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Exploring the effect of microtubule domains and various microtubule associated proteins on cellulose… Mulvihill, Adam 2016

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  Exploring	  the	  Effect	  of	  Microtubule	  Domains	  and	  Various	  Microtubule	  Associated	  Proteins	  on	  Cellulose	  Synthase	  Complex	  Velocity	  for	  Determining	  Anisotropic	  Growth	  in	  Arabidopsis	  thaliana   by  Adam Mulvihill  B.Sc. (Genetics & Plant Biology), University, of California Berkeley, 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE   in   The Faculty of Graduate & Postdoctoral Studies   (Botany)   THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)      October 2016 © Adam Mulvihill, 2016   ii ABSTRACT Previous	  research	  with	  anisotropy	  mutants,	  any1	  and	  mor1-­‐1,	  deficient	  in	  cellulose	  production	  and	  microtubule	  disruption	  respectively,	  linked	  the	  velocity	  of	  the	  cellulose	  synthase	  complex	  (CSC)	  to	  the	  crystalline	  structure	  of	  microfibrils	  in	  the	  wall	  for	  maintaining	  anisotropy	  during	  rapid	  elongation	  at	  elevated	  temperatures.	  CSCs	  have	  been	  visualized	  as	  being	  functional	  inside	  and	  outside	  of	  these	  microtubule	  domains	  and,	  in	  mor1-­‐1,	  it	  was	  observed	  that	  increased	  CSC	  tracking	  outside	  of	  microtubule	  domains	  may	  be	  the	  means	  by	  which	  anisotropy	  fails	  due	  to	  correlations	  with	  increased	  CSC	  velocity	  and	  crystallinity.	  We	  hypothesized	  based	  on	  these	  findings	  that	  microtubule	  association	  with	  the	  plasma	  membrane,	  spatial	  organization,	  and	  polymerization	  status	  may	  have	  a	  significant	  effect	  on	  CSC	  velocity	  and	  anisotropy	  in	  a	  variety	  of	  backgrounds	  and/or	  drug	  treatments.	    We	  explored	  the	  concurrent	  visualization	  of	  YFP-­‐CESA6	  labeled	  cellulose	  synthase	  complexes	  with	  RFP-­‐TUB6	  labeled	  microtubules	  with	  variable	  angle	  total	  internal	  reflectance	  fluorescence	  (near-­‐TIRF)	  microscopy.	  Not	  all	  genotypes	  had	  the	  proper	  RFP-­‐TUB6	  constructs,	  limiting	  our	  assessment	  to	  the	  same	  degree	  as	  previous	  research.	  This	  allowed	  for	  a	  more	  comprehensive	  analysis	  of	  CSC	  movement	  and	  velocity	  due	  to	  its	  increased	  resolution	  at	  specific	  optical	  sections.	  By	  comparing	  the	  results	  obtained	  from	  live-­‐cell	  imaging	  with	  near-­‐TIRF	  microscopy	  between	  a	  number	  of	  anisotropy-­‐compromised	  mutants,	  such	  as	  any1,	  mor1-­‐1,	  bot1,	  and	  oryzalin-­‐treated	  seedlings,	  and	  microtubule-­‐associated	  protein	  (MAP)	  genotypes	  with	  either	  enhanced	  (RIC1-­‐OX)	  or	  non-­‐ iii specific	  anisotropy	  defects	  (clasp-­‐1),	  we	  were	  able	  to	  obtain	  a	  more	  accurate	  depiction	  of	  the	  extent	  of	  microtubule	  influence	  on	  CSC	  activity.	    From	  these	  results,	  we	  were	  able	  to	  consider	  potential	  effects	  of	  microtubules	  on	  plasma	  membrane	  domains	  on	  CSC	  velocity	  during	  rapid	  elongation,	  as	  well	  as	  to	  discern	  potential	  new	  roles	  and	  effects	  of	  the	  MOR1	  protein.	  Analysis	  of	  oryzalin-­‐treated	  seedlings	  compared	  to	  the	  severely	  impaired	  double	  mutant,	  any1/mor1-­‐1,	  provided	  us	  with	  an	  awareness	  that	  the	  mechanisms	  controlling	  CSC	  activity	  are	  complex.	  With	  this	  understanding,	  our	  exploration	  into	  the	  effect	  of	  several	  MAPs	  on	  CSC	  velocity	  highlighted	  that	  microtubule	  polymerization	  and	  organization	  do	  not	  hold	  equal	  weight,	  and	  that	  a	  potential	  standard	  exists	  for	  CSC	  velocity	  and	  wall	  crystallinity,	  as	  exemplified	  in	  wild	  type	  and	  RIC1-­‐OX	  lines.	                  iv PREFACE  This	  dissertation	  is	  original,	  unpublished,	  independent	  work	  by	  the	  author,	  A.	  Mulvihill.	  	  	  Original	  project	  planning	  was	  decided	  by	  G.	  Wasteneys	  and	  M.	  Fujita	  prior	  to	  commencement.	  Project	  reevaluation	  was	  tasked,	  changed,	  and	  confirmed	  by	  the	  author,	  A.	  Mulvihill,	  and	  supervisors,	  G.	  Wasteneys	  and	  M.	  Fujita,	  following	  the	  publication	  of	  Xiao	  et	  al.	  2016.	  	  	   	   v TABLE	  OF	  CONTENTS  ABSTRACT…………………………………………………………………………………………………………………….ii PREFACE………………………………………………………………………………………………………………………iv TABLE	  OF	  CONTENTS……………………………………………………………………………………………………v LIST	  OF	  TABLES………………………………………………………………………………………………………….viii LIST	  OF	  FIGURES……………………………………………………………………………………………....................ix LIST	  OF	  ABBREVIATIONS…………………………………………………………………………………................xi ACKNOWLEDGEMENTS……………………………………………………………………………………................xii   Chapter	  1:	  General	  Introduction	  &	  Background	  ……………………………………………...................1  	   1.1	  Anisotropy	  and	  the	  Cell	  Wall…………………………………………….…………………..1 	   1.2	  Cellulose	  Synthesis	  &	  Structure…………………………………………….………………4 	   1.3	  Microtubules	  &	  Anisotropy…………………………………………….……………………..9 	  	  	  1.4	  Objectives…………………………………………….…………………...……………………….13   Chapter	  2:	  Microtubule	  Domain-­‐Specific	  Effects	  on	  Cellulose	  Synthase	  Complex	  Activity…………………………………………….…………………...…………………………......................................17  	   	   	   2.1	  Background…………………………………………….…………………...…………………….17 	   2.2	  Materials	  &	  Methods…………………………………………….…………………………….20 2.2.1	  Plant	  material	  &	  growth	  conditions	  ……………………………………...20 2.2.2	  Mutant	  backgrounds	  &	  reporter	  constructs…………………………..21 2.2.3	  Drug	  treatment	  &	  hormone	  mounting	  solution……………………..21 2.2.4	  Live-­‐cell	  imaging…………………………………………….…………………….22 2.2.5	  CSC	  velocity	  analysis	  …………………………………………….……………...23 2.2.6	  CSC	  &	  microtubule	  coincidence	  analysis………………………………..24 2.3	  Results…………………………………………….…………………...……………………………25  vi 2.3.1	  CSCs	  are	  mostly	  confined	  to	  microtubule	  domains…………………25 2.3.2	  Microtubule	  domains	  restrict	  CSC	  velocity	  in	  wild	  type	  during	  rapid	  cell	  elongation..	  ...............................................................................................26 2.3.3	  Microtubules	  fail	  to	  properly	  restrict	  CSC	  velocity	  in	  mor1-­‐1…..28 2.3.4	  Oryzalin	  disruption	  of	  cortical	  microtubule	  network	  does	  not	  impact	  mean	  CSC	  velocity…………………………………….………………………..31 2.4	  Discussion…………………………………………….…………………...………………………36   Chapter	  3:	  Assessing	  the	  Nature	  of	  Microtubule	  Disruption	  in	  the	  Reduction	  of	  Growth	  Anisotropy…………………………………………….…………………...…………………………………..40  3.1	  Background…………………………………………….…………………...…………………….40 	   	   	   3.2	  Materials	  &	  Methods…………………………………………….…………………...……….45 3.2.1	  Plant	  material	  &	  growth	  conditions	  ……………………………………..45 3.2.2	  Mutant	  backgrounds	  &	  reporter	  constructs………………………….46 3.2.3	  Drug	  treatment	  &	  hormone	  mounting	  solution…………………….46 3.2.4	  Live-­‐cell	  imaging…………………………………………….……………………46 3.2.5	  CSC	  velocity	  analysis	  …………………………………………………………...46 3.3	  Results…………………………………………….