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Study of wound healing and the involvement of the cytoskeleton in Vaucheria longicaulis variety macounii Tornbom, Laurie Jean 1992

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STUDY OF WOUND HEALING AND THE INVOLVEMENT OF THECYTOSKELETON IN Vaucheria longicaulis VARIETY macouniibyLAURIE JEAN TORNBOMB. SC., UNIVERSITY OF BRITISH COLUMBIA, VANCOUVER, B.C., 1977A THESIS SUBMITTED IN PARTIAL FULLFILMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTERS OF SCIENCEinTHE DEPARTMENT OF BOTANYWe accept this thesis as conformingto th required standardTHE UNIVERSITY OF BRITISH COLUMBIATornbom April 1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of  Ziri-a 1-1 yThe University of British ColumbiaVancouver, Canada/ Date(Signature) DE-6 (2/88)iiAbstractVaucheria longicaulis var. macounii is a coenocyticsiphonous member of the Division Chrysophyta. This alga grows inthe intertidal zone, and it is exposed to grazing damage by slugsand snails, as well as probable fragmentation and sand abrasiondue to wave action. V. lonqicaulis exhibits the capacity to heallarge transverse wounds, and this wound healing process is theobject of this study.The sequence of events leading to wound healing andresumption of growth are studied by light microscopy as well asthe use of differential interference contrast optics. Woundhealing has been divided into four stages. In Stage 1, a bi-membranous wound healing vesicle is rapidly extruded from thewound site. Loss of turgor pressure, as observed by theappearance of plasmolysis, is observed along the length of thefilament. In Stage 2, the wound healing vesicle is sealed offfrom the filament by the fusion of the plasma membrane andtonoplast at the wound site. Organelles then accumulate at themembranous barrier created by the sealing off procedure. This isfollowed by chloroplast retraction and return to the wound site,concurrent with restoration of turgor pressure in late Stage 2.In Stage 3 chloroplasts again retract and return to the woundsite, in preparation for Stage 4 that finally leads to theresumption of growth on the part of the healed filament.iiiThe effect of wound healing on organelle distribution hasbeen studied with the use of selective fluorochromes. Thedistribution of nuclei, identified by DAPI staining, remainsuniform throughout the wound region. CTC fluorescence along thewound site and wound vesicle cytoplasm implies the importance ofCa2+-based exocytosis in both wound healing vesicle expansion andits sealing off from the filament. Mitochondria and probablelipid bodies (designated RBIs) are identified by staining withDiOC6 and Rhodamine B, respectively. They are present in thewound healing vesicle cytoplasm and accumulate along the woundsite.The cytoskeleton is identified as strand-like cables ortracks by differential contrast microscopy. The cytoskeletontracks are oriented parallel to the length of the filament, andthe organelles are closely associated and appear to move alongthem. In uncut filaments, the different classes of organellesmove independently of one another and travel at different speeds.Chloroplasts, mitochondria, RBIs, and smaller organelles (SSIs)exhibit a bulk pattern of motion characterized by brief reversalsin their primary pattern of movement that do not affect theirfinal direction of travel. Nuclei move independently of eachother and do not reverse their direction of movement. In cutfilaments, cytoskeleton density and relationship to organelles inthe wound region during Stage 1 is similar to that in uncutfilaments, and extends into the wound healing vesicle.ivMitochondria size organelles move in bulk rapidly toward thewound site and into the wound healing vesicle. In Stage 2, anincrease in cytoskeleton density is observed in the wound region,and bulk movement of mitochondria and similar size organellestoward the wound occurs. Nuclei also move in bulkunidirectionally toward the wound. Organelle retraction from thewound is restricted to chloroplasts.Both uncut and cut filaments are treated with thecytoskeleton depolymerizers Cytochalasin B and Oryzalin todetermine the role(s) of microfilaments and microtubules inorganelle movement and the overall role of the cytoskeleton inthe wound healing process. In uncut filaments, cytoplasmicstreaming is severely reduced by both Cytochalasin B andOryzalin, with the greatest reduction observed followingCytochalasin B treatment. Work in cut filaments points to apreferential association between microfilaments and chloroplasts,mitochondria, and RBIs, and microtubules with nuclei during thewound healing process. Consequently, bulk movement ofchloroplasts and mitochondria (with temporary reversals) in uncutfilaments may be microfilament based, and the independent nuclearmovement may be microtubule based.TABLE OF CONTENTSABSTRACT ^TABLE OF CONTENTS ^LIST OF TABLES viiLIST OF FIGURES ^ viiiACKNOWLEDGEMENTS xiiINTRODUCTION ^ 1MATERIALS AND METHODS ^ 4TABLE I ^ 6RESULTS 7IDENTIFICATION AND DISTRIBUTION OF ORGANELLES IN UNCUTFILAMENTS ^ 7TABLE II 10WOUNDING AND WOUND HEALING STAGES ^ 11TABLE III ^ 14DISTRIBUTION OF ORGANELLES AND CYTOCHEMICAL CHARAC-TERIZATION OF THE EARLY STAGES OF WOUND HEALING ^ 15CYTOSKELETAL ORGANIZATION AND CYTOPLASMIC STREAMING ^ 18TABLE IV ^ 23STUDIES WITH CYTOSKELETAL DEPOLYMERIZING AGENTCYTOCHALASIN B ^ 24TABLE V ^ 29STUDIES WITH CYTOSKELETAL DEPOLYMERIZING AGENTORYZALIN ^ 30TABLE VI 34viSTUDIES WITH OTHER CYTOSKELETON ALTERING TREATMENTS ^ 35DISCUSSION ^ 36WOUND HEALING AND ORGANELLE DISTRIBUTION ^ 36STUDY OF CYTOSKELETAL-BASED ORGANELLE MOVEMENTIN WOUND HEALING ^ 45EFFECT OF CYTOCHALASIN B AND ORYZALINON WOUND HEALING ^ 51CYTOSKELETON BASED MOVEMENT OF ORGANELLES:POSSIBLE INTERPRETATIONS ^ 56CONCLUSIONS ^ 59FIGURES 61aLITERATURE CITED ^ 71LIST OF TABLESTABLE I. Specific information on the use of fluorochromes ^ 6TABLE II. Summary of the size and density of organelles invegetative filaments ^ 10TABLE III. Wound healing stages 14TABLE IV. Characteristics of organelle movement and speedof travel in untreated filaments of Vaucheria longicaulis var. macounii  23TABLE V. Characteristics of organelle movement, speed oftravel and aggregation status in Cytochalasin Btreated filaments of Vaucheria longicaulis var.macounii ^ 29TABLE VI. Characteristics of organelle movement, speed oftravel and aggregation status in Oryzalin treatedfilaments of Vaucheria longicaulis var. macounii.34vi iviiiLIST OF FIGURESFIGURE123Vacuole in Vaucheria filament ^ 616161Cortical cytoplasm in Vaucheria filament ^Chloroplasts, nuclei and smaller organelles ^4 DAPI fluorescence showing nuclei ^ 615 Osmiophilic inclusions (TEM) ^ 626 Rhodamine B positive inclusions (RBIs) ^ 627 Di0C6 positive inclusions (mitochondria) ^ 628 Endoplasmic reticulum (DiOC6 staining) 629 Stage 1: wound healing vesicle and loss of turgorpressure (plasmolysis), inset: wound healingvesicle structure ^ 6210 Closing of wound 6211 Sealed wound, Stage 2 ^ 6312 Membrane barrier between wound healing vesicleand filament ^ 6313 Organelle accumulation at the wound site ^ 6314 Chloroplast retraction ^ 6315 Accumulation of small organelles at the woundsite ^ 6316 Return of chloroplasts to the wound site,elimination of plasmolysis ^ 6317 Stage 3: Chloroplast retraction 63ix18 Stage 4: Resumption of growth ^ 6419 Stage 1: chloroplasts near wound site(autofluorescence) ^ 6420 Late Stage 2: chloroplast retraction(autofluorescence)^ 6421 Late Stage 2: chloroplast return to the wound site(autofluorescence)^ 6422^Stage 1: DAPI fluorescence, nucleardistribution ^ 6423^Stage 2: DAPI fluorescence, nucleardistribution ^ 6424^Stage 1: DiOC6 fluorescence, mitochondriaaggregation at wound site ^ 6425 Early Stage 2: DiOC6 fluorescence, mitochondriadistribution at wound site and in the cytoplasm ofwound healing vesicle ^ 6526 Late Stage 2: DiOC6 fluorescence, mitochondriaaccumulation at wound site ^ 6527 Stage 1: Rhodamine B fluorescence, RBIs in woundhealing vesicle cytoplasm ^ 6528^Early Stage 2: Rhodamine B fluorescence,accumulation of RBIs at the wound site ^ 6529 Late Stage 2: Rhodamine B fluorescence,accumulation of RBIs at the wound site^ 6530^Stage 1: CTC fluorescence ^ 6531^Stage 2: CTC fluorescence 6632^Stage 2: Calcofluor White fluorescence ^ 66x33 Cytoskeleton and associated organelles in uncutfilaments ^ 6734 Cytoskeleton tracks along the base of chloroplasts ^ 6735 Cytoskeleton tracks on the surface of achloroplast^ 6736 Opposite orientation of nuclei ^ 6737 Stage 1: Wound region cytoskeleton distribution ^ 6738 Stage 1: Cytoskeleton in wound healing vesicle ^ 6739 Stage 2: Increase in cytoskeleton density in woundregion ^ 6840 Stage 2: Accumulation of organelles near the woundsite and visualization of the cytoskeleton^ 6841 Cytochalasin B: uncut filament, undulatory patternof cytoskeleton and aggregation of smallorganelles^ 6842^Cytochalasin B: uncut filament, visualization ofthe cytoskeleton and relationship with organelles ^ 6843 Cytochalasin B: Stage 2, cytoskeleton in woundregion ^ 6844 Cytochalasin B: Stage 2, organelle distributionand orientation ^ 68-945 Cytochalasin B: non-wound region, organelleaggregation ^ 7046 Oryzalin: uncut filament, cytoskeletondistrubution. inset, organelle aggregation ^ 7047^Oryzalin: cut filament, Stage 2 ^ 70xi48 Oryzalin: non-wound region, organelledistribution ^ 70xi iAcknowledgementsThe author gratefully acknowledges the dependable financialsupport, guidance and patience of Dr Luis Oliveira, her thesissupervisor. She also acknowledges the generosity of Dr.Cavalier-Smith for the use of his Zeiss microscope. Help inareas of this work by Grant Chu, Danny Lin and James Bailey wasvalued. Advice and humor from fellow graduate students Lucy Peatand Brian Nichol have often cleared blurred vision.Most importantly, she thanks her partner, Graham, for hislove, humor, refreshing insights, and trust in her eventualcompletion.1IntroductionWound healing studies have been carried out in both higherplants and algae (Aist, 1976, Hardham and McCully, 1982, LaClaire, 1982a, Menzel, 1980, 1988). Wound healing can occur in avariety of ways depending on the organism and how extensive thedamage is. In plants composed of several tissues, wound healinggenerally involves necrosis of damaged cells, and restoration ofthe wound region takes place through division of intact cells.This response is seen in Pisum sativum (Hardham and McCully,1982), Nicotiana tabacum (Nims et al., 1967), as well as in algaesuch as Euchemia alvarezii (Azanza-Corrales and Dawes, 1989),Sargassum filipendula (Fagerberg and Dawes, 1976, 1977), andFucus vesiculosus (Fulcher and McCully, 1969). In contrast,filamentous multicellular algae such as Rhodochorton purpureum(Pearlmutter and Vadas, 1978), and Griffithsia Pacifica (Waalandand Cleland, 1974) repair damaged cells. Wound healing has beenextensively studied in siphonous members of the DivisionsChlorophyta (Order Caulerpales: Caulerpa sp., Bryopsis sp.,Udotea sp., and Order Siphonocladales: Ernodesmis verticillataand Boergesenia forbesii), and Charophyta (Chara sp. and Nitella sp.). These siphonous algae are exposed to wound damage throughgrazing, fragmentation in winter storms, and sand abrasion(Menzel, 1980 & 1988). These pose a serious risk to the survivalof the organisms, given their unique (coenocytic) type ofcellular organization.Wound healing has been studied by performing three types ofwounds: transverse cuts, puncture and pressure wounds. Siphonous2Chlorophytes such as Caulerpa sp. (Dreher et al., 1978, Goddardand Dawes, 1983), Bryopsis sp. (Burr and West, 1971),Acetabularia sp. (Menzel and Elner-Menzel, 1989c), and Ernodesmis verticillata (La Claire, 1987, 1989a) survive all three kinds ofwounds. Charophytes and higher plants survive puncture wounds(Nims et al., 1967, Aist, 1976, Foissner, 1988, Schackel et al.,1991,), but more extensive wounds result in cell death (Hardhamand McCully, 1982, Menzel, 1988).Wound healing work in siphonous Chlorophytes has delineateda series of stages (events) that occur in the majority of thespecies studied. First, a wound plug is extruded from the wound,concurrent with rapid contraction of the protoplasm away from thewound, and