a morphogen in N. crassa, based on the presence and concentration gradients at branching sites, is calcium. The presence of microfdaments in the Spitzenkorper is recorded at the ultrastructural level in some fungi such as Fusarium acuminatum (Howard, 1981). In N. crassa wild type both the Spitzenkorper and the apical actin accumulation coincide with the VSC. The actin accumulation may be part of the smaller zone of the apical vesicle cloud proposed by Grove and Bracker (1970) to correspond with the Spitzenkorper. In the C A P model, the apical actin accumulation is the core of the VSC. Closeness of the V S C to the apex in wild type results in the incorporation of vesicles mainly into the apical plasma membrane, and the absence of apical branches. In act the apical actin accumulation is either completely dislocated to a 1  subapical position, or split into two severely unequal parts due to the disruption of its connection with the plasma membrane at the hyphal tip. The subapical actin accumulation functions as a V S C and guides the distribution of vesicles to their final destination at the hyphal tip in the same manner as the V S C described by Bartnicki-Garcia et al. (1990). The subapical position of the actin accumulation in act results in the incorporation of vesicles 1  more evenly along the whole surface of the extensible cell wall and underlying plasma membrane in the hyphal tip. Other factors that can affect hyphal growth and branching when considering a V S C are the distance of the apical/subapical accumulation from the apex and the extent of hyphal  175  tip tapering. When the apical actin accumulation is at a distance from the tip less than half the diameter of the hypha, then a tip-high gradient of vesicle distribution will occur. At distances greater than half the diameter of the hypha, vesicles will be evenly distributed to the hyphal tip and isodiametric enlargement of the expandable tip or cessation of growth can be expected. A more tapered hyphal tip will have a smaller hyphal diameter, which will . determine a different arrangement of vesicle incorporation influencing the shape and branching pattern. The multiple effects of cold at the cellular and molecular level in various organisms, as well as the discovery that the cold sensitive crib-1 N. crassa mutant is defective in ribosome biosynthesis (Schlitt and Russell, 1974), suggest that the cold shock effect in N. crassa wild type is likely to be a result of a complex set of cold-induced changes. This does not exclude the possibility that actin filaments participate in adaptation to cold conditions. The mode of their participation could be through the performance of actin binding proteins controlled by signalling pathways. In human platelets chilling caused uncapping of fastgrowing actin filament ends, and severing of actin fdaments that resulted in the formation of filopodia and lamellipodia (Winokur and Hartwig, 1995). The same study showed that higher levels of internal [Ca ] that occurred during chilling enhanced the severing activity. A 2+  correlation between cold conditions, and frequency of branching is present in the unicellular seaweed Acetabularia acetabulum (Harrison, 1997). There is a drop in spacing of hairs in a whorl at low temperatures, most likely due to structural changes in the plasma membrane resulting in a lower diffusivity for membrane proteins. In N. crassa exogenously added C a  2+  prevents the morphological changes that occur as a response to temperature reduction, although it does not restore the normal growth rate (Kawano and Said, 2002). This suggests  176  that C a  2+  concentrations are disturbed during chilling in N. crassa, and that this disturbance  may be important in cold-induced morphological changes. The cold sensitivity of act is, on the other hand, exclusively a result of a single 1  mutation. The unique set of events after a downshift to the threshold temperature of 7°C appears to be an exacerbation of the polarity defect seen at 25°C. The loss and subsequent gain of polarity after a temperature downshift in act suggests actin is crucial for establishing 1  and maintaining polarity. The transient nature of the polarity loss indicates a transient change in the actin protein characteristics or the characteristics of an actin-interacting protein induced by chilling. A wider diameter of hyphae after regaining polarity can be explained if we incorporate an additional variable in our model-the extent of cell wall plasticity. Previous studies in human platelets showed that chilling causes changes in cell membranes (Ando et al., 1974) and C a  2 +  levels (Oliver et al., 1999). In N. crassa a less rigid cell wall and an  increase in intracellular [Ca ] are likely to occur at low temperatures. Both of these factors 2+  would allow incorporation of vesicles into subapical regions of the plasma membrane and cell wall. Together with a slower growth rate, the effect would be an enlarged hyphal diameter, which is what we observed. The currently accepted model for the distribution of nuclei in N. crassa includes the presence of a connection between the most distal nucleus and the hyphal apex (Plamann et al., 1994). The observation that nuclei are found closer to the apex in act than in wild type 1  indicates that the proposed connection may not involve microfdaments, or that a connection may be absent altogether. As an alternative, distal nuclei may be transported to their subapical position passively, by the bulk flow of the cytoplasm.  