………………………………………………..47 	   	   3.3.1	  Disruption	  of	  cortical	  microtubule	  network	  by	  oryzalin	  exacerbates	  any1	  CSC	  velocity	  phenotype……………………………………...47 	   	   3.3.2	  The	  mor1-­‐1/any1	  double	  mutant	  displays	  reduced	  seedling	  viability	  and	  CSC	  velocity…………………………………….....................................51 	   	   3.3.3	  Hypocotyl	  CSC	  velocities	  are	  elevated	  in	  clasp-­‐1	  mutant	  background	  …………………………………...................................................................56 	   	   3.3.4	  botero1	  displays	  unaltered	  mean	  CSC	  velocity	  compared	  to	  wild	  type	  values………...…………………………..................................................................58 	   	   3.3.5	  CSCs	  in	  RIC1-­‐OX	  exacerbate	  wild	  type	  response	  to	  rapid	  elongation	  by	  increasing	  velocity…………………………………………….…….60 	   	   	   3.4	  Discussion…………………………………………….…………………...……………………..63  vii   Chapter	  4:	  Summary	  &	  Future	  Directions……………………………………………………………....….67 	    REFERENCES………………………………………………...……………………………………………………………..72 APPENDICES………………………………………………...……………………………………………………………..80	  	   	   viii LIST	  OF	  TABLES   Table	  2.1	  	  	  Comparison	  of	  mean	  velocities	  of	  CSCs	  and	  percent	  association	  of	  CSCs	  with	  microtubule	  domains	  by	  confocal	  and	  near-­‐TIRF	  microscopy………………………….……………...25  Table	  2.2	  	  	  Comparison	  of	  mean	  velocities	  of	  CSCs	  and	  percent	  association	  of	  CSCs	  with	  microtubule	  domains	  by	  confocal	  and	  near-­‐TIRF	  microscopy	  in	  oryzalin	  treated	  seedlings………………………………………………………………………………………………………………………31	  	   	   ix LIST	  OF	  FIGURES   Figure	  1.1	  	  Summary	  of	  previous	  findings	  concerning	  CSC	  velocity,	  crystallinity,	  degree	  of	  anisotropy,	  and	  microtubule	  phenotypes……………………………………………………………………….15	  Figure	  2.1	  	  CSC	  velocity	  in	  microtubule	  domains	  and	  non-­‐microtubule	  domains……………...27 Figure	  2.2	  	  CSC	  velocity	  in	  microtubule	  domains	  and	  non-­‐microtubule	  domains	  in	  mor1-­‐1………………………………………………………………………………………………….……………………………….29  Figure	  2.3	  	  Comparison	  of	  CSC	  velocity	  in	  microtubule	  domains	  and	  non-­‐microtubule	  domains	  in	  wild	  type	  and	  mor1-­‐1…………………………………………………………………………………..30  Figure	  2.4	  	  CSC	  velocity	  in	  microtubule	  domains	  and	  non-­‐microtubule	  domains	  in	  2	  μM	  oryzalin………………………………………………………………………………………………………………………..32  Figure	  2.5	  	  CSC	  velocity	  in	  microtubule	  domains	  and	  non-­‐microtubule	  domains	  in	  20μM	  oryzalin……………………………………………………………………………………………….………………………34  Figure	  2.6	  	  Comparison	  of	  CSC	  velocity	  in	  microtubule	  domains	  and	  non-­‐microtubule	  domains	  in	  untreated,	  2μM	  oryzalin,	  and	  20μM	  treated	  seedlings	  …………………………..……..35   Figure	  3.1	  	  CSC	  velocity	  in	  any1	  mutant…………………………………………………..…………………….48  Figure	  3.2	  	  CSC	  velocity	  in	  the	  any1	  mutant	  with	  2μM	  oryzalin………………………………………49  Figure	  3.3	  	  CSC	  velocity	  in	  the	  any1	  mutant	  with	  20μM	  oryzalin………………………………….....50  Figure	  3.4	  	  The	  any1/mor1-­‐1	  double	  mutant……………………………………………………………...….52  Figure	  3.5	  	  CSC	  velocity	  in	  the	  any1/mor1-­‐1	  double	  mutant…………………………………………...54  Figure	  3.6	  	  Comparison	  of	  CSC	  velocity	  in	  wild	  type,	  untreated	  any1,	  any1/mor1-­‐1,	  any1	  +	  2μM	  oryzalin,	  any1	  +	  20μM	  oryzalin	  treated	  seedlings……………………………………………...…..55  Figure	  3.7	  	  CSC	  velocity	  in	  clasp-­‐1………………………………………………………………………………...57  Figure	  3.8	  	  CSC	  velocity	  in	  bot1………………………………………………………………………………….....59  x  Figure	  3.9	  	  CSC	  velocity	  in	  RIC1-­‐OX……………………………………………………………………………...61	    Figure	  3.1A	  	  Comparison	  of	  CSC	  velocity	  in	  wild	  type,	  clasp-­‐1,	  bot1,	  and	  RIC1-­‐OX	   seedlings…………………………………………………………………………………………………………………....62   Figure	  4.1	  	  CSC	  Velocity	  versus	  percent	  change	  in	  crystallinity	  from	  wild	  type……..............68	  	   	   xi LIST	  OF	  ABBREVIATIONS CESA	   	   	   Cellulose	  Synthase	  	  CLIP	   	   	   Cytoplasmic	  Linker	  Protein-­‐Interacting	  Domain  Col-­‐0	   	   	   Columbia	  ecotype	  of	  Arabidopsis	  thaliana  CSC	  	   	   	   Cellulose	  Synthase	  Complex	  	  CSR	  	   	   	   Class-­‐specific	  Region  DMSO	   	   	   Dimethyl	  Sulfoxide  EDTA	   	   	   Ethylene	  Diamine	  Tetra-­‐Acetic	  Acid	  	  EMS	   	   	   Ethyl	  methanesulfonate  GA	   	   	   	   Gibberellic	  Acid  GDP/GTP	   	   Guanosine	  Di/Triphosphate  GOF	   	   	   Gain-­‐of-­‐Function  LOF	   	   	   Loss-­‐of-­‐Function  MAP	   	   	   Microtubule	  Associated	  Protein  MT	  	   	   	   Microtubule	  	  P-­‐CR	   	   	   Plant-­‐Conserved	  Sequence  RGB	   	   	   Red/Green/Blue	    RFP	   	   	   Red	  Fluorescent	  Protein	  	  ROP	   	   	   Rho	  GTPAse  SD	   	   	   	   Standard	  Deviation  +TIPS	   	   	   Microtubule	  Plus-­‐End	  Tracking	  Proteins  TIRF	   	   	   Total	  Internal	  Reflection	  Fluorescence	  	  	  TOG	   	   	   Tumour	  Overexpressed	  Gene  TUB	   	   	   Beta	  Tubulin  UDP	   	   	   Uridine	  Diphosphate  WT	  	   	   	   Wild	  Type  YFP	  	   	   	   Yellow	  Fluorescent	  Protein	   xii ACKNOWLEDGMENTS  Three	  years	  as	  a	  graduate	  student	  has	  taught	  me	  more	  than	  I	  would	  ever	  have	  thought	  imaginable.	  Similar	  to	  many	  things	  in	  life,	  it	  is	  not	  possible	  to	  predict	  exactly	  how	  a	  situation	  or	  event	  may	  impact	  you.	  Thanks	  to	  Dr.	  Geoffrey	  O.	  Wasteneys,	  I	  was	  able	  to	  explore	  one	  of	  the	  most	  challenging	  and	  rewarding	  experiences	  of	  my	  life.	  I’ve	  had	  very	  few	  opportunities	  for	  such	  direct	  mentorship	  prior	  to	  moving	  to	  Vancouver	  to	  pursue	  this	  degree,	  so	  I	  am	  immensely	  grateful	  and	  appreciative	  to	  Dr.	  Wasteneys	  for	  his	  support	  and	  input	  over	  the	  duration	  of	  the	  program.	  His	  guidance	  was	  critical	  for	  me	  to	  make	  as	  much	  of	  the	  transition	  from	  student	  to	  professional	  as	  possible	  under	  our	  constraints.	  With	  difficulties	  ranging	  from	  his	  immediate	  injury	  upon	  my	  arrival,	  to	  some	  very	  bizarre	  and	  adverse	  health	  anomalies	  on	  my	  end,	  to	  getting	  scooped	  two	  thirds	  of	  the	  way	  through	  the	  intended	  program,	  it	  is	  only	  with	  great	  patience	  and	  care	  that	  we	  have	  made	  it	  through.	  His	  directness	  and	  ability	  to	  use	  logic	  to	  his	  greatest	  advantage	  are	  some	  skills	  I	  have	  immeasurably	  appreciated	  and	  attempted	  to	  adapt	  into	  my	  own	  repertoire.	  For	  this	  guidance,	  and	  for	  the	  endless	  opportunities	  to	  learn	  and	  grow	  in	  both	  knowledge	  and	  skills,	  I	  am	  eternally	  grateful.	    Additionally,	  I’d	  like	  to	  thank	  my	  committee	  members,	  Dr.	  Lacey	  Samuels	  and	  Dr.	  Harry	  Brumer,	  for	  their	  continued	  efforts	  during	  the	  pursuit	  of	  this	  degree.	  Their	  input	  at	  various	  stages	  was	  critical	  in	  me	  finding	  my	  footing	  as	  many	  of	  my	  experiences	  in	  this	  program	  were	  firsts	  for	  me.	  