eventual wound closure. Turgor pressure is lostconcomitantly with these events, and regained shortly thereafter.A cell wall is later deposited over the wound. Most of thestudies from which this information is garnered are based on TEMobservations that are restricted to a narrow region along thewound site (Dreher et al., 1978, Goddard and Dawes, 1983, Burrand West, 1971). These portray a static image of the woundhealing process at fixed time intervals. These studies andothers have yielded information on gross protoplasmrearrangement, but provide little information on the movement ofindividual organelles along the cytoskeleton during wound healing(Menzel, 1988). No information is available on movement ofdifferent classes of organelles following treatment withcytoskeleton inhibitors. Furthermore, this work has beenconducted solely on members of Divisions Chlorophyta andCharophyta, with no published studies on wound healing in3divisions such as the Chrysophyta, which contains large celledsiphonous species such as Vaucheria longicaulis.The objectives of this study are to determine the basicstructure of the V. longicaulis var macounii vegetative filament,and the sequence of events leading to wound healing, includinghow wounding affects its cellular organization. For thispurpose, studies are conducted on the patterns of distributionand movement of the major classes of organelles, including thespeed of movement. The role of the cytoskeleton in theseprocesses is also explored by determining its distribution andrelationship to the organelles. Furthermore, the effects ofcytoskeleton inhibitors Cytochalasin B (microfilament inhibitor)and Oryzalin (microtubule inhibitor) on cytoplasmic streaming andorganelle movement are also studied to help elucidate the role(s)of the major cytoskeleton components (microfilaments andmicrotubules) on the wound healing process.4Materials and Methods:Vaucheria longicaulis var macounii was collected from anintertidal region of North Vancouver, B.C., and grown on itsnatural substrate, supplemented with half strength Instant Ocean(Aquarium Systems, Inc., Eastlake, Ohio) medium supplemented withminor elements (Lewin, 1966) and soil extract. Cultures werekept at 10 C, with vita-lite fluorescent lighting providing 2000lux under a 16-8h light-dark photoperiod.Filaments of V. longicaulis var macounii selected formicroscopical studies were removed from actively growingcultures, and excised while immersed in half strength InstantOcean medium (Fitch and Oliveira, 1986) under a Leitz Diavertinverted microscope. Detailed observations of wound healing wereconducted on a Leitz Dialux 20EB compound light microscope, and aZeiss Axiophot microscope equipped with differential contrastoptics. All staining and fluorochrome work was conducted on athe Leitz Dialux 20EB microscope equipped with epifluorescenceoptics. Staining was done at room temperature, and treatmentswere applied to the filaments in half strength Instant Ocean (seeTable 1 for specific information). Neutral Red staining (aqueoussolution) was applied at 50 pg/ml and the material was rinsedbefore observation. Fixation for TEM was conducted as describedby Fitch and Oliveira (1986).Organelle density, determined from an average of 10filaments, was established by counting the number of organellesin the central, focussed portion of the filament. This area was5estimated to be 900 pm2 , and corresponds to a square measuring 30pm across and 30 pm in length.Cytochalasin B dissolved in 1% DMSO was applied at 200pg/ml, and Oryzalin dissolved in 1% acetone at 26 pg/ml.Colchicine (aqueous solution) was applied at up to 50mM, APMdissolved in 1% DMSO at up to 200 pM, and taxol dissolved in 1%DMSO at up to 50 pM.All chemicals were purchased from Sigma Chemical Co., exceptfor Oryzalin [4(Dipropylamino)-3,5-dinitrobenzenesulfonamide,synonym Surflan] which was a generous gift from Dow Elanco Co.,Greenfield, Indiana, APM [0-methy1-0-(4-methy1-6-nitropheny1)-N-isopropyl-phosphorothioamidate, synonym NTN-6867] that was agenerous gift from Mobay Corporation, Kansas City, Missouri, andtaxol, which was a generous gift from the National CancerInstitute, Bethesda, Maryland. When appropriate the propercontrols using 1% DMSO, acetone, or ethanol, were also carriedout. Oxytetracycline (OTC), a Ca 2+ insensitive probe (Wolniak etal, 1980, Wise and Wolniak, 1984), was used as a control for thelocalization of membrane-bound Ca 2+ by the CTC method.The pattern of organelle movement and the speed at whichdifferent classes of organelles travel were analyzed fromvideotaped material. Ten filaments were videotaped for eachexperimental set, and the distance travelled (free of temporaryreversals) by three organelles of each class was measured over atime period of at least one minute. The mean speed +SE for eachorganelle class was then calculated.Table 1Specific Information on use of FluorochromesChemical Target^excit emis^sol^conc^live/ rinse^(nm)^(nm) (Fg/m1) fixedDAPI^nuclei^372^456^aq^5.0^fixed^noDi0C6 mito 478^496^etoh^1.5^live noER^11 II II^5.0^fixed^noCTC^memb-Ca2+ 385 515^aq^51.5^live yesCalc. W cellulose^365^410 aq 1.0^live^eitherRhod. B^lipid bod. 560^580^aq^10.0^live yesabbreviations: excit = excitation wavelengthemis = emission wavelengthsol = solutionconc = concentrationDAPI = 4-6-diamidino-2-phenylindoleDiOC6 = 3,3'-dihexyloxacarbocyanine iodideCTC = chlorotetracyclineCalcofluor White = 2,2'-(1,2-ethenediy1)bis[5-[(4-[bis(2-hydroxyethyl)amino]-6-(phenylamino)-1,3,5-triazin-2-yl]amino]-benzene sulphonic acidRhodamine B = tetraethyldiamino-O-carboxy-phenyl-xanthenyl67ResultsIdentification and distribution of organelles in uncut filamentsVaucheria longicaulis var macounii is a coenocytic tip-growing filamentous alga. The generally unbranched filamentscontain a large central vacuole. This is easily observable afterstaining with Neutral Red, and it extends from the subapicalregion all the way down the length of the filament (Fig. 1).Between the tonoplast and the plasma membrane lies a thin layerof cytoplasm containing numerous organelles. This layer isnoticeably thicker in the apical zone, and it is only observableelsewhere in the filament upon focussing on the cortical regionof the cell (Fig. 2).The chloroplasts are the largest and most easilydistinguishable of the organelles. Although their numbers varyfrom filament to filament, the average density is 5 chloroplastsper 900 pm2 of cytoplasm. They measure approximately 13.9+0.5 pmin length and each contains a single disc-shaped pyrenoid (Py)measuring 6.1+0.2 pm in diameter. Chloroplasts are usuallyuniformly distributed and oriented with their long axis parallelto the length of the filament (Fig. 3).V. longicaulis filaments also contain numerous nuclei.Their average density is 15 nuclei/900 pm2 of cytoplasm. Theseare tear drop in shape and average 5.2+0.1 pm in length. Aprominent feature is the presence of one or two smallerorganelles, referred to in the text as nuclear associated bodies(NABS), at the broad end of the nucleus (Fig. 3, arrowheads).DAPI fluorescence illustrates the even distribution of nuclei8through most of the filament; they occur in groups in thesubapical region (Fig. 4).Numerous small sized inclusions, ranging from 1.0-1.5 pm inlength are also observed throughout the vegetative filaments,including the tip region (Figs. 2 and 3). The larger inclusions(1.5+0.1 pm) represent mitochondria and other structures similarin size to mitochondria. Although these are easily distinguishedby electron microscopy (Fig. 5), they are otherwise difficult toseparate without the use of appropriate cytochemical techniques.Electron microscopic observations show some of these inclusionsto be intensely osmiophilic which suggests a lipid composition.They occur at an average density of 17 inclusions/900 pm 2 ofcytoplasm. They accumulate Neutral Red (Fig. 2), and arepositively stained with Rhodamine B (Fig. 6). They are referredto in the text as Rhodamine B-positive inclusions (RBIs). DiOC6,a fluorescent stain without affinity for RBIs, is used tovisualize the distribution and determine the density ofmitochondria. Mitochondria also occur at an average density of17 mitochondria/900 Fm 2 of cytoplasm (Fig. 7). Dictyosomes areclosely associated with mitochondria in Vaucheria (Ott and Brown,1974, Fitch and Oliveira, 1986); hence their distribution mirrorsthat of the mitochondria. Smaller inclusions (1.0+0.1 pm) varyin density and distribution, and because no reliable cytochemicaltest could be found for their identification, they are lumpedtogether under the general designation of Small-Sized Inclusions(SSIs). A summary of all major organelles, their size, anddensity of occurrence in the cytoplasm is presented in Table 2.9Di0C6 fluorescence also reveals the existence of anendoplasmic reticulum system which extends along the length ofthe filament (Fig. 8), and into the tip region (Fig. 8, inset).This appears as a fine network of strand-like structures, whichpreferentially occupies the cortical cytoplasm of the filaments.Table 2Summary of the size and density of occurrence of major organellesin vegetative filaments of Vaucheria longicualis var macounii Organelle^Size (pm) ±SE^Density(# structures/30 pre)chloroplasts^13.9 ± 0.5^5pyrenoids 6.1 ± 0.2nuclei^5.2 + 0.1 15NAB* 2.0 + 0.1mitochondria^1.5 + 0.1^17RBI's**^1.5 + 0.1 17SSI's*** 1.0 + 0.1 variable* nucleus associated bodies, 1-2 per nucleus** Rhodamine B positive Inclusions*** Smaller-Sized Inclusions1011Wounding and Wound Healing StagesVegetative filaments were excised approximately 3 mm fromthe tip region, and wound healing was studied on the basal end ofthe resulting fragment. Four stages of wound healing wereidentified.In Stage 1, a globular vesicle emerges from the wound siteapproximately 1 minute after wounding (Fig. 9). The vesicleaverages 50.9+1.8 pm in diameter. Sometimes the vesicle deflatesand then always reinflates quickly. The vesicle exterior isadhesive, frequently adhering to the bottom of the petrie dish.At high magnification the vesicle appears to be delimited by twomembranes with a thin layer of cytoplasm occupying the space inbetween them (Fig. 9 inset, arrowheads). Based on the size rangeof the inclusions found in the cytoplasm of the wound vesicle,one can identify them as mitochondria, RBIs, and SSIs. Largerorganelles such as chloroplasts and nuclei are usually excludedfrom the wound vesicle cytoplasm. The vesicle seems to arisefrom a region of the cytoplasm underneath the wound site. Boththe plasma membrane (Fig. 10, arrows) and tonoplast (Fig. 10,arrowheads) of the filament are continuous with the membranes ofthe vesicle. This continuity extends to both the vacuole and thecytoplasm (Fig. 10 and inset). Loss of turgor pressure, asdetected by the appearance of plasmolysis, is seen throughout thefilament, including the tip region (Fig. 9). Signs ofplasmolysis begin to occur approximately 1.5 min after wounding.Stage 2 begins with the sealing off of the wound vesiclefrom the filament. This seems to take place through the merging12and eventual fusion of the plasma membrane and tonoplast near thewound site (Fig. 11, arrowheads). Figure 10 inset shows an earlystage in the narrowing of the space defined by these two membranesystems in preparation for fusion (arrowheads). After fusion, amembrane barrier separates the wound vesicle from the filament(Fig. 12), and a large accumulation of organelles develops at thewound site (Fig. 13). This is followed by a major displacementof larger organelles (mainly chloroplasts) away from the woundsite (Fig. 14). This phenomenon, characteristic of the laterpart of Stage 2, occurs at about 8 minutes after excision andleaves a clearing of optically bright cytoplasm in place thatextends approximately 50.0+4.6 pm from the wound site. This areaof the filament vacated by the larger organelles is designatedthe "wound region". The remainder of the filament is referred toas the "non-wound region". Plasmolysis is still apparent in boththe wound and non-wound regions of the excised filament (Fig.14). Approximately 17 min after wounding, the organelles returnto the wound site. This is recognizable by the increase in theoptical density of the wound region