177  Co-localization of actin with some nuclei was previously reported in Allomyces macrogynus, but no explanation has been offered for its presence (Srinivasan et al., 1996). Turian et al. (1992) noticed that cytochalasin B-treated conidia display R N A protein-rich intra- and perinucleolar dense granules. They therefore proposed that actin is involved in the mechanism for monopolar transport of pre-ribosomal R N A from the nucleoli to the cytoplasm. If this is true, then the actin plaques that co-localize with nuclei may mark the transport sites. The absence of co-localization in some nuclei may indicate the absence or reduction in the transport activity of nuclei. Another possibility is that the actin plaques at the nuclei represent attachment sites to a yet unidentified hyphal component mediated by actin. In conclusion, I propose that the mutation in act results in the disruption of the 1  vesicle flow towards the hyphal tip, and consequently an altered [Ca ] and gradient at the 2+  apex. This triggers a number of changes that together account for the act phenotype. Hence 1  the CAP model provides a clear role for actin in hyphal growth and branching.  6.2 Conclusions Based on the Actin RIP Mutant Characteristics  The RIP mutants obtained provide an excellent foundation for investigating the different aspects of actin involvement in the life cycle of N. crassa, particularly in hyphal morphogenesis. Several conclusions can be made based on the phenotypes of these mutants (Fig. 4.35 and Tab.4.6). In the vegetative cycle, actin is crucial for the initiation and maintenance of polarized growth of the germ tube, the switch from a non-branching germination tube to a branching hypha, as well as for the selection of a branch site, initiation and maintenance of branch and hyphal growth (Tab. 4.6). In the asexual cycle, actin could be  178  necessary to implement the switch to the conidiation cycle once the signal for the switch is generated. In the sexual cycle, the normal formation of perithecia and ascospores within asci cannot proceed if actin is altered. Although previous work in N. crassa suggested some of these actin functions (see Chapter I), most of the conclusions were based on experiments using actin inhibitors that could also have non-actin related effects. The evident absence of some categories of phenotypes in the actin RIP mutants may be due to the stringency of the screen, or to an invalid assumption about the involvement of actin in the particular cellular process. Proof for the former possibility is already emerging. In a recent study aimed at identifying genes contributing to cellular morphogenesis in N. crassa, an actin mutant with a different phenotype than any of the mutants from this study was described (Seiler and Plamann, 2003). The phenotype of this mutant falls into the category of predicted actin mutants with a defect in the selection of a polarization site during branching, resulting in multiple branches at a lateral branching site (Fig. 4.6). On the other hand, some stages of polarized growth seem to be controlled by cellular components other than actin. During germination in ./V. crassa, for instance, the selection of a germination site is most likely under the control of microtubules, because treatment with benomyl, an inhibitor of microtubule function, results in the formation of multiple germination tubes (Barja et al., 1993). However, as with actin inhibitors, these results need to be confirmed, and until then, actin involvement is still possible. To track down how the changes in actin can lead to aberrant phenotypes, the first steps were to look at the actin gene sequence information, and the actin distribution patterns in hyphae.  179  The sequence of the actin gene in the actin RIP mutants predicts which amino acids are substituted in the mutant actin protein (Fig. 6.2). The amino acid substitutions are at different positions not only in different categories of mutants, but also within the same category of mutants. The variety of positions at which amino acids are substituted in the mutant categories indicates the involvement of these amino acids in different functions. The presence of different substitutions within a category of mutants suggests the cooperation of different sites in the execution of an actin function. Tab. 6.1 shows a summary of evidence for ligand binding at the residues that are mutated in the actin RIP mutants (Sheterline et al., 1998). The ligands can be divided in three groups: ligands related to assembly of actin monomers into actin polymers (G-actin and nucleotide), a diverse group of actin binding proteins involved in the structure and function of actin, and toxins. Most of the information was collected from in vitro experiments, with few exceptions. In the next few paragraphs I will discuss some in vivo data supporting ligand binding, and what effects binding to each one of the ligand candidates may have in N. crassa. A change in an amino acid residue at a site that is proposed to be involved in selfassembly or nucleotide binding suggests a problem with G-actin assembly into F-actin fdaments in that mutant. This is a major deficit for the hypha, having in mind that the active form of actin for the majority of processes related to hyphal morphogenesis requires the presence of F-actin. Mutants from five out of six categories that were sequenced had mutations of this kind, suggesting that amino acid substitutions at different positions could have different effects on self-assembly. The different effects on self-assembly could, in turn, affect distinct processes in the hypha, resulting in distinct phenotypes of the mutants.  