While	  many	  tasks	  were	  arduous	  and	  telling	  of	  my	  inexperience,	  without	  the	  full	  efforts	  and	  critiques	  of	  my	  committee	  I	  would	  not	  be	  half	  as	  adept	  or	  prepared	  for	  the	  expectations	  that	  will	  be	  placed	  on	  my	  career	  in	  years	  to	  come.	  	   xiii 	  A	  special	  note	  goes	  out	  to	  the	  Wasteneys	  Lab	  members,	  and	  specifically	  senior	  member,	  Dr.	  Miki	  Fujita.	  Dr.	  Fujita	  was	  kind	  and	  balanced	  in	  many	  of	  her	  teaching	  moments	  with	  me	  and	  was	  a	  major	  contributor	  to	  my	  ability	  to	  move	  forward	  in	  difficult	  times.	  Her	  patience	  and	  calm	  demeanor	  were	  increasingly	  valuable	  as	  the	  pressures	  of	  this	  degree	  mounted.	  It	  is	  easy	  as	  an	  international	  student	  to	  feel	  isolated,	  but	  the	  Wasteneys	  lab	  over	  the	  years	  has	  as	  a	  whole,	  and	  individually,	  been	  able	  to	  provide	  me	  with	  moments	  of	  support	  and	  friendship.	  I	  would	  specifically	  like	  to	  thank	  Dr.	  Ryan	  Eng	  and	  Laryssa	  Hallet	  for	  providing	  me	  with	  great	  friendship	  and	  advice,	  both	  academically	  and	  personally.	  It	  was	  extremely	  valuable	  for	  me	  to	  see	  such	  wonderful	  examples	  of	  how	  to	  balance	  both	  one’s	  personal	  life	  and	  professional	  career,	  even	  if	  I	  could	  never	  live	  up	  to	  such	  standards.	  I’d	  like	  to	  also	  thank	  other	  members	  of	  the	  Wasteneys	  lab	  and	  adjacent	  labs	  for	  providing	  such	  a	  wonderful	  workspace	  and	  welcoming	  environment;	  Dr.	  Sylwia	  Jankowski,	  Dr.	  Katherine	  Celler,	  Dr.	  Yuan	  Ruan,	  Caitlin	  Donnelly,	  Sam	  Livingston,	  Dr.	  Teagen	  Quilichini,	  Yoichiro	  Watanabe,	  Miranda	  Meents,	  Karlson	  Pang,	  Eva	  Chou,	  Thamali	  Kariyawasam,	  Dr.	  Michelle	  Wang,	  and	  Evan	  Cronmiller.	    Through	  my	  experiences	  with	  learning	  many	  of	  the	  microscopy	  and	  imaging	  techniques	  necessary	  for	  my	  projects,	  I	  was	  lucky	  enough	  to	  have	  access	  to	  the	  Bioimaging	  Facility	  (BIF)	  and	  the	  amazing	  expertise	  of	  their	  team.	  I’d	  like	  to	  thank	  Garnet	  Martens,	  Derrick	  Home,	  Kevin	  Hodgson,	  and	  Brad	  Ross	  for	  all	  their	  help	  with	  the	  various	  TEM	  preparation	  techniques	  and	  imaging	  issues	  that	  presented	  themselves.	     xiv One	  of	  the	  most	  rewarding	  and	  enjoyable	  experiences	  I	  had	  as	  a	  graduate	  student	  was	  being	  a	  teaching	  assistant.	  I	  am	  so	  grateful	  to	  mentors	  Dr.	  Robin	  Young	  and	  Dr.	  Sunita	  Chowrira	  for	  being	  not	  just	  exceptional	  leaders,	  but	  also	  respectful	  and	  helpful	  in	  my	  own	  moments	  of	  stress.	  The	  experiences	  I	  had	  in	  Biology	  200	  and	  201	  were	  some	  of	  the	  most	  important	  for	  me	  in	  realizing	  my	  strengths,	  weaknesses,	  and	  how	  to	  appropriately	  problem	  solve.	    Of	  course,	  nothing	  would	  be	  possible	  without	  the	  assistance	  with	  administration	  that	  the	  staff	  and	  faculty	  of	  the	  Department	  of	  Botany,	  UBC,	  provided.	  A	  special	  thanks	  to	  Isabel	  Ferens,	  Veronica	  Oxtoby,	  Alice	  Liou,	  and	  Jessica	  Sui	  for	  their	  help	  in	  all	  my	  troubles	  being	  an	  international	  student	  and	  keeping	  balanced	  with	  finances	  and	  paperwork.  Lastly,	  I	  greatly	  want	  to	  thank	  my	  mothers	  back	  home	  in	  San	  Diego,	  Debra	  Mulvihill,	  Tamar	  Berg,	  Cynthia	  Mulvihill,	  and	  Rhonda	  Shapiro,	  as	  well	  as	  my	  friends	  and	  communities	  here	  in	  Vancouver.	  My	  parents	  single-­‐handedly	  give	  me	  the	  support	  and	  love	  to	  endure	  any	  struggle	  I	  face,	  and	  to	  pull	  me	  out	  of	  the	  depths	  of	  situations	  we	  can’t	  prepare	  for.	  When	  living	  thousands	  of	  miles	  away,	  I	  cannot	  express	  how	  moved	  I	  am	  by	  their	  continued	  devotion	  to	  my	  endeavors.	  One	  of	  the	  greatest	  struggles	  for	  me	  has	  been	  the	  isolation	  and	  work	  I’ve	  had	  to	  put	  into	  finding	  support	  systems	  while	  also	  maintaining	  a	  healthy	  relationship	  between	  work	  and	  life.	  I	  could	  not	  have	  been	  successful	  in	  the	  last	  few	  months	  of	  writing	  and	  stress	  without	  my	  boyfriend,	  Brett	  Hawksby,	  or	  my	  best	  friends	  Jordan	  Eggert,	  Fiorella	  Vallejo,	  Will	  McGuire,	  James	  Kramer-­‐Anderson,	  Luc	  Sanscartier,	  Kevin	  Orr,	  and	  Quentin	  Verra. 1 Chapter	  1:	  General	  Introduction	  &	  Background  1.1	  Anisotropy	  and	  The	  Cell	  Wall	   Plant	  survival	  is	  largely	  dependent	  on	  its	  light	  harvesting	  cellular	  constituents.	  For	  this	  to	  be	  possible,	  plants	  must	  reach	  the	  light	  from	  their	  stationary	  positioning	  underground	  as	  a	  seed	  by	  traversing	  through	  both	  soil	  and	  aerial	  domains.	  In	  land	  plants,	  the	  hypocotyl	  is	  the	  specialized	  organ	  that	  functions	  during	  germination	  to	  move	  the	  shoot	  apical	  meristem	  out	  of	  the	  soil.	  In	  dicots,	  such	  as	  Arabidopsis	  thaliana,	  the	  hypocotyl	  expands	  through	  coordinated	  cell	  elongation	  that	  begins	  near	  its	  apical	  base	  and	  progresses	  to	  the	  region	  just	  below	  the	  cotyledons.	  It	  is	  the	  focus	  of	  this	  thesis	  to	  assess	  this	  example	  of	  unidirectional	  expansion	  in	  plants.  The	  process	  by	  which	  plant	  organs	  grow	  unidirectionally	  is	  known	  as	  growth	  anisotropy.	  Building	  turgor	  pressure	  within	  the	  cell	  provides	  the	  physical	  force	  that	  stretches	  the	  wall	  (Cosgrove	  et	  al.	  1984),	  leading	  to	  expansion,	  but	  in	  order	  to	  grow	  anisotropically,	  the	  cell	  wall	  must	  have	  a	  physical	  structure	  that	  permits	  expansion	  to	  occur	  predominantly	  in	  one	  direction.	  The	  cell	  wall,	  a	  structural	  biological	  layer	  that	  surrounds	  the	  cell	  membrane,	  not	  only	  is	  the	  barrier	  with	  which	  the	  cell	  can	  modulate	  elongation	  (Lockhart	  et	  al.	  1965,	  Cosgrove	  et	  al.	  1985),	  but	  also	  confers	  resistance	  to	  environmental	  stresses,	  pathogens,	  and	  regulates	  many	  signalling	  mechanisms.	  Due	  to	  its	  dense	  polysaccharide	  content	  and	  structural	  rigidity,	  the	  cell	  wall	  also	  provides	  the	  plant	  with	  physical	  support.  In	  order	  to	  achieve	  unidirectional	  growth,	  the	  cell	  relies	  on	  both	  mechanical	  and	  chemical	  features	  of	  the	  cell	  wall	  that	  enables	  the	  cell	  to	  yield	  to	  the	  internal	  turgor	  pressure.	  The	   2 biological	  structure	  most	  responsible	  is	  cellulose	  and	  is	  utilized	  by	  a	  diverse	  number	  of	  species	  for	  cellular	  processes	  regarding	  physical	  and	  mechanical	  activity	  (Yamanaka	  et	  al.	  1989).	  In	  tandem	  with	  other	  wall	  constituents,	  such	  as	  hemicelluloses,	  pectin,	  and	  various	  wall	  proteins,	  ordered	  cellulose	  microfibrils	  are	  selectively	  separated	  during	  elongation	  (Preston	  et	  al.	  1952,	  Frey-­‐Wyssling	  et	  al.	  1953,	  McCann	  et	  al.	  1990).	  