cytoplasm. Large organellesare excluded from the immediate vicinity of the wound site,leaving an area of cytoplasm occupied by smaller organelles thatextends from the vacuole tonoplast to the plasma membrane at thewound site (Fig. 15, cy). The return of the larger organelles tothe wound site occurs simultaneously with a decrease and eventualdisappearance of plasmolysis throughout the excised filament(Fig. 16). The wound vesicle persists throughout this stage.However, the loss of the wound vesicle, leaving the wound seal13directly exposed to the medium (Fig. 15), does not seem to affectsubsequent stages of wound healing.In Stage 3, approximately 25 min after excision, theorganelles once more move away from the wound area (Fig. 17). Inthis case the retraction movement extends farther (84.0+.1 pm)from the wound site than in Stage 2. This is followed by thereturn of the organelles to the wound site, beginningapproximately 33 min after excision. The recovery from organelleretraction does not usually extend beyond 41 minutes afterwounding. Another important difference is that no signs ofplasmolysis occur during Stage 3. A substantial period of timethen ensues before resumption of growth is observed. During thisperiod designated as Stage 4, no reorganization of cytoplasm andorganelles is detected. Growth occurs approximately 43 hoursafter wounding, indicating that the process of wound healing hasbeen completed. Growth usually takes place both at the apicalend of the filament and at a new growth region established at anangle to the wound site (Fig. 18). The stages, their maincharacteristics and duration are summarized in Table 3.14Table 3Wound Healing Stages^Stage^Morphological Characteristics^Durationcut area^filament1^vesicle plasmolysis^0-4 minformation^ (1 min)*2 early wound^plasmolysis^4-21 minsealing (4 min)late^organelle^plasmolysis^(8 min)retractionfrom woundregionrecovery from^recovery from^(17 min)organelle^plasmolysisretraction3^organelle^no plasmolysis^21-41 minretraction (25 min)recovery from^no plasmolysis^(37 min)organelleretraction4^preparation^no plasmolysis^41 min-43 hrand resumptionof growth* The time in between brackets represents the point in each Stagewhen the morphological event is usually observed.15Distribution of Organelles and Cytochemical Characterization ofthe Early Stages of Wound HealingWound RegionStages 1 and 2 seem to be the most critical for filamentsurvival since their disturbance leads to permanent loss of thecapability of excised filaments to complete wound healing.Therefore a more detailed study of the pattern of organelledistribution in the wound and non-wound regions, as well ascytochemical characterization of the early stages of the woundhealing process, are reported here.Immediately after wounding, the majority of chloroplastsremain closely associated with the wound region (Fig. 19). Theyretract from the wound area during Stage 2 (Fig. 20), beforefinally returning to it at the end of this Stage (Fig. 21). Incontrast, DAPI fluorescence studies show that the distribution ofnuclei remains undisturbed throughout the wounded filaments andno retraction of nuclei from the wound region is observed (Fig.22 and 23).Di0C6 fluorescence shows that there is an accumulation ofmitochondria at the wound site shortly after wounding (Fig. 24).This increase in mitochondria in the wound region persists evenafter the wound vesicle is sealed off from the filament (Fig.25). This figure also shows that mitochondria are present in thewound vesicle cytoplasm. As healing proceeds, the mitochondrialaccumulation in the wound area increases (Fig. 26). Rhodamine Bfluorescence shows that RBIs also occur in the cytoplasm of the16wound vesicle (Fig. 27). As in the case of mitochondria,staining intensity increases during the healing process (Figs.28, 29). No retraction movements of mitochondria and RBIs aredetected. The behavior and distribution of SSIs appears to besimilar to that of the mitochondria and RBIs. The study of thedistribution of the endoplasmic reticulum in the wound regionwith DiOC6 has proved to be difficult due to the accumulation oflarge numbers of organelles that interferes with thevisualization process.Two important events one must expect to occur during theprocess of wound-healing are the repair of the surface membraneand cell wall at the healing site. NPN, a lipophilic stainutilized for the visualization of cytoplasmic membranedistribution (Wolniak et al, 1983), failed to stain the woundsite. However, CTC, an antibiotic known to fluoresce oninteraction with membrane-bound Ca 2+ (Caswell & Hutchinson,1971), shows the development of fluorescence starting with thewounding process. In Stage 1, the CTC fluorescence patternoccupies a large area of the wound region and extends into thewound vesicle. Overall, the fluorescence shows a reticulatepattern of distribution (Fig. 30). This contrasts with thefluorescence pattern observed after wound sealing that is morerestricted and uniform in occurrence Fig. 31). Treatment ofwounded filaments with Calcofluor White shows bright fluorescencein the wound region (Fig. 32). This pattern of fluorescenceremains unchanged during the healing process.Non-wound regionNo major changes in the distribution and density oforganelles were detected in the non-wound region of excisedfilaments compared to uncut filaments.1718Cytoskeletal Organization and Cytoplasmic Streaming:1. Uncut filamentsUnder differential contrast microscopy, the cytoskeletonappears as fine parallel cable-like strands (tracks) running thelength of the filament. Changes in the organization of thecytoskeleton tracks can occur locally due to the branching ofthese structures (Fig. 33, arrowhead). Chloroplasts, nuclei,mitochondria, RBIs, and SSIs appear to glide along thecytoskeletal tracks with which they are closely associated.Small organelles use one, often the same, track (Fig. 34,arrows). Large organelles, particularly chloroplasts, aresurrounded by several tracks (Fig. 35, arrowheads). Whether ornot the movement of larger organelles occurs through interactionwith a single or multi-track system is more difficult toestablish, although some observations seem to suggest theexistence of a preferential interaction between each chloroplastand a single cable-like strand of the cytoskeleton (Fig. 34,arrowheads).Cytoplasmic streaming proceeds as a continuous flow oforganelles and other cytoplasmic inclusions. The predominantdirection of movement is parallel to the length of the filament.All organelles move from the basal to the apical region of thefilament, and then return to the basal region in endless motion.Chloroplasts, mitochondria, RBIs, and SSIs move as a group (inbulk), and in coordination with one another within each categoryof organelles. These organelles exhibit a pattern of motioncharacterized by brief reversals in their primary direction of19movement that do not affect the final net direction of travel.In contrast, nuclei move independently and in opposite directionsin relation to one another. This is easily detected inmicrographs due to the fact that nuclei always travel with theirpointed end first, NAB region last, and often nuclei in closeproximity to each other can be seen oriented in oppositedirections (Fig. 36, arrows). Nuclei exhibit a pattern of motioncharacterized by the absence of reversals on the primarydirection of movement.The speed of movement differs among the various types oforganelles. Chloroplast flow occurs at an average speed of36.0+3.2 pm/min, while nuclei move rapidly with an average speedof 101.7+7.7 pm/min. The movement of mitochondria and RBIsaverages 63.6+5.2 pm/min. SSIs move at 170.4+7.3 pm/min.2. Cut FilamentsWound regionIn Stage 1, the organization and density of the cytoskeletonis similar in appearance to that in uncut filaments (Fig. 37).The cytoskeleton tracks extend into the wound vesicle cytoplasm.Numerous inclusions (organelles) are observed in closeassociation with the cytoskeleton tracks (Fig. 38). Overall, theinteraction of the cytoskeleton with the organelles seemsunchanged.In Stage 2, there is an increase in the number ofcytoskeleton tracks in the wound healing region. This increasein cytoskeleton density can be seen to extend approximately 50-70pm away from the wound area into the filament during the period20of organelle retraction. These cytoskeletal elements aremoderately convoluted (Fig. 39). The increase in cytoskeletondensity persists through the end of Stage 2, although it becomesmore difficult to visualize due to the accumulation of organellesduring the recovery phase from organelle retraction (Fig. 40).No changes are observed in the interaction of organelles with thecytoskeleton tracks.With wounding, changes occur both in the pattern and speedof movement of the organelles. The flow of chloroplasts isnearly halted (2.6+0.3 pm/min) in Stage 1 and early Stage 2. Inlate Stage 2, bulk movement of chloroplasts away from the woundsite and back again occurs without brief reversals in theirprimary direction of travel. This movement occurs atapproximately 12.9+1.0 pm/min. The movement of nuclei changesfrom independent to bulk following wounding. During Stage 1 andearly Stage 2, the movement of nuclei is unidirectional towardsthe wound region and proceeds at an average speed of 39.6+2.0pm/min. In contrast, in late Stage 2, the movement of nucleibecomes bidirectional. Nuclei move away from the wound site onone side of the cell at 37.1+2.4 pm/min, and return to it throughthe other side of the cell at the same speed. In both instances,nuclei exhibit bulk flow without reversals in the primarydirection of movement. Mitochondria and RBIs follow the samedirection, type, and pattern of motion as the nuclei duringStages 1 and 2. However, these organelles move more rapidly thanthe nuclei, at 74.0+5.1 pm/min in Stage 1 and early Stage 2, andat 66.3+4.6 pm/min in late Stage 2. SSI's appear to move incoordination with and at the same speed as mitochondria and RBIs.21Non-Wound Region:The organization of the cytoskeleton tracks in the cytoplasmfar from the wound region appears similar to that in uncutfilaments. However, both the pattern and speed of movement ofthe organelles are different from those in uncut filaments.These changes are largely coincident with the development ofplasmolysis along the length of the wounded filaments.Initially, chloroplast movement stops as plasmolysis increases.Approximately 15 minutes after cutting, when no further increasein plasmolysis occurs, these organelles exhibit bulk movement atan average speed of 8.5+0.6 pm/min. Their pattern of motion ischaracterized by the absence of reversals on the primarydirection of movement. With the gradual disappearance ofplasmolysis toward the end of Stage 2, the speed of this bulkmovement of chloroplasts increases to 15.3+0.8 pm/min. Thepattern of motion is unchanged. Nuclei move in coordination withthe chloroplasts, and no longer exhibit independent motion inopposite directions. Consequently, changes in nuclear speedmirror those of the chloroplasts. In contrast, the movement ofmitochondria and RBIs is independent from those of chloroplastsand nuclei, and unaffected by the degree of plasmolysis.Mitochondria and RBIs exhibit bulk movement, characterized by apattern of motion free of reversals in the primary direction oftravel, and at an average speed of 52.4+4.0 pm/min. SSIs appearto move in conjunction with mitochondria and RBIs.22A summary of the speed and major characteristics oforganelle movement and distribution in uncut and cut filaments ispresented in Table 4.23Table 4 - Characteristics of organelle movement and speed of travel inuntreated filaments of Vaucheria longicaulis var. macouniiUncut Filaments Cut FilamentsWound Region Non - wound regionOrganelle Characteristicsof movementSpeedpm/minStage Characteristicsof movementSpeedpm/minPiaamolysielevelCharacteristicsof movementSpeedp.m/minChloro-plastsBulk,Complex36.0± 3.21 Bulk, Simple 2.6 ± 0.3Increasing Bulk, Simple 0 -> *8.5 ± 0.6early2Bulk, Simple 2.6 ± 0.3late Bulk, Simple 12.9 ± 1.0 Decreasing Bulk, Simple 15.3 ± 0.8Nuclei Independent,Simple101.7± 7.71 Bulk, Simple 39.6 ± 2.0Increasing Bulk, Simple 0 -> *8.5 ± 3.2early2Bulk, Simple 39.6 ± 2.0late Bulk, Simple 37.1 ± 2.4 Decreasing Bulk, Simple 15.3 ± 0.8Mit0Ch.