180  The cold-sensitivity in mutant 20.2C (act ) may be due to polymerization defects 20  related to cold temperature. In the yeast actin protein, residues 265-268 form a hydrophobic pocket at the interface of two subunits on opposite strands of the actin filament (Chen et al., 1993). A mutant with a L266D substitution shows a cold-sensitive polymerization defect because of the destabilization of this hydrophobic interaction at cold temperatures (Chen et al., 1993). In the N. crassa actin RIP mutant 20.2C (act ), the two sites with substituted 20  residues are in subdomain II. This subdomain faces the opposing strand of the F-actin filament (Sheterline et al., 1998), making it possible that the phenotype at cold temperatures is due to a defect in actin polymerization. Myosin from myofibrils of vertebrate muscle cells is a motor protein comprised of six polypeptide chains: two heavy chains, and two pairs of light chains (Alberts et al., 1989). This myosin is a member of class II, or conventional, myosins. An increasing number of myosin classes were added to this group of myosins in the last years, all of which have the capability to interact with actin filaments (Mooseker, 1993). The interaction with actin filaments enhances the ATPase activity of myosin, and the energy from A T P hydrolysis is transformed into mechanical work resulting in movement towards the + end of the actin filament. Some classes of myosins associate with cell membranes. Myosins'are prime candidates for actin-binding proteins with a function in hyphal morphogenesis. The forcegenerating property could have a function in the transport of vesicles along actin filaments, and the membrane-binding property could be important for polarization related events. In yeast, three classes of myosins were identified: class I, class II and class V (Brown, 1997). Null mutants and mutants with altered myosins have defects in cell separation, budding polarity, endocytosis and cell fate. Other mutants have wild type phenotypes, indicating  181  redundancy. The functions that are disrupted, leading to the defects at the phenotypic level, are all related to transport of different components within the cell and between the bud and mother cell (Brown, 1997). The sequence of the N. crassa genome allows predictions of gene function based on homology to genes from other organisms. Myosin mutants were not described in N. crassa, although there are at least three myosin homologues in the genome. In yeast, a R183A substitution in actin at a site predicted to bind myosin did not have any visible effect on the phenotype (Wertman et al., 1992). However, this mutant shows resistance to lantruculin-A, confirming that a qualitative change did occur (Ayscough et al., 1997). In another budding yeast mutant a D24A amino acid replacement confers a reduced range of temperatures at which cells can grow, and temperature sensitivity to both high and low temperatures (Wertman et al., 1992). The phenotype at threshold temperatures was not described. A D24H actin mutation in Dictyostelium discoideum resulted in loss of motility accompanied with loss of heavy meromyosin ATPase activation in an assay (Johara et al., 1993). In vertebrate muscle myofibrils actin binds to tropomyosin and troponin, proteins that activate the myosin ATPase in a Ca -dependent manner (Spudich and Watt, 1971; Alberts et 2+  al., 1989). Tropomyosin is a polypeptide homodimer with six to seven actin-binding sites (Sheterline et al., 1998). It binds to actin filaments longitudinally, stabilizing them (Alberts et al., 1989). Troponin is a complex of three polypeptides, troponins T, I, and C. Troponin T (T stands for tropomyosin-binding) binds to tropomyosin, bringing the complex close to the actin filament. Binding to the actin filament is the responsibility of troponin I (I stands for inhibitory). This binding prevents the attachment of myosin to actin filaments, even in the presence of C a  2 +  molecules. Troponin C (C stands for calcium-binding) confers C a 2+  182  sensitivity to the complex. In the presence of C a  2 +  troponin C allows myosin to bind to actin  fdaments. In Drosophila melanogaster, a P307L mutation resulted in mild abnormalities in flight muscle fibres (An and Mogami, 1996). The abnormalities were less severe than expected, because a change in proline was predicted to result in changes in actin folding. While N. crassa has at least one homologue of tropomyosin, homologues of the troponin peptides are absent. The similaritiy between troponin C and calmodulin makes it very appealing to speculate that troponin may functionally be replaced by calmodulin, and that