A	  number	  of	  models	  (Roelofsen	  et	  al.	  1958,	  Green	  et	  al.	  1960,	  Brummel	  &	  Hall	  et	  al.	  1985,	  Ortega	  et	  al.	  1985,	  Talbott	  et	  al.	  1992,	  Carpita	  &	  Gibeaut,	  1993)	  have	  been	  theorized	  to	  explain	  the	  means	  by	  which	  the	  microfibrils	  are	  ordered,	  modified,	  and	  separated,	  but	  none	  have	  been	  entirely	  confirmed.	  It	  is	  known	  that	  cellulose	  microfibrils	  in	  elongating	  cells	  are	  transversely	  ordered	  at	  the	  onset	  of	  their	  deposition	  in	  elongating	  cells	  (Takeda	  &	  Shibaoka	  et	  al.	  1981,	  Richmond	  et	  al.	  1980,	  Sellen	  et	  al.	  1983,	  Sugimoto	  et	  al.	  2000).	  This	  transverse	  array	  of	  microfibrils	  is	  posited	  to	  be	  critical	  for	  constraining	  growth	  along	  one	  axis.	  As	  new	  cellulose	  is	  produced,	  the	  inner	  layers	  are	  displaced	  outward	  and	  separate	  from	  one	  another	  into	  less	  uniform	  patterns	  (Wardrop	  et	  al.	  1956,	  Sugimoto	  et	  al.	  2001).	  It	  is	  unclear	  whether	  the	  synthesis	  of	  the	  microfibril	  defines	  its	  localization	  and	  relationships	  with	  other	  wall	  constituents,	  or	  vice	  versa.	  In	  order	  to	  test	  this,	  experiments	  have	  tackled	  the	  synthesis,	  regulation,	  and	  modification	  of	  these	  polysaccharides	  in	  relation	  to	  achieving	  anisotropy	  (Pauly	  et	  al.	  1999).	    During	  the	  initiation	  of	  elongation,	  acidification	  of	  the	  cell	  wall	  promotes	  enzymatic	  and	  other	  wall	  editing	  activity	  (Rayle	  et	  al.	  1970,	  Cosgrove	  et	  al.	  1989,	  Rayle	  et	  al.	  1992).	  Expansins	  (McQueen-­‐Mason	  et	  al.	  1992,	  McQueen-­‐Mason	  et	  al.	  1995)	  are	  a	  class	  of	  proteins	  that	  are	  involved	  in	  this	  acid	  response	  and	  have	  been	  shown	  to	  edit	  polysaccharides	  after	   3 deposition	  to	  direct	  the	  slippage	  of	  microfibrils	  during	  elongation.	  Along	  with	  enzymes	  such	  as	  endotransglycosylases	  (Nishitani	  et	  al.	  1982,	  Fry	  et	  al.	  1995),	  the	  cell	  has	  the	  means	  to	  edit	  the	  synthesis,	  deposition,	  and	  post-­‐deposition	  of	  wall	  material	  to	  direct	  turgor	  driven	  growth.	  This	  provided	  not	  only	  the	  basis	  for	  postulating	  post-­‐deposition	  wall	  modification	  as	  a	  means	  for	  controlling	  wall	  growth	  but	  also	  an	  emphasis	  on	  roles	  for	  non-­‐cellulosic	  wall	  components.	  Interactions	  with	  polysaccharides,	  such	  as	  xyloglucans	  and	  pectins,	  and	  modifications	  of	  the	  wall	  are	  thought	  to	  be	  tied	  to	  the	  binding	  quality	  of	  glucan	  monomers	  in	  a	  cellulose	  chain,	  otherwise	  known	  as	  microfibril,	  or	  wall,	  crystallinity.	  This	  structural	  characteristic	  of	  microfibrils	  is	  determined	  by	  the	  tightness	  of	  bonding	  between	  hydroxyl	  groups	  and	  is	  thought	  to	  biologically	  impact	  anisotropy	  (Benziman	  et	  al.	  1980,	  Carpita	  &	  Gibeaut	  et	  al.	  1993,	  Cousins	  &	  Brown	  et	  al.	  1997a/b).	    Forward	  genetic	  screens	  and	  the	  resulting	  extensive	  literature	  have	  identified	  anisotropy	  defects	  specifically	  pertaining	  to	  perturbations	  in	  cellulose	  synthesis	  (Arioli	  et	  al.	  1998,	  Scheible	  et	  al.	  2001,	  Burn	  et	  al.	  2002,	  Fujita	  et	  al.	  2013),	  hormonal/drug	  imbalance	  (Shibaoka	  et	  al.	  1972,	  Takeda	  et	  al.	  1981,	  Vaughn	  et	  al.	  2001),	  compromised	  integrity	  of	  wall	  constituents	  other	  than	  cellulose	  (Park	  et	  al.	  2012,	  Peaucelle	  et	  al.	  2015),	  and/or	  microtubule	  disruption	  (Whittington	  et	  al.	  2001,	  Baskin	  et	  al.	  2001,	  Burk	  et	  al.	  2002).	  These	  mutants	  and	  various	  experimental	  techniques	  can	  result	  in	  a	  number	  of	  phenotypes	  including	  disordered	  wall	  microfibrils	  (Nicol	  et	  al.	  1998,	  Zuo	  et	  al.	  2000,	  Sugimoto	  et	  al.	  2001),	  changes	  in	  amount	  of	  cellulose	  produced	  or	  cellulose	  crystallinity	  (Fisher	  et	  al.	  1998,	  Robert	  et	  al.	  2004,	  Fujita	  et	  al.	  2011/2013)	  and	  abnormal	  cellulose	  synthesis	   4 patterns	  by	  the	  production	  machinery	  (Anderson	  et	  al.	  2010,	  Fujita	  et	  al.	  2011),	  the	  Cellulose	  Synthase	  Complex	  (CSC).  During	  times	  of	  rapid	  elongation,	  displacement	  velocity	  of	  CSCs	  increases,	  while	  wall	  crystallinity,	  as	  measured	  by	  X-­‐ray	  diffraction,	  decreases	  (Fujita	  et	  al.	  2011).	  This	  suggests	  that	  these	  are	  both	  requirements	  for	  maintaining	  anisotropy	  as	  growth	  increases	  but	  they	  have	  yet	  to	  be	  strongly	  associated	  with	  any	  elongation	  models	  as	  a	  related	  pair.	  Therefore,	  associations	  between	  the	  velocity	  of	  the	  complex	  and	  the	  crystallinity	  of	  the	  wall	  are	  under	  active	  investigation	  for	  at	  least	  partially	  determining	  proper	  anisotropy	  during	  rapid	  elongation.	    1.2	  Cellulose	  Synthesis	  and	  Structure	  	  Cellulose	  is	  a	  polysaccharide	  of	  repeating	  β-­‐(1,4)-­‐glucose	  monomers	  inverted	  180°	  from	  one	  another	  and	  crystallized	  into	  a	  fibrillar	  form	  called	  a	  microfibril	  (Saxena	  et	  al.	  1995,	  Koboyashi	  &	  Shoda	  et	  al.	  1995).	  These	  microfibrils	  average	  approximately	  3nm	  in	  width	  (Chanzy	  et	  al	  1979,	  Thomas	  et	  al.	  2013)	  and	  are	  of	  variable	  length	  in	  the	  wall.	  From	  within	  the	  cytoplasm,	  monomers	  are	  harvested	  from	  UDP-­‐glucose	  and	  added	  to	  the	  reducing	  end	  of	  the	  growing	  polymer	  (Saxena	  et	  al.	  1995,	  Peng	  et	  al.	  2002).	  The	  polysaccharide	  is	  produced	  by	  the	  CSC,	  which	  is	  made	  up	  of	  individual	  CesA	  proteins	  estimated	  to	  total	  between	  18-­‐24	  in	  number	  (Thomas	  et	  al.	  2013).	  The	  CSC	  forms	  a	  rosette	  shape	  with	  a	  number	  of	  lobe-­‐like	  structures	  (Brown	  &	  Montezinos	  et	  al.	  1976,	  Mueller	  &	  Brown	  et	  al.	  1980,	  Kimura	  et	  al.	  1999)	  containing	  variable	  combinations	  of	  CesA	  isoforms	  (Perrin	  et	  al.	  2001).	  The	  protein	  structure	  of	  members	  of	  the	  CesA	  family	  is	  fairly	  similar,	  containing	   5 highly	  conserved	  sequences	  (DDDQxRw)	  thought	  to	  be	  involved	  in	  the	  catalytic	  action	  of	  the	  enzymes,	  as	  well	  as	  highly	  variable,	  species-­‐specific	  domains,	  the	  functions	  of	  which	  are	  less	  well	  understood	  (Pear	  et	  al	  1996,	  Holland	  et	  al.	  2000,	  Richmond	  &	  Somerville	  et	  al.	  2000).	  The	  structure	  of	  these	  isoforms	  has	  been	  elucidated	  using	  a	  number	  of	  algorithms	  comparing	  the	  structure	  of	  the	  bacterial	  synthase’s	  catalytic	  domain	  (CatD)	  with	  those	  in	  plant	  species	  (Olek	  et	  al.	  2014).	    By	  removing	  the	  plant-­‐conserved	  sequence	  (P-­‐CR)	  and	  the	  class-­‐specific	  region	  (CSR),	  Olek	  et	  al.	  (2014)	  made	  comparisons	  with	  the	  bacterial	  catalytic	  domains.	  This	  revealed	  strong	  homologies	  within	  the	  catalytic	  domains	  necessary	  for	  binding	  UDP,	  UDP-­‐Glucose,	  and	  the	  non-­‐reducing	  cellobiosyl	  unit	  of	  the	  cellulose	  chain	  being	  produced.	  