+RBIsBulk,Complex63.6 ± 5.21 Bulk, Simple 74.0 ± 5.1Increasing Bulk, Simple 52.4 ± 4.0early2Bulk, Simple 74.0 ± 5.1late Bulk, Simple 66.3 ± 4.3 Decreasing Bulk, Simple 52.4 ± 4.0Definitions of Characteristics of MovementBulk Movement:^The organelles move as a unit.Independent Movement: The organelles move separately from one another.Simple Movement:^Movement without reversals on the primary direction of travel.Complex Movement:^Movement with brief reversals in the primary direction ofmovement that do not affect the final net direction of travel.* Denotes the occurance of an initial motionless ( 0 Am/min ) phase followed bymovement at the speed indicated.24Studies with Cytoskeleton Depolymerizing Agents1. Effects of the microfilament depolymerizing agent CytochalasinB on the cytoskeleton and wound healingUncut filamentsConcentrations of 100 and 150 Fg/ml Cytochalasin B weretested, but failed to produce clear cut results. Treatment with200 Fg/ml Cytochalasin B yielded almost immediate reduction inthe speed of cytoplasmic streaming, and the response remainedunaltered during exposures of up to 2 hours. Most of the studieson the effects of Cytochalasin B were carried out at aconcentration of 200 Fg/m1 and the observation time lasted forapproximately 20 minutes.The cytoskeleton tracks continue to be identifiable ascable-like strands following Cytochalasin B treatment. Thedensity of these tracks remains unchanged. However, the tracksoften assume an undulating pattern of configuration only rarelyseen in untreated filaments (Fig. 41). The relationship betweenthe tracks and the organelles also remains unaltered, withmovement occurring along the tracks (Figs. 41, 42).Cytochalasin B treatment results in a major slowdown ofchloroplast movement (1.8+0.3 Fm/min). This occurs withoutalteration in their distribution, orientation (Fig. 42), ordirection of travel compared to untreated filaments.Chloroplasts continue to move in bulk, with a pattern of motion25characterized by brief reversals in the primary direction ofmovement that do not affect the final net direction of travel.The movement of the majority of the nuclei is severely andalmost immediately affected by the treatment. These nucleiretain the ability for independent motion over short distances.However, they are incapable of moving around chloroplasts orconglomerates of smaller organelles, and frequently come to restin between them (Fig. 42, arrows). Under these circumstances,their movement is extremely slow (1.8+0.3 pm/min) and restrictedto that of the structures they are associated with. A smallportion, no more than 10%, of the nuclei remain freely mobile.These nuclei continue to travel independently of each other,often moving in opposite directions. Their pattern of movementis characterized by many stops and starts along the way, with anaverage speed of 23.9+2.9 pm/min.The majority of the mitochondria and RBIs show a tendency toconglomerate into well defined groups of smaller organelles.SSIs also appear to become part of these aggregates oforganelles. Organelle conglomerates are often formed in thevicinity of the chloroplasts (Fig. 42, arrowheads). The primarydirection and speed of movement of these aggregates of organellesis the same as that of the chloroplasts with which they areassociated. A smaller portion of mitochondria and RBIs do notbecome organized into conglomerates. These exhibit independentmovement characterized by brief reversals in their primarydirection of travel. Their speed of movement averages 4.6+0.3pm/min.26Cut filamentsWound regionThe development of the wound vesicle as well as theappearance of plasmolysis during Stage 1 remains unaltered withCytochalasin B (200 pg/ml) treatment. The same is true of theprocess of wound sealing, the accumulation of organelles at thesealing barrier, and the movements of retraction and recoveryfrom retraction, coincident with the subsiding of plasmolysis,exhibited by the chloroplasts during Stage 2. However,chloroplast retraction and recovery takes approximately 3.5 timeslonger than in untreated cut filaments. In Stages 1 and 2, thecytoskeleton remains similar to that in untreated cut filamentsin terms of density, undulating configuration, and in itsrelationship to the organelles (Fig. 43).In Stage 1 and early Stage 2 chloroplast distribution andthe direction and speed of travel are similar to those inuntreated cut filaments. They show bulk movement and a patternof motion free of reversals on the primary direction of travel.In late Stage 2, the chloroplasts move away from the wound sitein bulk (Fig. 43), and then back again. The chloroplasts move atapproximately 3.7+0.5 pm/min, while maintaining the same patternof motion characterized by the absence of reversals on theprimary direction of travel. Chloroplasts, particularly in thevicinity of the wound site, frequently come to rest at an angleoblique to the length of the filament (Fig. 44). Theseorganelles move at about half the speed of normally alignedchloroplasts.27In Stage 1, nuclei move in bulk toward the wound site. Thisoccurs without reversals in their main direction of travel, at aspeed of approximately 39.6+2.0 pm/min. In early Stage 2, themajority of the nuclei cease independent movement and gathertogether with smaller organelles into conglomerates. These occurnear chloroplasts, and move in coordination with them. Thiscondition persists through late Stage 2 (Fig. 43, arrowheads).During Stage 2 some nuclei remain independent. These nuclei movewith their NAB region first, or in an orientation oblique to thelength of the filament (Fig. 44, arrowheads). They travel inbulk, at an average speed of 6.8+1.3 pm/min, and with a patternof motion characterized by many stops and starts along the way.These non-aggregated nuclei, like those in untreated cutfilaments, travel unidirectionally toward the wound region inearly Stage 2, and assume a bidirectional (to and from the wound)flow later during this Stage.Mitochondria and RBIs in Stage 1 move toward the wound sitein bulk, with a speed and pattern of movement similar to those inuntreated cut filaments. This pattern of flow continues for thefirst 3-4 minutes of early Stage 2. The movement then stops,leaving the mitochondria and RBIs aggregated together with nucleiand accumulated near the chloroplasts (Fig. 44). Theseorganelles move in conjunction with the chloroplasts at 3.7+0.5pm/min. This condition persists into late Stage 2. SSIs appearto move and congregate with mitochondria and RBIs.28Non-wound region:In contrast to untreated cut filaments, chloroplasts andnuclei remain motionless during plasmolysis and the recoverythereof. As plasmolysis develops, mitochondria and RBIs move inbulk at 15.7+1.2 pm/min and with a pattern of motioncharacterized by the absence of reversals on their primarydirection of travel. As plasmolysis subsides, aggregates ofmotionless mitochondria, RBIs, and nuclei form close to thechloroplasts (Fig. 45). SSIs appear to gather with mitochondriaand RBIs.A summary of the speed and major characteristics oforganelle movement and distribution in uncut and cut filamentstreated with Cytochalasin B is presented in Table 5.2qTable 5 - Characteristics of organelle movement, speed of travel andaggregation status in Cytochalasin B treated filaments ofVaucheria longicaulis var. macouniiUncut Filaments Cut FilamentsWound Region Non - wound regionOrganelle Characteristics Ag. Speed Stage Characteristics Ag. Speed 91ssmslysls Characteristics Ag. Speedof movement 4 pm/min or movement * Am/min level of movement * gm/min1 Bulk, Simple N 2.6 i 0.3Chloro-plastsBulk,Complex N1.8t 0.3Increasing Motionless N Motionlessearly Bulk, Simple N 2.6 i 0.32late Bulk, Simple N 3.7 ± 0.5 Decreasing Motionless N MotionlessNucleiBulk,Complex A1.8t 0.31 Bulk, Simple N 39.6 i 2.0increasing Motionless N Motionlessearly Bulk, Simple A 2.6 i 0.32late Bulk, Simple A 2.6 ± 0.3 Decreasing Motionless N-A MotionlessMitoch.+RBIsBulk,Complex A1.8± 0.31 Bulk, Simple N 74.0 ± 5.1increasing Bulk, Simple N 15.7 ± 1.2early2Bulk, Simple N-A 74.0 ± 5.1late Bulk, Simple A 3.7 ± 0.5 Decreasing Bulk, Simple N-A 15.7 i 1.2Definitions of Characteristics of Movement Bulk Movement:^The organelles move as a unit.Independent Movement: The organelles move independently of one another.Simple Movement:^Movement without reversals on the primary direction of travel.Complex Movement:^Movement with brief reversals in the primary direction ofmovement that do not affect the final net direction of travel.* Ag. = "Aggregration Status" ie:A :^AggregrationN :^Non AggregatingN-A: Changes from N to A302. Effects of the microtubule depolymerizing agent Oryzalin onthe cytoskeleton and wound healing:Uncut filaments:Concentrations of 8 and 17 pg/ml Oryzalin were tested, butfailed to produce clear cut responses. Treatment with 26 pg/mlOryzalin yielded nearly immediate responses, which remainedunaltered during exposures of up to 2 hours. Oryzalin wasapplied at 26 pg/ml for approximately 20 minutes in subsequentstudies.The cytoskeleton remains visible as cable-like strands(tracks) following Oryzalin treatment. The orientation anddensity of tracks remains unchanged (Fig. 46). Organellescontinue to move along these tracks, and the relationship betweenthe tracks and the organelles remains unaltered (Fig. 46).Cytoskeleton tracks continue to surround the chloroplasts (Fig.46, inset).Oryzalin treatment results in an almost immediate reductionin the speed of chloroplast movement to 13.5+1.6 pm/min. As inthe case of untreated filaments, chloroplasts exhibit bulk flowwith brief reversals in their primary direction of movement thatdo not affect the final net direction of travel. Thedistribution and orientation of chloroplasts in relation to thelongitudinal axis of the filament, as well as the net directionof travel, remain unaltered from the situation observed inuntreated filaments (Fig. 46).31Nuclei cease independent movement. They all gather togetherwith mitochondria and RBIs near the chloroplasts to formconglomerates which move in synchrony and exhibit the samepattern of motion as the chloroplasts they are associated with(Fig. 46, arrowheads). SSIs also appear to become part of thesegroups of organelles. Some mitochondria remain free and exhibitbulk movement, with brief reversals in their primary direction oftravel. These organelles move at a speed of 16.9+1.7 pm/min.Cut filaments:Wound regionThe development of the wound vesicle and the appearance ofplasmolysis during Stage 1 remain unaltered with Oryzalin (26pg/ml) treatment. The same is true of the process of woundsealing, the accumulation of organelles by the wound site, andthe movement of retraction and recovery from retraction exhibitedby the organelles coincident with the subsiding of plasmolysis.However, the wound healing process is approximately 2 timesslower than that in untreated excised filaments. During Stages 1and 2 the cytoskeleton in the wound region is similar, both indensity and relationship with the organelles, to that inuntreated wounded filaments (Fig. 47).In Stage 1 and early Stage 2 chloroplast distribution,orientation, and speed are similar to that in untreated cutfilaments. These organelles show bulk movement, without32reversals in the primary direction of travel. In late Stage 2,the chloroplasts move in bulk away from the wound site atapproximately 8.5+1.1 pm/min (Fig. 47). Their movement occurswithout reversals on the primary direction of travelapproximately 70% of the time. However, during the remaining 30%of the time, they exhibit a pattern of motion characterized bybrief reversals in the primary direction of movement that has noeffect on the final direction of travel.The majority of the nuclei gather into groups nearchloroplasts and move with them in Stages 1 and 2 (Fig. 47). Asmall portion of the nuclei remain independent. These move withtheir NAB end first, or in an orientation oblique to the lengthof the filament (with the NAB in varying positions) as inCytochalasin B treated filaments. They travel in bulk at anaverage speed of 18.4+1.7 pm/min, with a pattern of motioncomposed of many stops and starts along the way. In both Stages1 and 2, their net direction of flow is the same as that inuntreated cut filaments.During Stage 1 mitochondria and RBIs move in bulk toward thewound site, with a speed, pattern of motion, and distributionsimilar to that in untreated cut filaments. During early Stage 2the speed of movement gradually declines, but the distribution ofthe majority of mitochondria and RBIs remains unchanged. In lateStage 2, these organelles come to a halt and remain virtuallymotionless throughout the remainder of this Stage. A smallportion of mitochondria and RBIs aggregate around thechloroplasts and move with them during early and late Stage 2.33SSIs appear to move and come to rest close to the mitochondriaand RBIs.Non-wound region:As in Cytochalasin B treated cut filaments, chloroplasts andnuclei remain immobile during plasmolysis and the recovery fromit. This is in contrast with mitochondria and RBIs, whichcontinue to move. Their movement occurs without reversals in theprimary direction of flow at 40.3+4.4 pm/min, and it stops asplasmolysis diminishes. Under these circumstances, nuclei,mitochondria, and RBIs can be seen clustered and motionless nearchloroplasts (Fig. 48). SSIs appear to move and aggregate withmitochondria and RBIs.A summary of the speed and major characteristics oforganelle movement and distribution in uncut and cut filamentstreated with Oryzalin is presented in Table 6.Table 6 - Characteristics of organelle movement, speed of travel andaggregation status in Oryzalin treated filaments ofVaucheria longicaulis  var. macouniiUncut Filaments Cut FilamentsWound Region Non - wound regionOrganelle Characteristicsof movementAg.