calmodulin may be involved in activity control of the actin-myosin complex. Profdin binds to G-actin or terminal monomers of F-actin (Schmidt and Hall, 1998). It stimulates the exchange of ADP with A T P in actin monomers and by doing this facilitates actin polymerization. However, binding to terminal F-actin monomers prevents polymerization. Profdin has a phosphoinositol-4,5-bisphosphate-binding site, indicating the regulation of its function by phosphoinositide signalling. A profdin homologue exists in N.  crassa. The binding site for DNase is at the pointed end of actin filaments (Sheterline et al., 1998). Once DNase is bound to actin, it is inactivated (Sheterline et al., 1998). It is not clear how binding to DNase could affect hyphal morphogenesis, so it is possible that the site implicated in the interaction with DNase in other organisms may have additional functions in  N. crassa. Hartwig (1994) gave an extensive overview of the spectrin superfamily that consists of three subfamilies. Spectrin, a-actinin, and dystrophin are members of the spectrin subfamily; filamin, ABP (actin-binding protein), and T A B P (thyroid actin-binding protein) comprise the ABP subfamily; fimbrin and adducin make up the fimbrin subfamily. The  183  proteins from this group are large proteins, and are teteramers (spectrin), homodimers (aactinin, ABP, filamin) heterodimers (adducin), or monomers (fimbrin). The main function of proteins of this superfamily is to cross link actin fdaments with each other, and with other proteins (Hartwig, 1994, and references therein). Proteins that act as a bridge between actin filaments bind to the sides of actin filaments with their termini, e.g. spectrin, or along their length, e.g. fimbrin. Other proteins from the spectrin superfamily link actin filaments with integral membrane glycoproteins, e.g. dystrophin, or with plasma-membrane bound receptors, e.g. filamin and ABP. Spectrins also have sites that bind to proteins such as ankyrin, indicating roles in anchoring actin networks to integrin receptor-containing complexes. Another interesting aspect is the control of proteins from the spectrin superfamily through C a , calmodulin, and phosphoinositide signalling pathways. Proteins from the 2+  fimbrin subfamily are phosphoproteins, and therefore additionally have the potential of being controlled by kinases and/or phosphatases from signalling pathways. Identifying and confirming connections of this kind in N. crassa in the future would significantly contribute in lighting up the black box that currently exists between signal generation by internal and external stimuli, and the visible ultracellular, cellular, and phenotypic response to the signal. The caldesmon family of proteins and calponin bind to actin and regulate contraction in smooth muscle. Caldesmons are elongated molecules with a globular head containing actin-binding sites and calcium regulatory sites (Sheterline et al., 1998). It is proposed that caldesmon and calponin inhibit the ATPase activity of the actin-myosin complex, and that binding of [Ca ]-calmodulin releases the inhibition (Graceffa and Jancsd, 1991; Mezgueldi 2+  et al., 1992; Bonnet-Kerrache and Momet, 1995). Proteins similar to caldesmon or calponin have not yet been identified in N. crassa.  184  Gelsolin binds to actin filaments, severs them, and then binds to the + ends of the new actin fragments (Alberts et al.). It is activated in the presence of elevated [Ca ] and contributes to the formation of new actin nucleation sites. A requirement for this kind of action is present at parts of the cell selected for expansion. Phalloidin binding sites were identified in vitro at amino acid positions 117,119 and 355 in the actin gene of rabbit skeletal muscle (Vandekerckhove et al., 1985). Phalloidin is a phallotoxin produced by the toadstool Amanita phalloides that binds to the + ends of actin filaments and prevents further polymerization, thus stabilizing the filament (Korn, 1982; Cooper, 1987). In vivo studies in S. cerevisiae showed that amino acid substitutions at positions 158,177, and 179 of the yeast actin gene resulted in the loss of phalloidin-binding (Drubin et al., 1993; Belmont et al., 1999). Besides resistance to phalloidin binding, the yeast mutant with these two substitutions had additional defects, such as in increase in the randomness of bud scars in diploids, and an altered actin organization at elevated temperatures. The additional defects indicate the involvement of sites at which the residues were substituted in functions other than phalloidin binding. Myosin, tropomyosin, and gelsolin all bind at the same sites as phalloidin, and may be responsible for the additional effects (Sheterline et al., 1998). The actin distribution patterns provided another way of deducing the connection between changes in actin and the resulting phenotype. In the actin RIP mutants in which the number of tips with a specific actin distribution was quantified, even though the actin distribution in the majority of hyphal tips was the same as in the wild type, all of the mutants had a wider range of different actin distributions (Tab. 4.5). The shift from one apical actin accumulation to another distribution pattern indicated that the change in the actin protein