All	  of	  these	  similarities	  which	  mapped	  to	  the	  catalytic	  domain	  and	  activity	  of	  the	  bacterial	  synthases,	  were	  further	  related	  to	  several	  other	  species	  based	  on	  homology	  found	  in	  their	  catalytic	  core.	  Based	  on	  algorithmic	  values	  from	  previous	  research,	  this	  analysis	  suggested	  the	  presence	  of	  a	  general	  nucleotide	  binding	  fold	  in	  many	  of	  the	  sequence	  templates	  (Zhang	  &	  Skolnick	  et	  al.	  2005).	  They	  further	  showed	  that	  plant	  CesAs	  not	  only	  dimerize	  via	  their	  Zinc-­‐finger	  domains,	  as	  was	  previously	  shown	  in	  Kurek	  et	  al.	  (2002),	  but	  also	  via	  their	  catalytic	  domains,	  which	  were	  observed	  to	  unexpectedly	  bind	  two	  UDP-­‐Glucose	  monomers	  per	  monomer,	  or	  four	  per	  dimer.	  This	  is	  in	  direct	  contrast	  to	  many	  other	  models	  by	  placing	  emphasis	  on	  the	  basic	  structural	  unit	  of	  the	  complex	  as	  two	  CesA	  proteins,	  either	  in	  hetero-­‐	  or	  homodimer	  conformations.	  In	  order	  to	  determine	  the	  location	  of	  the	  plant-­‐	  (P-­‐CR)	  and	  class-­‐specific	  (CSR)	  protein	  domains,	  a	  reassessment	  of	  sequence	  homology	  provided	  several	  potential	  models,	  the	  most	  likely	  of	  which	  places	  the	  P-­‐CR	  and	  CSR	  on	  the	  lateral	   6 flanks	  of	  the	  catalytic	  domain,	  where	  the	  CSR	  may	  occupy	  a	  small	  space	  of	  the	  catalytic	  domain	  for	  assistance	  in	  monomer/dimer	  substrate	  binding	  (Olek	  et	  al.	  2014).	  Future	  work	  is	  still	  necessary	  to	  determine	  the	  specifications	  for	  hetero-­‐	  vs.	  homodimer	  preferences	  in	  comprising	  the	  CSC	  and	  how	  this	  relates	  to	  what	  is	  already	  known	  about	  CesA	  synthesis	  and	  delivery	  to	  the	  plasma	  membrane.	    The	  CesAs	  are	  synthesized	  in	  the	  Endoplasmic	  Reticulum	  and	  assembled	  either	  before	  or	  during	  transport	  to	  the	  plasma	  membrane	  (Haigler	  &	  Brown	  et	  al.	  1986,	  Kurek	  et	  al.	  2002).	  In	  the	  primary	  cell	  wall,	  CESA1,	  CESA3,	  and	  CESA6	  are	  considered	  to	  be	  the	  major	  protein	  components	  of	  the	  CSC	  and	  the	  main	  cellulose	  synthesizing	  proteins	  in	  the	  primary	  wall	  (Fagard	  et	  al.	  2000,	  Williamson	  et	  al.	  2001a,	  Scheible	  et	  al.	  2001,	  Burn	  et	  al.	  2002).	  CESA6	  expression,	  in	  particular,	  is	  upregulated	  during	  times	  of	  rapid	  elongation	  but	  may	  still	  retain	  reduced	  or	  redundant	  function	  during	  other	  activities	  (Fagard	  et	  al.	  2000).	  Although	  some	  CesAs,	  such	  as	  CESA2,	  CESA5,	  CESA6,	  and	  CESA9,	  are	  considered	  semi-­‐redundant,	  research	  has	  yet	  to	  elucidate	  the	  individual	  relevance	  of	  the	  isoforms	  and	  their	  selective	  packaging	  into	  CSCs.	  	  	  Since	  CESA6	  is	  upregulated	  during	  rapid	  elongation	  of	  dark-­‐grown	  hypocotyl	  cells,	  it	  has	  become	  a	  focal	  point	  of	  CSC	  research	  for	  following	  CSC	  dynamics	  by	  live-­‐cell	  imaging	  when	  fused	  to	  the	  fluorescent	  reporter	  protein	  citrine-­‐YFP	  (yellow	  fluorescent	  protein).	  Similar	  to	  microfibrils,	  it	  has	  been	  shown	  that	  trajectories	  of	  the	  CSC	  are	  transverse	  to	  the	  growth	  axis	  and	  mirror	  microtubules	  aligned	  underneath	  for	  determining	  proper	  elongation	  (Paredez	  et	  al.	  2006).	  Dynamicity	  of	  the	  complex	  is	  best	  observed	  in	  dark-­‐grown	  seedling	   7 hypocotyls	  as	  it	  mimics	  the	  seedling	  response	  to	  being	  beneath	  soil.	  These	  reporter	  constructs	  linked	  to	  the	  CesA	  isoforms	  allow	  visualization	  of	  CSC	  movement	  and	  provide	  insight	  into	  the	  means	  by	  which	  microfibril	  orientation	  and	  synthesis	  are	  determined.	    Modification	  of	  the	  CSC	  has	  identified	  that	  both	  the	  synthesis	  and	  crystallization	  of	  the	  microfibril	  are	  concurrent	  processes	  determined	  by	  the	  CSC	  (Cousins	  &	  Brown	  et	  al.	  1997	  a/b,	  Benziman	  et	  al.	  1980)	  that	  can	  be	  disrupted	  to	  influence	  anisotropy	  (Sugimoto	  et	  al.	  2001,	  Fujita	  et	  al.	  2013).	  Synthesis	  and	  crystallization	  are	  also	  separate	  processes	  (Benziman	  et	  al.	  1980)	  as	  shown	  by	  work	  with	  Calcofluor	  white,	  a	  fluorescent	  brightener,	  which	  hydrogen	  bonds	  to	  the	  glucan	  chains,	  preventing	  the	  proper	  assembly	  of	  microfibrils	  without	  impeding	  synthesis.	  Models	  predict	  that	  these	  concurrent	  processes	  displace	  the	  CSC	  from	  its	  position	  within	  the	  plasma	  membrane	  and	  the	  portion	  of	  polysaccharide	  it	  just	  produced	  (Diotallevi	  et	  al.	  2007).	  Crystallinity	  of	  a	  microfibril	  is	  not	  consistent	  along	  or	  throughout	  the	  polysaccharide,	  providing	  pockets	  or	  zones	  of	  more	  and	  less	  amorphous	  cellulose	  (Hayashi	  et	  al.	  1994).	  Hemicellulose	  and	  other	  wall	  matrix	  components	  are	  thought	  to	  interact	  more	  specifically	  with	  amorphous	  zones	  of	  cellulose,	  defining	  a	  number	  of	  models	  for	  microfibril	  separation	  in	  the	  wall	  (Brummel	  &	  Hall	  et	  al.	  1985,	  McCann	  et	  al.	  1993,	  Zhao	  et	  al.	  2014).	  	  	  Even	  though	  the	  precise	  means	  by	  which	  crystallization	  occurs	  is	  unknown,	  it	  is	  expected	  that	  microfibril	  crystallinity	  is	  linked	  to	  movement	  of	  the	  CSCs	  potentially	  through	  the	  physical	  forces	  of	  the	  catalytic	  pore	  of	  the	  CESAs	  (Cousins	  &	  Brown	  et	  al.	  1997	  a/b).	  Since	  CSC	  movement	  is	  linked	  to	  its	  synthesis,	  then	  determining	  crystallinity	  at	  the	  same	  time	   8 may	  define	  it	  as	  rate-­‐limiting.	  It	  is	  possible	  that	  not	  only	  the	  presence	  of	  other	  wall	  polysaccharides	  and	  proteins	  could	  affect	  the	  crystallization	  process	  of	  cellulose	  microfibrils,	  but	  that	  proteins,	  like	  KOR1,	  may	  play	  a	  distinct	  role	  in	  these	  relationships	  by	  building	  a	  “scaffold”	  that	  the	  CSC	  can	  build	  upon	  (Sato	  et	  al.	  2001).	  Distinguishing	  between	  synthesis	  and	  crystallization	  is	  fundamental	  for	  modeling	  functional	  relationships	  in	  anisotropy	  and	  explaining	  mutant	  phenotypes.  During	  times	  of	  rapid	  elongation,	  the	  cellular	  response	  in	  terms	  of	  CSC	  velocity	  and	  crystallinity	  for	  maintaining	  anisotropy	  has	  been	  examined	  experimentally	  (Fujita	  et	  al.	  2011).	  	  At	  29°C	  compared	  to	  21°C,	  CSC	  velocity	  increases	  fourfold	  in	  response	  to	  increased	  growth	  kinetics,	  whereas	  overall	  crystallinity	  of	  the	  wall	  decreases.	  A	  variety	  of	  cellulose-­‐deficient	  mutations	  have	  been	  generated	  with	  varying	  severity	  and	  phenotypes	  related	  to	  anisotropy,	  complex	  velocity,	  and/or	  microfibril	  crystallinity	  (Arioli	  et	  al.	  1998,	  Scheible	  et	  al.	  2001,	  Fagard	  et	  al.	  2000).	  