*Speedyin/minStage Characteristicsof movementAg.*Speeditm/minPiesmeiysislevelCharacteristicsof movementAg.*Speedy.m/minChloro-plastsBulk,Complex N13.5i 1.61 Bulk, Simple N 2.6 i 0.3Increasing Motionless N Motionlessearly Bulk, Simple N 2.6 i 0.32late Bulk, Simple N 8.5 ± 1.1 Decreasing Motionless N MotionlessNuclei Bulk,Complex A13.5± 1.61 Bulk, Simple A 2.6 ± 0.3Increasing Motionless N Motionlessearly2Bulk, Simple A 2.6 ± 0.3late Bulk, Simple A 8.5 ± 1.1 Decreasing Motionless N-A MotionlessMitoch.+RBIsBulk,Complex A13.5I 1.61 Bulk, Simple N 74.0 ± 5.1Increasing Bulk, Simple N 40.3 ± 4.4early2Bulk, Simple N 74.0 *5g).1(reducinlate Motionless N 0 Decreasing Bulk, Simple N-A 40.3 ± 4.4Definitions of Characteristics of MovementBulk Movement:^The organelles move as a unit.Independent Movement: The organelles move independently of one another.Simple Movement:^Movement without reversals on the primary direction of travel.Complex Movement:^Movement with brief reversals in the primary direction ofmovement that do not affect the final net direction of travel.* Ag. = "Aggregration Status" ie:A :^AggregrationN :^Non AggregatingN -A: Changes from N to A353. Effects of the microtubule depolymerizing agents Colchicineand APM, and microtubule stabilizing agent Taxol:Colchicine was applied to filaments at 50 mM, the maximumconcentration employed by Blatt et al, (1980b), but no changes incytoplasmic streaming or cytoskeleton were observed. APM, aherbicide, applied at 30pM, 100 pM, and 200 pM, showed variablereduction of cytoplasmic streaming, with no change incytoskeletal structure. These results were less reproduciblethan those with Oryzalin. Taxol application at 25 pM and 50 pMresulted in no observable changes in cytoplasmic streaming orcytoskeleton.36DiscussionWound healing and organelle distributionWound healing of full width transverse wounds leads to fullrecovery and normal function in Vaucheria longicaulis var.macounii and siphonous Chlorophyta (Burr and West, 1971, Dreher,et al., 1978, La Claire, 1982a & b, Goddard and Dawes, 1983,Mariani-Colombo and Postai, 1984, O'Neil and La Claire, 1988).However, this is not the case for the Charophytes, in which largewounds invariably lead to cell death (Foissner, 1988, Menzel,1988).The first step in wound healing recovery in V. longicaulis provides short term protection for the organism. This is assumedto be related to the prevention of loss of cytoplasm andpenetration of pathogenic microorganisms into the cell (Menzel,1988). One major mechanism has been identified regarding shortterm wound protection. In V. longicaulis, Caulerpa sp., Bryopsissp., Udotea petiolata, Struvea anastomosans, and Chamaedoris sp.a wound healing vesicle or an external wound plug is formed fromthe intact cytoplasm underlying the wound region seconds afterwounding (Dreher et al., 1978, La Claire, 1982a, Goddard andDawes, 1983, Mariani-Colombo and Postai, 1984, Menzel, 1988).Different mechanisms occur in the majority of the organismsbelonging to the Order Siphonocladales. In these algae, no woundplug is produced in response to damage. The cytoplasm andvacuole remain in direct contact with the surrounding mediumuntil the wound is closed. This seems to be accomplished, atleast in some of these algae, by the contraction of the cytoplasm3 7away from the wound region. In other species, the protoplasmdivides into numerous viable protoplasts, presumably by localizedcontractions. The result is similar to the segregative celldivision observed in these algae, and yields many new plants uponregeneration from the wounding process (La Claire, 1982a & b,O'Neill and La Claire, 1988, Foissner, 1988).The wound healing vesicle in V. longicaulis seems to arisefrom a region of the cytoplasm underneath the wound site. It ismade up of plasma membrane and tonoplast, with a thin layer ofcytoplasm occupying the space in between the two membranesystems. The cytoplasm of the wound vesicle is continuous withthat of the filament, and it contains numerous mitochondria andRBI's, but few larger organelles. In contrast, in Caulerpa,Udotea, and Bryopsis, an External Wound Plug (EWP) of vacuole-derived material is extruded that undergoes sol to geltransformation upon contact with seawater (Dreher et al., 1978,Menzel, 1988). It has no membrane structure, contains noorganelles, and lacks continuity with the cytoplasm after gelling(Burr and West, 1971, Dreher el al, 1978, Menzel, 1988).The wound healing vesicle in V. longicaulis swells toapproximately 50 um in diameter about 1 minute after excision.This indicates the occurrence of extremely rapid repair of thesevered plasma membrane and tonoplast at the wound site, followedby the rapid growth of these two membrane systems to produce thewound healing vesicle. The repair of severed membranes is notwell understood (Menzel, 1988). However, it most likely involvesfusion of vesicles, probably of dictyosomal origin, with theintact portion of the severed membrane systems. The fact that38large numbers of small inclusions (possibly SSIs) appear toaccumulate at the wound site concurrently with the first signs offormation of the wound healing vesicle is consistent with thisinterpretation. CTC is a Ca 2+-sensitive fluorochrome widelyutilized in the visualization of the distribution of membrane-bound Ca2+ (Caswell and Hutchinson, 1971). CTC fluorescence atthe wound site during Stage 1 occupies a broader area anddisplays a distribution pattern more complex than those observedduring Stage 2. These observations indicate that a largeaccumulation of membranous material occurs at the wound sitesimultaneously with wound healing vesicle formation. CTCfluorescence also demonstrates the presence of membrane-boundCa2+ which is necessary to trigger membrane fusion (Sudhof andJahn, 1991). Consequently, the visualization of CTC fluorescenceoccupying a large area of the wound site and extending into thewound healing vesicle suggests the occurrence of extensiveexocytotic activity in association with its formation. Theaccumulation of mitochondria and RBIs (probable lipid source) atthe wound site and in the cytoplasm of the wound healing vesiclepoints to the existence of an efficient mechanism for ATPproduction in support of this high rate of exocytotic activity.The formation of the wound healing vesicle in V. longicaulis occurs concurrently with a loss of turgor pressure (plasmolysis)throughout the excised filament. At the same time, vacuolarcontents appear to be squeezed into the wound healing vesicle.This interpretation is consistent with the observation that whenthe wound healing vesicle breaks apart during Stage 1, it israpidly reformed and reinflated. A similar situation occurs in3 9other large celled algae, including Caulerpa simpliciuscula, C.ashmeadii,and Udotea petiolata in which turgor pressureadjustments appear to be a major force behind repeated extrusionsof EWP material (Dreher et al., 1978, Goddard and Dawes, 1983,Mariani-Colombo and Postai, 1984, Menzel, 1988). In contrast, noloss of turgor pressure is reported in wound healing studies ofseveral members of the Order Siphonocladales. The process ofwound healing in many of these algae involves retraction of theprotoplast away from the wound (La Claire, 1982a). Thiseffectively reduces the volume of the cytoplasm, and it has theopposite effect of the situation observed in V. longicaulis.Stage 2 begins with the sealing off of the wound healingvesicle from the wound region of the excised filament. InAcetabularia sp. and Ernodesmis verticillata sealing of the woundoccurs by rapid centripetal closure of the wound region material,due to cytoskeletal contraction, around the central vacuole(Menzel, 1988, Menzel and Menzel-Elsner, 1989c, La Claire,1989a). In Caulerpa sp., wound sealing requires in excess of 30min and is the result of vesicle coalescence (Dreher et al.,1978, Goddard and Dawes, 1983). In V. longicaulis, the sealingprocess involves the fusion of both the plasma membrane andtonoplast at the wound site and takes place approximately 4minutes after excision (Blatt et al., 1980).In coordination with the wound closure, there is a permanentcontraction of the protoplasm 1-6 mm or more from the wound sitein Bryopsis sp., Acetabularia sp., and Ernodesmis verticillata(Burr and West, 1971, La Claire, 1982b, Menzel, 1988, Menzel andElser-Menzel, 1989c, Goddard and La Claire, 1991). This40situation contrasts with that observed in V. longicaulis whereretraction of organelles, not cytoplasm, occurs only after thesealing process is completed, and it is largely restricted to atemporary repositioning of chloroplasts away from the wound site.This retraction movement of organelles associated with woundhealing and their subsequent return to the wound region severalminutes later occurs in other algae. In Caulerpa sp.,chloroplasts and most other organelles retract 1-2 mm from thewound immediately upon wounding and return to the wound site overa 2 to 11 hour period (Dreher et al., 1978, Goddard and Dawes,1983). In Nitella flexis, the area surrounding small puncturewounds also becomes devoid of chloroplasts. Chloroplastretraction from the wound area in Caulerpa sp. is considered tobe due to a combination of physical, osmotic, and/or chemicalstresses (Menzel and Elsner-Menzel, 1989a). Wounding by excisiondoes impose intense physical, and osmotic stresses, as well asprobable chemical stress, and these could account for thetemporary retraction of organelles observed in V. longicaulis.The return of organelles to the wound region may then be possibleonly after the healing process has proceeded long enough torelieve the effects of stress. The fact that the return oforganelles to the wound region is only observed toward the end ofStage 2 is supportive of such an interpretation. In contrastwith other algae, however, retraction of organelles in V.longicaulis does not occur immediately after wounding, but ratherafter wound sealing is completed and it involves only thechloroplasts. This suggests that stress, whether of a physicalnature or otherwise, is not the most likely cause of retraction.41The retraction of chloroplasts from the wound site coincides withthe collection of large quantities of mitochondria and RBIs inthis region.Accumulation of large numbers of mitochondria in woundedareas is observed in Caulerpa simpliciuscula (Dreher et al.,1978) and Udotea petiolata (Mariani-Colombo and Postai, 1984).Vesicles with electron-dense contents (similar to V. longicaulisRBIs) are also found to accumulate in the wound region of Udotea petiolata (Mariani-Colombo and Postai, 