did  185  affect its distribution, but that the change was not as severe as in act , in which the majority 1  o f hyphae had an actin distribution pattern that was different than in the w i l d type. The presence o f an apical Spitzenkdrper observed in most o f the mutants, with the exceptions of the colonial mutants and mutants with severe polarity defects, shows that hyphal growth is not compromised. In instances where growth ceased, or was very slow, the Spitzenkdrper was absent, as expected. In some actin mutants, the presence of two apical actin accumulations prior to the formation of a dichotomous branch provides circumstantial evidence that division of the apical actin accumulation may control a dichotomous branching event. This statement has to be accepted with caution, because in fixed cells it is not possible to determine with certainty i f the tips with a double apical actin accumulation would continue with the formation o f a dichotomous branch. More information needs to be gathered from live cells, before conclusions can be made with confidence. The gap between actin changes and distribution at one side, and the mutant phenotype at the cellular and colony level still remains. The C A P model that best fits the act  1  mutant  can partly fill this gap. In the light of this model, the category E mutants with severe polarity defects could be defective in the maintenance o f the V S C structure assembly. A t sites where polarity was lost, large actin accumulations were never observed, and the Spitzenkdrper was absent, indicating that the flow of vesicles was not organized. The C A P model predicts a 2+  change in calcium levels in this case, because the vesicles, being the main source o f C a , are more evenly distributed throughout the zone that lost polarization. The levels o f inositol phosphate that the vesicles are exposed to may also be altered, since phospholipase C that generates phophoinositides may not be activated efficiently due to the reduced cell wall 186  expansion rate. A threshold level of actin concentration or a modified actin binding function may be required, because some hyphae grow before the polarity disorder sets in. The actin function proposed to be defective in this group of mutants could be a structural assembly step at the V S C that is disassembled at threshold levels of actin concentration or actin binding function. Altered calcium concentrations could result in the colonial phenotype of the category G mutants. In these mutants the slow growth rate and high frequency of branching is connected with a dichotomous branching pattern. It is somewhat confusing that the actin distribution in the majority of hyphae is the same as in wild type, indicating that the actin core of the V S C is at a position that should allow a C a  2 +  concentration and gradient expected  to give a lateral branching pattern. One possible reason for the persistence of dichotomous branching may be a lower calcium concentration due to a reduced supply of vesicles to the tip favouring a more even fusion of vesicles throughout the hyphal tip surface. At the same time, the calcium concentrations may be high'enough to prevent the dislocation of the actin cap and actin V S C core to a subapical position. Category H mutants with a higher branching frequency can also be explained with the CAP model by having a lower calcium concentration in the apical region, and different relative concentrations of calcium in the apical vs. subapical regions. The calcium concentration difference is expected to be higher in lateral branching, Increasing apical dominance, and lower in dichotomous branching, lowering apical dominance and allowing dichotomous apical branching. The CAP model rationalizes most of the effects found in the actin mutants generated in this study. Although the fit is not perfect, and allows improvement, it is the best model that  187  can be devised based on information available for processes that pertain to hyphal morphogenesis in N. crassa.  6.3 Conclusions Based on act Double Mutant Characteristics 1  The search for interactions involving actin is at an early stage in N. crassa. Not many structural or regulatory components directly relevant for hyphal morphogenesis are identified. The ones that are known are few, and connections that link them are lacking or incomplete. In most of the mutants the genes at the mutated loci are not identified or sequenced. In the one case (fr) in which the gene was identified, its function is not clear, although the involvement in C a  2+  and M n  2 +  homeostasis and signalling has been proposed  (Sone and Griffiths, 1999). In yeast, CDC1 mutants have a depolarized actin cytoskeleton coupled with Golgi retention and reduced vacuolar inheritance (Rossanese et al., 2001). This finding points to a role for CDC1 in actin organization and organelle movement. These results, together with the proposed central position offr in pathways deduced from epistatic interactions (Gavric and Griffiths, 2003), suggest that fr may act upstream of act in TV. crassa. At the same time both act and fr have pleiotropic effects, which may provide an explanation for the additional characteristics of the double mutant phenotype. The presence of epistatic interactions in all the tested double mutants shows that actin interacts with a number of different proteins in different ways. This is in agreement with the predicted multiple functions of actin in diverse processes in the cell, and provides an explanation for the variety of phenotypes encountered in the actin RIP mutants.  