Those	  most	  useful	  are	  constitutively	  active	  but	  without	  being	  seedling	  lethal,	  such	  as	  any1,	  a	  mutant	  in	  the	  catalytic	  portion	  of	  CESA1	  (Fujita	  et	  al.	  2013).	  The	  missense	  allele	  was	  isolated	  from	  EMS	  screens	  for	  defective	  microtubule	  patterns	  in	  leaf	  epidermal	  cells	  and	  is	  completely	  recessive.	    Easily	  identified	  by	  their	  reduced	  growth	  rate	  and	  globular	  trichome	  phenotype,	  any1	  mutants	  display	  altered	  cellulosic	  phenotypes	  compared	  to	  wild	  type,	  which	  has	  helped	  to	  define	  the	  relationship	  between	  crystallinity	  and	  velocity.	  Since	  the	  any1	  mutant	  does	  not	  alter	  the	  overall	  cellulose	  content	  in	  inflorescence	  stems	  or	  CSC	  trajectories	  and	  microfibril	  alignment	  in	  etiolated	  hypocotyls,	  isotropic	  expansion	  can	  be	  attributed	  to	  a	  reduction	  in	   9 CSC	  velocity	  and	  microfibril	  crystallinity.	  This	  reduction	  in	  cellulose	  crystallinity	  is	  associated	  with	  disorganization	  of	  microfibrils	  in	  the	  outer	  wall	  of	  epidermal	  cells	  and	  illustrates	  that	  anisotropy	  is	  highly	  dependent	  on	  processes	  other	  than	  just	  the	  amount	  and	  initial	  organization	  of	  cellulose	  synthesis.	  This	  highlights	  the	  value	  of	  exploring	  how	  CSC	  velocity	  and	  cellulose	  crystallinity	  define	  growth	  anisotropy	  in	  plant	  cell	  walls.	     1.3	  Microtubules	  and	  Anisotropy	  Microtubules	  are	  cylindrical	  polymers	  that	  serve	  the	  cell	  in	  a	  vast	  array	  of	  functions	  and	  are	  made	  up	  of	  GTP-­‐bound	  alpha	  and	  beta	  tubulin	  subunits	  (Luduena	  et	  al.	  1977,	  Nagales	  et	  al.	  1998).	  These	  subunits	  form	  heterodimers	  in	  the	  cytoplasm	  and	  readily	  self-­‐assemble	  into	  microtubule	  structures	  25nm	  in	  diameter	  (Ledbetter	  et	  al.	  1963,	  Hardham	  et	  al.	  1978)	  consisting	  of	  generally	  13	  protofilaments	  (Tilney	  et	  al.	  1973,	  Chrétien	  et	  al.	  1992,	  Job	  et	  al.	  2003).	  The	  cell	  is	  able	  to	  manipulate	  the	  ratio	  of	  monomer	  to	  polymer	  in	  the	  cytoplasm	  via	  the	  critical	  concentration;	  the	  concentration	  at	  which	  polymer	  begins	  to	  disassemble	  into	  dimers	  (Karr	  et	  al	  1979).	  In	  doing	  so,	  cellular	  demands	  can	  call	  upon	  the	  action	  of	  extensible	  and	  transient	  polymer	  networks	  for	  processes	  such	  as	  intracellular	  trafficking	  (Chabin-­‐Brion	  et	  al.	  2001),	  cytokinesis	  (Pickett-­‐Heaps	  et	  al.	  1964),	  and	  organelle	  movement	  and	  positioning	  (Dustin	  1964).	    During	  anisotropic	  growth	  regulation	  in	  dark-­‐grown	  hypocotyls,	  microtubules	  mimic	  the	  patterning	  of	  CSC	  movement	  (Laskowski	  et	  al.	  1990)	  and	  microfibrils	  in	  the	  wall	  by	  appearing	  transverse	  to	  the	  growth	  axis	  (Hepler	  et	  al.	  1974,	  Yuan	  et	  al.	  1994).	  CSCs	  have	   10 been	  shown	  via	  live-­‐cell	  imaging	  to	  move	  both	  within	  and	  outside	  of	  microtubule	  domains	  (Paredez	  et	  al.	  2006),	  suggesting	  a	  potentially	  microtubule-­‐independent	  means	  of	  modulating	  the	  functionality	  of	  the	  CSC.	  In	  dark-­‐grown	  cells,	  exposure	  to	  light	  reorients	  the	  microtubule	  network	  to	  a	  more	  longitudinal	  direction	  (Iwata	  et	  al.	  1989).	  Studies	  on	  the	  indirect	  effects	  of	  microtubule	  organization	  (Sedbrook	  et	  al.	  2004)	  and	  polymerization	  (Gardiner	  et	  al.	  2003,	  Debolt	  et	  al.	  2007)	  on	  cellulose	  synthesis	  have	  shown	  conflicting	  results.	  Microtubule	  disorganization	  did	  not	  prevent	  the	  reestablishment	  of	  parallel	  microfibrils	  following	  their	  disruption	  with	  the	  cellulose	  synthesis	  inhibitor	  DCB	  (Himmelspach	  et	  al.	  2003),	  yet	  it	  was	  shown	  that	  CSC	  trajectories	  do	  follow	  the	  disordered	  microtubule	  pattern	  (Fujita	  et	  al.	  2011).	  These	  finding	  suggest	  that	  cellulose	  microfibrils	  can	  be	  reorganized	  after	  their	  initial	  synthesis	  and	  deposition	  by	  CSCs	  and	  that	  there	  is	  more	  to	  be	  determined	  about	  the	  relationship	  of	  cellulose	  synthesis	  with	  microtubules.	    Considering	  that	  microtubules	  form	  polymers	  that	  are	  highly	  dynamic	  structures,	  modulations	  in	  dynamicity,	  polymer	  content,	  and	  organization	  could	  all	  influence	  cellulose	  synthesis	  and	  anisotropy.	  Normally,	  microtubule	  polymers	  are	  able	  to	  grow	  and	  shrink	  from	  both	  ends	  in	  vitro	  (Mitchison	  et	  al.	  1984/1987,	  Hush	  et	  al.	  1994),	  with	  one	  end	  acting	  much	  more	  dynamically.	  Termed	  the	  plus-­‐end,	  it	  defines	  a	  majority	  of	  microtubule	  cellular	  activity,	  whereas,	  in	  vivo,	  the	  other,	  termed	  the	  minus-­‐end,	  has	  reduced	  dynamicity	  due	  to	  often	  being	  embedded	  in	  the	  pericentriolar	  areas	  surrounding	  the	  nucleus	  (Tassin	  et	  al.	  1999,	  Doxsey	  et	  al.	  2005).	  	  In	  order	  to	  be	  both	  dynamic	  and	  stable,	  microtubule	  polymers	  form	  a	  GTP	  cap	  at	  the	  plus	  end	  that	  regulates	  stability	  of	  the	  polymer	  (Howard	  et	  al.	  1986,	  Caplow	  et	  al.	  1996).	  Beta-­‐tubulin	  subunits	  reversibly	  bind	  GTP,	  which	  undergoes	   11 hydrolysis	  after	  being	  bound,	  leading	  to	  a	  less	  stable	  conformation	  within	  the	  polymer.	  This	  process,	  however,	  is	  often	  outpaced	  by	  subunit	  addition,	  resulting	  in	  the	  formation	  of	  a	  GTP	  cap,	  until	  the	  critical	  concentration	  is	  reached;	  a	  situation	  that	  is	  defined	  by	  slow	  subunit	  addition	  and	  the	  failure	  to	  maintain	  the	  dwindling	  cap.	  As	  monomer	  concentration	  decreases	  and	  tubulin	  dimers	  are	  less	  readily	  added	  to	  the	  plus	  end,	  the	  cap	  is	  lost,	  thus	  exposing	  the	  remaining	  GDP-­‐bound	  subunits	  within	  the	  polymer.	  This	  form	  is	  far	  less	  stable	  and	  the	  microtubules	  therefor	  begin	  to	  depolymerize,	  which	  releases	  tubulin	  subunits.	  This	  delicate	  balance	  of	  polymer	  to	  monomer,	  known	  as	  the	  critical	  concentration,	  is	  a	  defining	  characteristic	  of	  microtubule	  dynamics	  and	  associations	  with	  other	  proteins.  The	  critical	  concentration	  confers	  the	  plus	  end	  with	  the	  ability	  to	  rapidly	  respond	  and	  to	  maintain	  interactions	  with	  a	  number	  of	  cellular	  constituents,	  including	  microtubule-­‐associated	  proteins	  (MAPs).	  These	  proteins	  can	  be	  grouped	  based	  on	  how	  they	  interact	  with	  microtubules.	  They	  are	  often	  categorized	  by	  their	  ability	  to	  provide	  stability	  or	  to	  cross-­‐link	  microtubules	  (Burk	  et	  al.	  2001,	  Timauer	  et	  al.	  2000),	  as	  well	  as	  by	  their	  specific	  distribution	  on	  microtubules	  and	  the	  direction,	  if	  any,	  they	  move.	  Many	  MAPs	  are	  considered	  microtubule	  plus-­‐end	  tracking	  proteins	  (+TIPS),	  such	  as	  EB1	  (Tirnauer	  et	  al.	  2001),	  because	  they	  associate	  predominantly	  with	  the	  plus	  end,	  performing	  their	  functions	  under	  highly	  dynamic	  conditions.	  