1984). In V. longicaulis the accumulation of mitochondria and RBIs along the wound sitereaches a peak during Stage 2. The close physical relationshipand simultaneous accumulation of mitochondria and RBIs at thewound site point to an intimate functional relationship betweenthese two organelles. The morphological characteristics andelectron density indicate that RBIs are storage bodies forlipids. These may represent a major energy source for themitochondrial synthesis of ATP, particularly important for V.longicaulis, an alga that does not synthesize starch (Bold andWynne, 1985). The ATP produced would likely be used fordifferent aspects of wound healing such as rebuilding thecytoskeleton, repair through exocytosis (as indicated by CTCstaining) of the membranous cellular components, proteinsynthesis, and synthesis of cell wall materials required for thewound healing process.The fluorochrome Calcofluor White, established as a probefor the localization of cellulose (Herth and Schnepf, 1980), wasused to detect the development of a new cell wall over the woundsite in V. longicaulis. Calcofluor White yielded fluorescence42during Stage 2. It is possible that cell wall deposition couldbegin as soon as Stage 2. This occurs within a surprisinglybrief period of time in the course of protoplasmic retractions inthe early stages of wound healing in Bryopsis hypnoides (Burr andWest, 1971). However, in other siphonous algae, cell walldeposition is reported to start only after extended periods oftime from excision. In Caulerpa ashmedii the new cell wall isformed in 2-6 hours (Goddard and Dawes, 1983), in C.simpliciuscula in 12 hours (Dreher et al., 1978), and in Udotea 4days are required for this process to occur (Mariani-Colombo andPostai, 1984). Calcofluor White also reacts with polysaccharidesother than cellulose (Herth and Schnepf, 1980). Therefore, it isunclear from these studies whether Calcofluor White staining ofV. longicaulis is indicative of cellulose deposition, orrepresents detection of other polysaccharides.Nuclear distribution as seen by DAPI fluorescence ishomogeneous throughout the wound region during Stages 1 and 2, incontrast with the distribution of other organelles. This uniformdistribution of nuclei in V. longicaulis is maintained despitecontinuous movement of nuclei during Stages 1 and 2 (see secondpart of discussion). The constant presence of nuclei in thisregion points to the importance of this organelle's presenceduring initial stages of wound healing in V. longicaulis.Nuclear distribution during wound healing has not been studied inother siphonous algae. However, numerous nuclei are found alongthe wound plug in Caulerpa ashmedii (Aist, 1976, Goddard andDawes, 1983). In Tradescantia wounds, as well as in otherplants, nuclei in intact cells neighboring the wounded ones move4 3close to the cell wall facing the wounded cells (Schnepf andVolkmann, 1974), indicating the existance of a direct, yetundetermined, involvement in wound healing recovery.Plasmolysis is completely eliminated by the timechloroplasts return to the wound region. In Caulerpa simpliciuscula, fluctuations in turgor pressure accompany thereturn of the cytoplasmic organelles to the wound site (Dreher etal., 1978). Turgor pressure appears to be regulated bycompensatory ion fluxes in large celled algae (Glass, 1983).Regaining the ions and water, lost to the vesicle (or medium)during the early phase of wound healing, most likely leads to therestoration of membrane potential and osmotic regulation.Osmotic regulation in siphonous algae is achieved by changingconcentrations of Na+ , K+ , and Cl- , especially in the vacuole(Kirst and Bisson, 1979). Bryopsis plumosa and Caulerpa sp..expend ATP to pump Cl - into the vacuole, and Na + is passivelyaccumulated (Kirst and Bisson, 1979). Although no research onosmotic regulation has been done on V. longicaulis, these factorshave been implicated in turgor pressure regulation in a widevariety of algae (Kirst and Bisson, 1979).Throughout plasmolysis and recovery thereof, organelledistribution in the non-wound region remains uniform. Theuniform distribution of chloroplasts most likely supportsefficient photosynthesis throughout this stressful period ofwound healing recovery. The uniform distribution of nuclei,mitochondria, and RBIs in this region promotes metabolic andfunctional stability, undoubtedly necessary for filamentsurvival. The same situation is seen in Udotea petiolata during44wound recovery, where normal cytoplasmic organization exists afew mm away from the wound site (Mariani-Colombo and Postai,1984).During Stage 3, a second retraction of chloroplasts from andsubsequent return to the wound region occurs in V. longicaulis.This is not seen in other algae where wound healing studies havebeen conducted. Its role in the wound healing process remainsundetermined. Approximately 43 hours after excision, resumptionof growth occurs in V. longicaulis. The time interval requiredfor growth resumption varies from under 24 hours in Bryopsis (Burr and West, 1971) to 2 months in Udotea petiolata (Mariani-Colombo and De Carli, 1980). In Siphonocladus growth occurs 2-3days after wounding (La Claire, 1982a). In V. longicaulis growthis observed as a lateral outgrowth of the filament wall justbelow the wound site, as well as at the apical region. Thesituation is similar to that observed in Bryopsis hypnoides (Burrand West, 1971) and Udotea petiolata (Mariani-Colombo and DeCarli, 1980). The resumption of growth indicates that woundhealing recovery is complete.45Study of cytoskeleton-based organelle movement in wound healingUncut filamentThe two major components of the cytoskeleton in plants arethe microfilaments and the microtubules (Williamson, 1986, LaClaire, 1989b). Differential interference contrast microscopycannot distinguish between them, although structures observed inprotoplasts of Vaucheria sessilis were tentatively identified asmicrofilaments by Blatt et al. (1980). Observations of thecytoskeleton in Vaucheria longicaulis by differentialinterference contrast microscopy show the cytoskeleton to beorganized as cable-like strands or "tracks" similar to thoseobserved in other algae (Sabnis and Jacobs, 1967, Menzel andSchliwa, 1986a, La Claire, 1987, Menzel, 1987), including otherspecies of Vaucheria (Ott and Brown, 1972, Blatt and Briggs,1980, Blatt et al., 1980). Fluorescence microscopy of V.longicaulis shows microfilament and microtubule distributionsimilar to these tracks (Peat, personal communication). Kacharand Reese (1988) pointed out that in Chara and Nitella there issimilarity between the distribution of cytoskeleton tracks andthe endoplasmic reticulum (ER). However, the ER in V.longicaulis is far more branched, and the pattern of branchingand distribution of the ER are significantly different from thoseof the cytoskeleton tracks.In V. longicaulis well defined interactions exist betweenthe cytoskeleton tracks and the cytoplasmic organelles. Smallerorganelles such as mitochondria and RBIs can be seen bydifferential contrast microscopy to be closely associated with a46single track. Using both differential contrast and fluorescencemicroscopies, mitochondria appear to be associated withmicrotubules in Bryopsis plumosa (Menzel and Schliwa 1986a), andwith microfilaments in angiosperm pollen tubes (Heslop-Harrisonand Heslop-Harrison, 1989a). In V. longicaulis, largerorganelles, such as nuclei and chloroplasts, are seen to interactwith several cytoskeleton strands, although one of these strandsseems to develop a preferential interaction with each one ofthese organelles. A similar situation between cytoskeletontracks, and nuclei and chloroplasts is observed in V. litorea (Ott and Brown, 1974) and Bryopsis plumosa (Menzel and Schliwa1986a).The movement of cytoplasm and organelles in siphonous algaeis parallel to the long axis of the filament and mirrors theorganization of the cytoskeleton tracks (Sabnis and Jacobs, 1967,Menzel and Schliwa, 1986a, La Claire, 1987, Menzel, 1987). Inmany of these organisms, the cytoplasm between plasma membraneand tonoplast is functionally divided into an outer stationaryectoplasm and an inner streaming endoplasm. This is seen inCaulerpa taxifolia, Udotea petiolata, and the Charophyta (Sabnisand Jacobs, 1967, Williamson, 1975, Mariani, 1984, Nagai andHayama, 1979, Menzel, 1987, Grolig et al., 1988, Menzel andElsner-Menzel, 1989a). In Caulerpa taxifolia, Nitella sp., andChara sp., the chloroplasts remain motionless in the ectoplasm,while other organelles move in the endoplasm. In the OrderSiphonocladales, the cytoplasm is divided into organelle-freeectoplasm and organelle-rich endoplasm, but no cytoplasmicstreaming occurs (La Claire, 1984a, 1987). In contrast, in V.47longicaulis, V. sessilis (Blatt and Briggs, 1980) and V. litorea (Ott and Brown 1974), as well as Bryopsis plumosa (Menzel andSchliwa, 1986a) and Acetabularia sp., (Menzel and Elsner-Menzel,1989c), there is no apparent partitioning of cytoplasm intophysical layers, and organelle movement occurs throughout theavailable cytoplasm (Menzel and Schliwa, 1986a, Ott and Brown,1974).Overall, depending on the organism, movement of individualorganelles can be either synchronized or independent.Synchronized movement of all the organelles, except immobilechloroplasts, is seen in Chara sp. and Nitella sp. (Williamson,1975, Kachar and Reese, 1988). In V. longicaulis, theChlorophytes, and angiosperm pollen tubes, different classes oforganelles move independently of one another (Menzel and Schliwa,1986a, Heslop-Harrison and Heslop-Harrison, 1989a). In theseorganisms, different classes of organelles also exhibit distinctpatterns and speeds of movement (Kamiya, 1962, Koop andKeirmayer, 1980, Menzel and Schliwa, 1986a, Heslop-Harrison andHeslop-Harrison, 1987, 1989a & b), as is the case observed in V.longicaulis.The mechanisms behind the variations in organelle speed andpattern of motion are only partially understood. The speed ofmovement along microfilaments in angiosperm pollen tubes wasshown to be unrelated to the size of the organelles (Heslop-Harrison and Heslop-Harrison, 1989a). In Physarum polycephalum,the direction of endoplasmic flow reverses every few minutes, andit is accompanied by alterations in the structure of thecytoskeleton that appear to correspond with rapid microfilament48disassembly and reassembly (Williamson, 1984). The reversals inthe direction of movement seen with chloroplasts, mitochondria,and RBIs in V. longicaulis, and chloroplasts and mitochondria inB. plumosa (Menzel and Schliwa, 1986a), point to a reorganizationof the cytoskeleton and flexibility in the interaction betweenthe cytoskeleton and these organelles. The cytoskeleton tracksbranch and change configuration rather rapidly in V. longicaulis.These changes imply that the occurrence of reorganization in thecytoskeleton may lead to organelles switching between neighboringtracks. This, as suggested for V. sessilis by Blatt et al.