188  It is interesting that similar mutant phenotypes, e.g. a colonial phenotype, were observed in mutants from the large N. crassa mutant collection as well as in actin mutants. A similar phenotype of two single mutants may indicate the involvement of the two mutated genes in the same pathway, or interaction between the two genes. However, there are problems in interpretation of these similarities. One is that the identity of the mutated genes of the majority of the mutants from this mutant collection is not clear. The Neurospora genome project is currently in the process of creating a genome wide knockout collection that should identify a subset of these mutants that are null.  6.4 Future Work  Although most of the predicted phenotypes were observed in the actin mutants, there are more phenotypes that were not. Some of these predicted phenotypes would be particularly interesting, e.g. mutants with branches at unusual sites such as at septae. Conditional lethal and temperature sensitive actin mutants would also be very useful, allowing lethal alleles to be investigated at threshold temperatures. Data collected from the new actin mutants establish the involvement of actin in certain processes directly related to tip growth and branching in fdamentous hyphae. Several lines of evidence have been compiled and require experimental follow up. Quantification of actin amounts, and polymerization efficiency tests are one possible course of action. The CAP model needs to be tested and validated on a variety of mutants. The concept of the CAP model includes several basic constituents including a calcium gradient, actin distribution, and Spitzenkorper presence. Calcium concentrations at the hyphal tips of the  189  actin mutants need to be determined during apical growth and branching. In most of the RIP mutants, the calcium concentrations at the hyphal tip should be different than in wild type. The detailed characteristics of these constituents need to be determined, spatially and temporally localized, the details need to be recorded and quantified in diverse conditions. An alternative way to rationalize branching events is in the context of reactiondiffusion theory. The Brusselator two-morphogen activation-depletion model, particularly suitable for generating patterns with radial symmetry, shows promise for providing a mechanism for dichotomous and apical branching. For lateral branching, the GiererMeinhardt model, with the capacity to maintain single concentration peaks, appears to be appropriate. These models are promising because they can provide a solution to how one or more polarization sites are generated, starting from smallfluctuationsin the concentrations of cell constituents. Several findings in N. crassa have the potential of fitting into one of the reaction-diffusion models. A strong candidate for a morphogen is calcium, based on the presence and concentration gradients of calcium at hyphal tips. In addition, the shift from lateral to dichotomous branching in some of the actin RIP mutants is coupled with the change in the shape and size of the hyphal tip. Changes in these parameters can result in the change in the number of branches that are formed if reaction-diffusion theory is applied. An example of such a change can be found in Larix x leptoeuropaea where the number of cotyledons is linearly related to the diameter of the apical surface of the embryo (Harrison and von Aderkas, 2004). These possible links suggest that the application of the concepts of reactiondiffusion theory is an avenue worth exploring. Actin is a constituent of cellular structures usually in the form of F-actin that is associated with actin binding proteins. Interaction of actin with actin-binding proteins, and  190  their spatial and temporal positions need to be determined. It is these cellular structures that engage in function of the cell leading to an effect that will shape the hyphae. The hyphae and their characteristics are then orchestrated to give the colonies their characteristic appearance. The nature and functional versatility of the actin renders the actin mutants a valuable source for discovering not only process related to hyphal morphogenesis, but also a multitude of other essential or non-essential processes necessary for the mycelium to survive and thrive in its environment.  191  cj  la .a  CJ  "o  d o a  00  o  C3  CJ  0£  § C3  1.3  o CJ  60 CXI  C  f  d  •E  '1  o  cj a.  49  2 S OH  od  X  o  CJ  II  Sal B  g .«  3  til  s  s ssS  S  S  S  « s S s a a> > •s  S  ss  •a  4»  ° 5 S A  PM  et  ON  o  CN  CO  co  u  o  >/->  CN  CS 9  2  IS s  cl o  B  a  'a ill  s  C3 M  ' i  o en  ^  'as ^ O  u.  o  CCJ  CJ  DO  eS  '•a 00 ,  B  B  2 '3  ZT «  eg  co  "  .2  JS = M  g  IS  a*  «  ~  Q  o „  £ 60  o  •©  ft  <+H  —i  a>  S3 *  192  a  —  'o C  H  •s  3  50 o  Figure 6.1. Model of hyphal tip growth and branching. Hyphal tip of (A) wild type and (B) act . 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