Several	  MAPs	  and/or	  associated	  subunits	  are	  explored	  in	  this	  thesis	  including	  MOR1	  (below),	  CLASP	  (Ambrose	  et	  al.	  2007),	  BOTERO1	  (Bichet	  et	  al.	  2001),	  &	  RIC1	  (Fu	  et	  al.	  2005)	  [Chapter	  3],	  several	  of	  which	  are	  considered	  plus-­‐end	   12 tracking	  and	  have	  roles	  in	  microtubule	  stability,	  dynamics,	  orientation,	  and	  polymerization	  and	  have	  mutational	  defects	  in	  anisotropy.	    MOR1,	  MICROTUBULE	  ORGANIZATION	  1,	  is	  a	  microtubule-­‐associated	  protein	  that	  has	  been	  shown	  to	  impact	  cellular	  processes	  including	  cell	  division	  (Twell	  et	  al.	  2002)	  and	  elongation	  (Fujita	  et	  al.	  2011)	  due	  to	  its	  role	  in	  microtubule	  organization.	  By	  screening	  EMS	  mutagenized	  seedlings	  for	  disrupted	  microtubule	  patterns	  with	  immunofluorescence	  microscopy,	  the	  mutant	  allele	  mor1-­‐1	  was	  isolated	  (Whittington	  et	  al.	  2001).	  It	  was	  found	  that	  the	  gene	  of	  MOR1,	  which	  contains	  53	  exons,	  encodes	  a	  217	  kD	  protein.	  The	  mutation	  in	  mor1-­‐1	  was	  mapped	  to	  a	  HEAT	  repeat	  5	  located	  within	  the	  TOG	  domain	  located	  closest	  to	  the	  N-­‐terminus.	  HEAT	  repeats	  were	  postulated	  and	  confirmed	  as	  the	  sites	  for	  microtubule	  interaction	  by	  solely	  expressing	  the	  two	  N-­‐terminal	  TOG	  domains,	  which	  contain	  the	  HEAT	  repeats	  and	  are	  named	  after	  the	  human	  ortholog,	  tumour	  overexpressed	  gene	  (TOG)	  (Charrasse	  et	  al.	  1998).	  This	  mutation	  is	  temperature	  sensitive,	  indicating	  that	  its	  phenotype	  is	  only	  manifested	  conditionally	  at	  elevated	  temperatures	  (29°C)	  and	  therefore	  provides	  an	  inducible	  system	  with	  which	  to	  assess	  its	  disruption	  of	  anisotropy.  At	  29°C,	  microtubules	  in	  the	  mor1-­‐1	  mutant	  fail	  to	  properly	  organize	  as	  a	  consequence	  of	  defects	  in	  polymerization,	  as	  well	  as	  maintain	  the	  dynamicity	  of	  the	  polymers.	  Specifically	  the	  microtubules	  in	  mor1-­‐1	  grow	  and	  shrink	  more	  slowly,	  and	  spend	  more	  time	  in	  the	  state	  of	  pause,	  which	  is	  when	  microtubules	  have	  no	  net	  growth	  or	  shrinkage	  (Kawamura	  &	  Wasteneys	  et	  al.	  2008).	  This	  may	  interfere	  with	  the	  functionality	  of	  other	  MAPs	  and	  other	  implicated	  processes,	  such	  as	  cellulose	  synthesis.	  Due	  to	  the	  reduced	  microtubule	   13 dynamics,	  polymer	  content,	  and	  organization,	  cell	  shape	  is	  more	  isotropic	  in	  mor1-­‐1	  compared	  to	  wild	  type,	  indicating	  a	  relationship	  between	  the	  microtubule	  disruption	  found	  in	  mor1-­‐1	  and	  anisotropy.	  Further	  investigations	  (Fujita	  et	  al.	  2011)	  identified	  changes	  in	  CSC	  velocity	  and	  wall	  crystallinity	  in	  mor1-­‐1	  as	  likely	  contributors	  to	  altered	  cellular	  elongation.	  Proteins	  such	  as	  CELLULOSE	  SYNTHASE	  INTERACTING1/POM2	  (Li	  et	  al.	  2012)	  and	  the	  recently	  discovered	  cellulose	  synthase	  companion	  proteins	  (CC;	  Endler	  et	  al.	  2016)	  have	  been	  associated	  with	  microtubules	  as	  well	  as	  the	  CSC	  and	  there	  is	  supporting	  evidence	  that	  microtubules	  determine	  some	  aspect	  of	  positioning	  (Gutierrez	  et	  al.	  2009)	  and/or	  modification	  of	  the	  orientation,	  crystallinity,	  or	  abundance	  of	  cellulose	  synthesis,	  with	  proteins	  such	  as	  POM2	  and	  MOR1	  being	  involved.	  It	  has	  also	  been	  shown	  that	  the	  disruption	  of	  cellulose	  synthesis	  can	  alter	  microtubule	  organization	  (Himmelspach	  et	  al.	  2003,	  Liu	  et	  al.	  2016). This	  thesis	  aims	  to	  address	  these	  issues	  and	  those	  raised	  in	  a	  number	  of	  other	  studies	  regarding	  the	  diverse	  relationships	  between	  microtubules	  and	  cellulose	  synthesis.  1.4	  Objectives	  Studies	  on	  mor1-­‐1	  (Fujita	  et	  al.	  2011)	  and	  on	  any1	  (Fujita	  et	  al.	  2013)	  previously	  drew	  conclusions	  about	  the	  relationship	  between	  cellulosic	  crystallinity	  and	  proper	  growth	  anisotropy	  during	  rapid	  elongation	  by	  correlating	  crystallinity	  with	  CSC	  velocity.	  Compared	  to	  wild	  type	  at	  29°C,	  mor1-­‐1	  seedlings	  displayed	  increased	  CSC	  velocity	  and	  wall	  crystallinity	  that	  failed	  to	  reduce,	  while	  any1	  seedlings	  had	  a	  reduction	  in	  both.	  The	  velocity	  data,	  however,	  was	  obtained	  by	  averaging	  CSC	  displacement	  velocities	  measured	  within	  and	  outside	  of	  microtubule	  domains	  and	  therefore	  could	  not	  assess	  the	  direct	  impact	  of	   14 microtubules	  on	  CSC	  velocity.	  This	  makes	  any	  inference	  on	  relationship	  between	  CSC	  velocity	  and	  cellulose	  crystallinity	  difficult.	  Furthermore,	  assessments	  of	  CSC	  coincidence	  with	  subtending	  microtubules	  indicated	  potential	  discrepancies	  between	  CSC	  alignment	  in	  wild	  type	  and	  mor1-­‐1	  cells.	  Coincidence	  analyses	  with	  confocal	  microscopy	  indicated	  a	  possibility	  that	  mor1-­‐1’s	  disruption	  of	  the	  microtubule	  network	  could	  have	  increased	  the	  abundance	  of	  CSCs	  synthesizing	  cellulose	  outside	  of	  microtubule	  domains	  (Fujita	  et	  al.	  2011).	  How	  exactly	  microtubule	  disruption	  is	  affecting	  CSC	  velocity	  and	  microtubule	  alignment,	  however,	  remains	  unclear.  The	  goal	  of	  this	  thesis	  was	  to	  assess	  the	  impact	  of	  CSC	  association	  with	  microtubules	  on	  CSC	  activity	  and	  to	  measure	  CSC	  velocity	  in	  anisotropy-­‐defective	  mutants	  to	  compare	  and	  contrast	  with	  previous	  studies’	  assertions.	  Specifically,	  in	  wild	  type,	  anisotropic	  expansion	  is	  associated	  with	  a	  decrease	  in	  crystallinity	  and	  a	  corresponding	  increase	  in	  CSC	  velocity	  while	  in	  loss-­‐of-­‐anisotropy	  mutants	  mor1-­‐1	  and	  any1,	  there	  is	  a	  direct	  positive	  correlation	  between	  changes	  in	  crystallinity	  and	  CSC	  velocity	  (Fig.	  1.1).	  The	  proposed	  experiments	  are	  expected	  to	  test	  whether	  these	  correlations	  also	  occur	  in	  other	  loss-­‐of-­‐function	  (LOF)	  anisotropy	  mutants.	  Since	  crystallinity	  is	  potentially	  a	  defining	  characteristic	  of	  growth	  anisotropy,	  understanding	  the	  relationship	  between	  it	  and	  CSC	  velocity	  in	  a	  variety	  of	  genotypes	  will	  help	  to	  elucidate	  or	  refute	  this	  concept.	  To	  achieve	  these	  goals,	  live-­‐cell	  imaging	  using	  near-­‐TIRF	  (Total	  Internal	  Reflection	  Fluorescence)	  microscopy	  was	  used	  to	  visualize	  CSC	  movement.	  	  This	  technique	  was	  selected	  because	  it	  could	  follow	  CSC	  activity	  with	  improved	  resolution	  compared	  to	  previous	  confocal	  techniques,	  allowing	  CSCs	  in	   15 microtubule	  domains	  to	  be	  accurately	  distinguished	  from	  CSCs	  outside	  microtubule	  domains,	  as	  well	  as	  allowing	  for	  a	  more	  precise	  analysis	  of	  YFP-­‐CESA6	  kymographs.	  	  	  