(1980), would account for reversals in the direction of movement,with the concomitant generation of variation in the speed andpattern of motion.Wound regionDuring Stage 1, by comparison with uncut filaments, thewound region cytoskeleton remains largely unchanged in terms ofboth its density and relationship of the cytoskeleton tracks withthe organelles. The extension of the cytoskeleton into the woundhealing vesicle cytoplasm indicates the occurrence of a rapid anddirected polymerization of the cytoskeleton at the wound site.The wound healing vesicle is formed and expands rapidly. Thisoccurs in conjunction with the unidirectional movement ofmitochondria, RBIs, and SSIs along the cytoskeleton towards thewound site and into the wound healing vesicle. Consequently, thecytoskeleton plays a major role in wound healing vesicleformation, and its extension into this vesicle appears to be49fundamental for the successful implementation of the process ofwound healing recovery. Plasmolysis seen along the filament atthis time may be due to cytoskeletal contraction that squeezesthe vacuole contents into the wound healing vesicle to expand it.Chlorotetracycline (CTC) studies show that Ca2+ , at leastmembrane-bound Ca2+ , accumulates in the wound region, possiblydue to the free entry of medium Ca2+ through the wound. Ca 2+ isa well known regulator for depolymerization and repolymerizationof both microtubules and microfilaments, as well as the cessationof cytoplasmic streaming in plants (Dustin, 1984, Hepler andWayne, 1985; La Claire, 1989a & b). The free entry of Ca 2+following wounding fails to result in depolymerization ofmicrotubules in Ernodesmis verticillata, and microfilaments inAcetabularia cliftoni (Menzel and Elsner-Menzel, 1989c, Goddardand La Claire, 1991). Similarly, it is interesting to observethat the increase in Ca 2+-dependent CTC fluorescence seen in V.longicaulis does not appear to depolymerize the cytoskeleton inthe wound region or impair the polymerization of the cytoskeletoninto the wound healing vesicle. The reduced speed of travel ofchloroplasts and nuclei, and the observed changes in theirpattern of motion may by due in part to myosin sensitivity toCa2+ (Peat, personal communication, Williamson, 1984), and/or tothe sensitivity of cytoskeleton associated proteins (Rao, 1992).Furthermore, reduced rates of organelle movement commonlyaccompany loss of turgor pressure (Kamiya, 1962).In Stage 2, the increase in cytoskeletal density(polymerization) in the wound region is similar to that observedin E. verticillata shortly after wounding (Goddard and La Claire,501991). In wounded E. verticillata, levels of Ca 2+ that commonlystop cytoplasmic streaming in other plant cells (Hepler andWayne, 1985, La Claire, 1989a) promote assembly of microfilamentsin the wound region (Goddard and La Claire, 1991). Theseelevated levels of Ca2+ also stimulate contraction ofmicrofilaments and cytoplasm at the wound site in both E.verticillata and Acetabularia cliftoni (Menzel and Elsner-Menzel,1989c, Goddard and La Claire, 1991). The increase incytoskeleton density in the wound region in V. longicaulis leadsto the retraction movement of chloroplasts, while selectivelyproviding for the massive transport of mitochondria (andassociated dictyosomes), RBIs, and SSIs to the wound site. Theseorganelles appear to provide the materials and energy necessaryfor the sealing off of the wound site from the wound healingvesicle. This occurs through the fusion of the tonoplast andplasma membrane, as well as the deposition of a new cell wall atthe wound site (see first part of the discussion). In late Stage2, concomitant with the disappearance of plasmolysis, thechloroplasts return to the wound site. This event, together withthe regaining by nuclei and smaller organelles of the pattern ofmovement similar to those observed in uncut filaments, indicatethat normal ionic balances leading to the restoration of turgorpressure have been reestablished (Kamiya, 1962, Kirst and Bisson,1979).51The Effect of Cytochalasin B and Oryzalin on Wound HealingUncut filamentCytochalasin B is well established as a rapid depolymerizingagent of microfilaments in many animal cells (Williamson, 1978,Williamson and Hurley, 1986). However, the cytoskeleton of somecells, including plants, appear to be more resistant toCytochalasin B action; hence the extent of actualdepolymerization is variable (Williamson 1978, Williamson andHurley, 1986, Palevitz, 1988). Algal cells are noted for theirresistance to Cytochalasin B (La Claire, 1984b), even afterprolonged exposures to this compound (Williamson and Hurley,1986). A wide range of Cytochalasin B concentrations (1-200ug/ml) have been applied to algae and higher plants, resulting invariable levels of cytoplasmic streaming inhibition andmicrofilament depolymerization. These include prevention offertilization tube formation in Chlamydomonas (La Claire, 1984b),frequent disruption of microfilament organization but noreduction of chloroplast movement in Bryopsis plumosa (Menzel andSchilwa, 1986b), and inhibition of cytoplasmic streaming withoutalteration of microfilament structure in Chara carrolina (Williamson and Hurley, 1986). The undulating configuration ofthe cytoskeleton observed in certain regions of Vaucheria longicaulis is also detected in A. mediterranea followingCytochalasin B treatment (Koop and Kiermeyer, 1980). A similarsituation observed in Zinnia root tip cells is considered to be aprecursor of depolymerization of the cytoskeleton (Cleary andHardham, 1988). The unaltered appearance of the majority of the52cytoskeleton in V. longicaulis following Cytochalasin B treatmentpoints to a lack of total depolymerization of microfilaments, asobserved in Acetabularia mediterranea (Koop and Keirmayer, 1980),although local depolymerization is suggested by the undulatoryconfiguration of the cytoskeleton. The aggregation of nuclei andsmaller organelles following Cytochalasin B treatment alsoindicates localized occurrence of cytoskeleton reorganization,possibly depolymerization.Oryzalin has not been used before in the study of thecytoskeleton of siphonous algae, but it has been observed toinhibit microtubule assembly in plant seedlings (Strachan andHess, 1983). In Chlamydomonas reinhardtii Oryzalin shortensregenerating flagella (Quader and Filner, 1980), and thiscompound depolymerizes microtubules in Funaria hygrometrica protonema (Wacker et al., 1988). Visualization of thecytoskeleton in V. longicaulis following Oryzalin treatmentpoints to a lack of extensive microtubule depolymerization inresponse to this treatment. However, most of our treatments lastonly 20 min, a time interval generally considered insufficient toachieve extensive microtubule depolymerization (Cleary andHardham, 1988). The occurrence of localized microtubuledepolymerization is suggested by the formation of aggregates oforganelles following treatment with Oryzalin. A similarphenomenon is observed following APM treatment in Ernodesmis verticillata, where fluorescence microscopy showed pools ofdepolymerized tubulin amidst the organelle aggregates (La Claire,1987).53Both Cytochalasin B and Oryzalin treatments result insignificant reductions in the speed of travel of chloroplasts,nuclei, mitochondria, and RBIs. These results suggest theparticipation of both microfilaments and microtubules inorganelle movement. Similar results have been described inBryopsis plumosa (Menzel and Schliwa, 1986b) and Acetabularia mediterranea (Koop and Keirmeyer, 1980, Menzel and Elsner-Menzel,1989c). In V. longicaulis, Cytochalasin B has a greater effecton cytoplasmic streaming and organelle movement than Oryzalin,which indicates greater involvement of microfilaments thanmicrotubules as part of its motor apparatus. The loss ofindependent movement exhibited by the majority of nuclei,mitochondria, RBIs, and SSIs after Cytochalasin B and Oryzalintreatment again demonstrates that both microtubules andmicrofilaments are involved in organelle movement, and it mayalso indicate alteration in the action of motor molecules and/orcytoskeleton associated proteins (Rao et al., 1992, Menzel andSchliwa, 1986b).Cut filaments:The wound region cytoskeleton, its extension into the woundhealing vesicle, as well as the relationship with organellesremain unaltered following Cytochalasin B and Oryzalintreatments. Rapid and selective movement of mitochondria, RBIs,and SSIs toward the wound site and into the healing vesiclecytoplasm is not inhibited by Cytochalasin B or Oryzalinapplications. Microfilaments and microtubules appear to escape54local depolymerization, as may be deduced from the lack ofaggregation shown by these organelles during Stage 1.Neither wound sealing nor the increase in cytoskeletaldensity observed in Stage 2 is inhibited by Cytochalasin B orOryzalin. However, in contrast with Stage 1, aggregation ofmitochondria occurs with Cytochalasin B treatment, but notOryzalin. This indicates a preferential role of microfilamentsin mitochondrial movement, as appears to be the case inangiosperm pollen tubes (Heslop-Harrison, 1989a). Nuclearaggregation is observed following both Cytochalasin B andOryzalin treatments, and it is especially strong with Oryzalin.This indicates the participation of both microfilaments andmicrotubules in the transport of nuclei towards the wound site,with greater dependence on microtubules. This is in agreementwith observations of Ott and Brown (1972) showing the existenceof close association between nuclei and microtubules in V.litorea, as well as with the work of Meindl (1983), Meindl(1986), Wordeman et al. (1986) with the nuclei of desmids anddiatoms. Changes in orientation exhibited by non-aggregatednuclei after Oryzalin treatment are similar to those seenfollowing colchicine treatment of Acetabularia mediterranea (Koopand Kiermeyer, 1980), and supports the idea that microtubulesplay a preferential role in the movement and orientation ofnuclei (Ott and Brown, 1972). Altered chloroplast orientationfollowing Cytochalasin B treatment suggests that microfilamentsplay a preferential role in chloroplast movement. This may notbe true for all organisms, since treatment of Bryopsis plumosa 55with microtubule inhibitors results in a similar change inchloroplast orientation (Menzel and Schliwa, 1986b).Overall, these observations suggest that the preferentialinvolvement of microfilaments and microtubules in the movement ofdifferent classes of organelles plays a major role in woundhealing. The apparent preferential interaction of microfilamentswith both mitochondria (and organelles of similar size) andchloroplasts, may explain why these organelles exhibit temporaryreversals in their main direction of travel in uncut filaments.Temporary reversals in the main direction of travel indicate thepossibility that microfilament bundles contain individual trackswith reverse polarity, as suggested by Blatt et al. (1980). Thelack of movement reversal displayed by these organelles followingwounding indicates possible changes in the polarity oforganization of cytoskeleton,and/ or alteration of associatedproteins and myosin due to their sensitivity to Ca2+ (Williamson,1984, 1986). The apparent preferential role of microtubules innuclear movement may explain the organelle's independent patternof movement in uncut filamets, as well as their rapid speed oftravel. The change to bulk nuclear movement toward the woundsite in Stage 1 and early Stage 2 may also signal use of adifferent type of molecular motor (kinesin vs dynein) (La Claire,1989b).56Cytoskeleton based movement of organelles: possibleinterpretationsIn animal nerve cells, organelle movement has been shown tooccur along microtubules via motor molecules such as kinesin anddyenin (Vale et al, 1985, La Claire, 1989b). In plant cells,most cytoplasmic streaming appears to occur along microfilaments,with myosin serving as the motor molecule (La Claire, 1989b).However, in a small number of plant cells, such as the siphonousalgae Bryopsis sp. and Caulerpa sp., organelles appear to movealong microtubules (Menzel and Schliwa, 1986b, Williamson, 1986,Menzel and Elsner-Menzel, 1989b). In V. longicaulis, the resultssuggest that the movement of chloroplasts, mitochondria, andsimilar size organelles is primarily microfilament based, with apossible guiding role by the microtubules. A similar cooperativerelationship between microfilaments and microtubules wasdescribed by La Claire (1984b) in Ernodesmis verticillata duringwound contraction. In contrast, nuclear movement in V.longicaulis appears to be preferentially microtubule based, withmicrofilaments playing an, as yet, undetermined role.Although we do not know how changes in the pattern and speedof organelle movement occur in cut filaments vs uncut filamentsin V. longicaulis, alteration of ionic levels has been shown toaffect the cytoskeleton and many proteins associated with it(Pollard et al, 1984, Williamson, 1984). With wounding and theloss of turgor pressure, normal concentrations of ions such asCa2+ , Na+ , K+ , and Cl - appear to be temporarily altered (Kirstand Bisson, 1979, Menzel, 1989c). Ionic levels were determined57by Pollard et al. (1984) to be crucial in the cross-linking ofMAP 2 to actin. Altered MAP 2 cross-linking between microtubulesand microfilaments could be responsible for the reduced rate ofchloroplast and nuclear movements observed following wounding.Indeed, the interaction between microfilaments and microtubulesrequired to produce organelle movement was suggested to occurthrough microtubule associated proteins, MAPs (Menzel, 1989d),and MAP 2 has been shown to associate with actin (Pollard et al,1984).CTC fluorescence demonstrates the presence of membrane-boundCa2+ at both the wound site and in the wound healing vesicle.These observations suggest that increased levels of cytosolicCa2+ occur following wounding. In Chara sp., myosin, the motorprotein associated with microfilaments, is sensitive to elevatedlevels of Ca2+ (Williamson, 1984). The action of myosin in V.longicaulis may then be altered by Ca 2+ intrusion into the woundsite from the surrounding medium. This could change the patternand speed of movement of chloroplasts, mitochondria, and similarsize organelles following wounding. Furthermore, Ca 2+ is knownto control the rate of polymerizaton and depolymerization ofmicrofilaments and microtubules (Picton and Steer, 1982, Goodwinand Trainor, 1985). Wound based Ca 2+ entry may then result inthe reorganization of the cytoskeleton, as observed in Ernodesmisverticillata (La Claire, 1982b) and Acetabularia cliftoni (Menzeland Elsner-Menzel, 1989c). In V. longicaulis, reorganization ofthe cytoskeleton in the wound region, resulting in increasedcytoskeleton density, may be the result of the increase in [Ca 2+ ]and lead to altered patterns and speeds of organelle movement.58Microtubules are often composed of a number of tubulin isotypes,which in turn interact with a variety of MAPs (Vale, 1987).Olmstead (1986) reports that MAP composition in interphase cellschanges constantly. These changes could lead to differences inmicrotubule behavior affecting organelle transport (Vale, 1987),and they may participate in changes in nuclear movement observedin wounded filaments of V. longicualis.59ConclusionsIn its intertidal environment, V. loncricaulis is exposed tofrequent grazing and fragmentation, and as a result its singlecelled construction requires an efficient wound healingmechanism. The rapid production of a protective bimembranouswound healing vesicle, in concert with the extension of thecytoskeleton and selective organelle movement into its cytoplasmduring Stage 1, appears to be unique among the siphonous algae.The sealing off of the wound healing vesicle from the wound sitecytoplasm observed in Stage 2, although similar in speed to woundclosure in Brvopsis plumosa and Acetabularia cliftoni (Menzel,1988, Menzel and Elsner-Menzel, 1989c), is also unique as a woundhealing event among algae. Finally, the accumulation oforganelles along the wound in conjunction with the increase incytoskeleton density, and the restitution of turgor pressure lostat the time of wounding, create the conditions leading to thedeposition of a new cell wall at the wound site.The fundamental processes of wound healing leading to theearly stages of filament survival are not inhibited byCytochalasin B or Oryzalin. The differential aggregation oforganelles following application of cytoskeleton inhibitorspoints to the importance of both microfilaments and microtubulesin organelle movement. Microfilaments appear to participate to agreater extent in the movement of chloroplasts, mitochondria,RBIs, and SSIs, while microtubules display a preferentialinvolvement in nuclear movement. These interactions seem toaccount for the accumulation of mitochondria and similar size60organelles at the wound site, while chloroplasts remain the onlyorganelles capable of undergoing retraction movement away fromthe wound region. Nuclei, possibly through their preferentialinteraction with microtubules, are able to establish a pattern ofmovement leading to a more or less uniform distributionthroughout the wound healing process.An important observation that needs to be addressed in moredetail in future studies concerns the increase in cytoskeletondensity observed during Stage 2 of the wound healing process.Wound-based Ca2+ entry was shown to result in cytoskeletalreorganization in Ernodesmis verticillata (La Claire, 1989a) andAcetabularia cliftoni (Menzel and Elsner-Menzel, 1989c). Morecomprehensive studies with cytoskeletal inhibitors to preventwound healing would clarify the role of the cytoskeleton in thisphenomenon. Preincubation of filaments with cytoskeletalinhibitors before wounding would promote better understanding ofthe role of the cytoskeleton in wound healing vesicle formation,and in wound healing in general. The fluorochrome Fluo-3, asensitive cytosolic Ca 2+ indicator (Williams et al., 1990),should also be employed to detect the presence of this ion in thewound region following wounding. Excision of filaments in Ca 2+-free medium could help clarify the role of this ion in the woundhealing process, including the increase in cytoskeletal densityseen in Stage 2.FIGURES 1, 2, 4: bar = 40 pm^FIG 3: bar = 15 pm61FIG 1FIG 2FIG 3Neutral Red staining of the large central vacuole in V.longicaulis var. macounii.Filament of V. longicaulis var macounii stained withNeutral Red, organelles are largely restricted to thecortical cytoplasm.Differential contrast microscopy of part of a filamentof V. longicaulis var macounii. Chloroplasts, nuclei,and a variety of small inclusions are easily observedthroughout the cytoplasm.FIG 4 DAPI fluorescence showing the distribution of nucleithroughout the vegetative filament of V. longicaulis var macounii. Nuclei tend to occur in groups in theapical region of the filament.1262FIGURES 5-10: bar = 40 pmFIG 5^TEM showing some of the major organelles found in avegetative filament. Some inclusions are denselyosmiophilic, suggesting a lipid composition.FIG 6^Rhodamine B fluorescence of small inclusions (RBIs) isindicative of lipid composition.FIG 7^Distribution of mitochondria in a vegetative filamentFIG 8utilizing DiOC6 fluorescence.Organization of the endoplasmic reticulum (ER)determined by Di0C6 fluorescence; inset shows theorganization of the ER in the tip region of thefilament.FIG 9 Wound healing Stage 1. Wounded filament showingformation of wound healing vesicle and loss of turgorpressure (plasmolysis). Inset shows that the woundhealing vesicle structure is composed of the externalplasma membrane (arrow), internal tonoplast(arrowhead), with a thin layer of cytoplasm andorganelles in between.FIG 10^Narrowing of the wound site in preparation for fusionbetween the plasma membrane and tonoplast; inset showsfurther narrowing at wound site..,a63FIGURES 11-17: bar = 40 pmFIG 11^Early Stage 2. Appearance of the wound healing vesicleshortly after membrane fusion. Observe the accumulationof organelles along the wound site.FIG 12^Early Stage 2. Detail of the membrane barrierseparating the wound healing vesicle from the filamentat the wound site.FIG 13^Early Stage 2. Accumulation of organelles at the woundsite subsequent to the sealing off of the wound site.FIG 14^Late Stage 2. Chloroplast retraction from the woundsite. Loss of turgor pressure (plasmolysis) continuesto be evident throughout the cut filament.FIG 15^Late Stage 2. Exclusion of large organelles from thewound site leaves the cytoplasm between tonoplast andplasma menbrane occupied largely by small organelles.FIG 16^Late Stage 2. Return of chloroplasts to the wound siteand elimination of plasmolysis (turgor pressureregained).FIG 17^Stage 3. Second retraction of chloroplasts from thewound site. No loss of turgor pressure (plasmolysis) isobserved during this stage.11^ 141 5121664FIGURES 18-24: bar = 40 pmFIG 18^Stage 4. Wound healing is complete, and resumption ofgrowth occurs at the wound site.FIG 19^Stage 1. Autofluorescence shows chloroplasts allignedalong the wound site.FIG 20^Late Stage 2. Autofluorescence shows retraction ofchloroplasts from the wound site.FIG 21^Late Stage 2. Autofluorescence shows return ofchloroplasts to the wound site.FIG 22^Stage 1. DAPI fluorescence shows even distribution ofnuclei in wound and non-wound regions.FIG 23^Stage 2. DAPI fluorescence shows even distribution ofnuclei in wound and non-wound regions.FIG 24^Early Stage 2. DiOC6 fluorescence shows limitedaccumulation of mitochondria at the wound site.(c4a22•ta23•e* 4• 0.^••.11111■10118242065FIGURES 25-30: bar = 40 pmFIG 25^Early Stage 2. DiOC6 fluorescence shows mitochondria inthe wound healing vesicle cytoplasm, the separation ofwound healing vesicle from wound site, and theaccumulation of mitochondria at the wound site.FIG 26^Late Stage 2. DiOC6 fluorescence shows increasedmitochondrial accumulation at the wound site.FIG 27^Stage 1. Rhodamine B fluorescence shows the presence ofRBIs in the wound healing vesicle cytoplasm.FIG 28^Early Stage 2 (Rhodamine B fluorescence). Noticelimited accumulation of RBIs at the wound site.FIG 29^Late Stage 2 (Rhodamine B fluorescence). Observe thelarge accumulation of RBIs at the wound site.FIG 30^Stage 1. CTC fluorescence showing the reticulatepattern of membrane bound Ca2+ distribution at thewound site and in the wound healing vesicle.66FIGURES 31-32: bar = 40 pmFIG 31^Stage 2: CTC fluorescece reveals a more restrictedpattern of membrane bound Ca2+ at the wound site and inthe wound healing vesicle.FIG 32^Stage 2: Calcofluor White fluorescence in the woundregion.(060.67FIGURES 33-38: bar = 40 FmFIG 33 Differential contrast microscopy of the cytoskeleton inan uncut filament of V. longicaulis var macounii.Observe the orientation of the cytoskeleton tracksparallel to the longitudinal axis of the filament andits close association with organelles.FIG 34FIG 35FIG 36FIG 37FIG 38Preferential interaction between each chloroplast and asingle strand of cytoskeleton (arrowhead). Smallorganelles seem to be travelling along the singlecytoskeleton track (arrows).Chloroplasts are surrounded by several cytoskeletontracks (arrowheads).Two nuclei (arrows) show opposite NAB orientation inrelation to one another.Cut filament (Stage 1). Wound region cytoskeleton andrelationship to organelles. Notice the similarity inappearance to uncut filaments.Cut filament (Stage 1). Cytoskeleton tracks extend intothe wound healing vesicle cytoplasm(L-1FIGURES 39-44: bar = 40 pm68FIG 39FIG 40FIG 41Late Stage 2. Increase in cytoskeleton density in thewound region of a cut filament is easily visualizedupon chloroplast retraction.Late Stage 2. Accumulation of organelles in the woundregion makes visualization of the cytoskeletondifficult.Cytochalasin B treatment (uncut filament). Thecytoskeleton exhibits an undulatory pattern.Aggregation of small organelles is also observed(arrowhead).FIG 42 Cytochalasin B treatment (uncut filament).Visualization of the cytoskeleton and relationship withorganelles. Distribution varies along the length of thefilament, with aggregation of small organellesoccurring in certain areas.FIG 43^Cytochalasin B treatment, cut filament, late Stage 2.The undulating pattern of cytoskeleton is observed aschloroplasts retract.FIG 44^Cytochalasin B treatment, cut filament, late Stage 2.Misalignment of chloroplasts, aggregation of69mitochondria, RBIs and SSIs, and altered orientation ofnuclei are observed in the wound region.3970FIGURES 45-48: bar = 40 pm^FIG. 46 inset: bar = 15 pmFIG 45^Cytochalasin B treatment, cut filament, non-woundregion. Observe the aggregation of smaller organellesin the close proximity of chloroplastsFIG 46^Oryzalin treatment, uncut filament. The cytoskeletonremains largely unchanged in its organization andrelationship to organelles (inset). Aggregation ofnuclei, mitochondria, RBIs, and SSIs are seenelsewhere.FIG 47^Oryzalin treatment, cut filament, late Stage 2. Thecytoskeleton continues to be visible, with an unchangedrelationship to organelles. Notice the tendency ofnuclei to form aggregates, while mitochondria remainisolated from one anotherFIG 48^Oryzalin treatment, cut filament, non-wound region.Nuclei, mitochondria, RBIs, and SSIs are seen clusterednear chloroplasts71REFERENCES CITEDAist, J.R. (1976) Papillae and Related Wound Plugs of PlantCells. Annual Review of Phytopathology 14, 145-163Azanza-Corrales, R. & Dawes, C.J. (1989) Wound healing inCultured Eucheuma alvarezii var. tambalang Doty. Botanica Marina32, 229-234.Bershadsky, A.D. & Vasiliev, J.M. (1988) Cytoskeleton. PlenumPress, N.Y.Blatt, M.R. & Briggs, W.R. 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