Figure	  1.1|	  Summary	  of	  previous	  findings	  concerning	  CSC	  velocity,	  crystallinity,	  degree	  of	  anisotropy,	  and	  microtubule	  phenotypes	  at	  29°C.	  Wild	  type	  and	  mor1-­‐1	  velocity	  &	  crystallinity	  data,	  and	  bot1	  and	  RIC1-­‐OX	  crystallinity	  data	  from	  Fujita	  et	  al	  (2011).	  any1	  data	  from	  Fujita	  et	  al.	  (2013),	  clasp-­‐1	  crystallinity	  data	  from	  Fujita	  (unpublished).	  	  Project	  one	  (chapter	  2)	  compares	  CSC	  movement	  within	  and	  outside	  microtubule	  domains	  in	  wild	  type,	  mor1-­‐1	  (in	  which	  cellulose	  crystallinity	  fails	  to	  be	  reduced;	  Fujita	  et	  al.	  2011)	  and	  in	  wild	  type	  after	  treatment	  with	  the	  microtubule-­‐destabilizing	  drug	  Oryzalin	  due	  to	  the	  previous	  findings	  in	  Fujita	  et	  al	  (2011)	  indicating	  an	  increase	  in	  CSCs	  moving	  without	  microtubule	  domain	  association.	  The	  intention	  of	  this	  project	  is	  to	  discern	  roles	  for	  microtubule	  association	  and	  MOR1	  in	  determining	  CSC	  velocity.	  Project	  two	  (chapter	  3)	  further	  explores	  the	  relationship	  between	  microtubule	  dynamics	  and	  organization,	  crystallinity,	  and	  CSC	  velocity	  by	  investigating	  combinations	  of	  the	  any1	  mutant	  with	  mor1-­‐ 16 1	  and	  oryzalin	  to	  assess	  the	  opposing	  relationships	  observed	  in	  CSC	  velocity	  and	  crystallinity	  of	  the	  two	  mutants	  (Fujita	  et	  al.	  2013)	  [Fig	  1.1].	  Continuing	  the	  focus	  of	  associated	  protein	  influence	  on	  CSC	  velocity,	  I	  investigated	  microtubule-­‐associated	  protein	  functionality	  and/or	  disrupted	  microtubules	  in	  three	  genotypes	  from	  which	  wall	  crystallinity	  data	  had	  been	  previously	  determined	  (Fujita	  et	  al	  2011,	  Fujita	  unpublished	  data);	  clasp-­‐1	  (Ambrose	  et	  al.	  2007),	  bot1	  (Bichet	  et	  al.	  2001),	  and	  RIC1-­‐OX	  (Fu	  et	  al.	  2005)[Fig	  1.1].	  This	  provides	  a	  further	  opportunity	  to	  explore	  the	  diverse	  relationships	  between	  changes	  in	  microtubule	  dynamics	  and	  CSC	  velocity	  and	  comprises	  the	  rest	  of	  the	  results	  found	  in	  Chapter	  3.	  	  	   	   17 Chapter	  2:	  Microtubule	  Domain-­‐Specific	  Effects	  on	  Cellulose	  Synthase	  Complex	  Activity  2.1	  Background: Due	  to	  its	  impact	  on	  cell	  elongation,	  I	  investigated	  the	  means	  by	  which	  microtubule	  perturbation	  in	  mor1-­‐1	  affected	  CSC	  velocity	  by	  microtubule	  association	  compared	  to	  wild	  type.	  It	  is	  known	  that	  microtubules	  often	  parallel	  or	  mirror	  cellulose	  synthesis	  (Paredez	  et	  al.	  2006)	  and	  are	  likely	  involved	  in	  the	  transport	  and	  recycling	  of	  CSCs	  (Gutierrez	  et	  al.	  2009,	  Crowell	  et	  al.	  2009).	  Fluorescent	  reporters	  of	  CESA6	  and	  TUB6	  allowed	  for	  visualization	  of	  both	  CSCs	  and	  microtubules	  in	  wild	  type	  and	  mor1-­‐1	  (Fujita	  et	  al.	  2011).	  CSC	  velocity	  was	  shown	  to	  increase	  in	  mor1-­‐1	  relative	  to	  the	  wild-­‐type	  control	  at	  29°C,	  correlating	  microtubule	  dynamics	  with	  activity	  of	  the	  CSC.	  These	  values	  were	  taken	  as	  an	  average	  of	  velocities	  of	  complexes	  moving	  both	  within	  and	  outside	  of	  microtubule	  domains	  and,	  as	  such,	  only	  gave	  a	  general	  idea	  of	  CSC	  activity	  under	  the	  disrupted	  microtubule	  dynamics	  characteristic	  of	  the	  mor1-­‐1	  mutant.  Two	  other	  pieces	  of	  evidence	  provided	  greater	  insight	  into	  how	  microtubule	  polymerization	  defects	  of	  the	  mor1-­‐1	  mutant	  affect	  cellulose	  synthesis.	  It	  was	  determined,	  through	  X-­‐ray	  diffraction	  measurements,	  that	  cell	  wall	  crystallinity	  failed	  to	  decrease	  and	  that	  CSCs	  tended	  to	  be	  less	  coincident	  with	  microtubules	  in	  mor1-­‐1	  mutants	  at	  restrictive	  temperature	  (Fujita	  et	  al.	  2011).	  These	  findings	  were	  the	  basis	  for	  the	  projects	  that	  follow	  because	  they	  suggested	  that	  the	  disruption	  of	  microtubules	  could	  impact	  cellulose	  structural	  integrity	  and	  thereby	  affect	  the	  ability	  of	  cellulose	  microfibrils	  to	  control	  anisotropic	  wall	  expansion	  (Wasteneys	  2004;	  Wasteneys	  &	  Fujita	  2006).	  Whether	  CSC	   18 velocity	  and	  crystallinity	  are	  tied	  to	  the	  plasma	  membrane	  domains	  associated	  with	  microtubules,	  microtubule-­‐associated	  protein	  functionality,	  or	  both	  is	  the	  main	  goal	  of	  this	  project.	    Due	  to	  its	  impact	  on	  anisotropy	  and	  similar	  appearance	  of	  microtubule	  disruption,	  oryzalin	  has	  been	  considered	  an	  appropriate	  drug	  for	  comparison	  with	  the	  mor1-­‐1	  mutation,	  allowing	  further	  investigation	  into	  the	  roles	  of	  microtubule	  domains.	  Previous	  research	  has	  been	  inconclusive	  on	  the	  effect	  of	  oryzalin.	  It	  has	  been	  shown	  that	  CSC	  bidirectionality	  (Chen	  et	  al.	  2010)	  and	  trajectory	  orientation	  (Li	  et	  al.	  2012)	  can	  be	  affected,	  but	  results	  on	  CSC	  velocity	  (DeBolt	  2007a,	  Li	  et	  al.	  2012)	  are	  unclear	  and	  effects	  on	  crystallinity	  are	  untested.	  At	  low	  concentrations,	  microtubules	  in	  oryzalin-­‐treated	  seedlings	  appear	  similar	  to	  those	  seen	  in	  mor1-­‐1;	  partially	  depolymerized	  and	  mostly	  disorganized.	  The	  expectation	  from	  this	  could	  be	  a	  mimicking	  of	  the	  increased	  mor1-­‐1	  velocity	  profile,	  but	  the	  means	  by	  which	  the	  polymers	  are	  being	  disrupted	  in	  each	  situation	  are	  quite	  different.	    Unlike	  mor1-­‐1,	  which	  disrupts	  microtubules	  by	  functioning	  less	  effectively	  at	  regulating	  microtubule	  dynamics	  than	  its	  wild-­‐type	  allele,	  oryzalin	  binds	  M	  loops	  in	  the	  tubulin	  subunits	  which	  (Hugdahl	  et	  al.	  1993),	  through	  weakening	  of	  the	  interprotofilament	  bonds,	  is	  thought	  to	  impair	  polymer	  formation	  and	  leads	  to	  depolymerization	  (Morejohn	  et	  al.	  1987).	  Therefore,	  comparing	  the	  effects	  of	  oryzalin	  and	  mor1-­‐1	  can	  determine	  if	  the	  effect	  of	  reduced	  microtubule	  polymer	  mass	  is	  the	  same	  regardless	  of	  the	  mechanism	  by	  which	  the	  disruption	  occurs.	  	  To	  assess	  whether	  oryzalin	  and	  mor1-­‐1	  would	  differ	  in	  their	  effect	  on	  CSC	  velocity,	  I	  first	  compared	  the	  velocities	  and	  domain	  attribution	  in	  oryzalin-­‐treated	   19 samples	  at	  increasing	  oryzalin	  concentration.	  If	  results	  were	  to	  show	  similar	  effects	  on	  CSC	  activity,	  then	  the	  impact	  of	  microtubules	  on	  the	  CSC	  could	  be	  considered	  to	  be	  a	  product	  of	  the	  relationship	  just	  with	  the	  microtubule	  polymer,	  potentially	  via	  a	  singular	  linking	  domain.	  Alternatively,	  CSC	  velocity	  might	  be	  sensitive	  to	  specific	  means	  of	  microtubule	  disruption.	  This	  would	  characterize	  the	  CSC	  as	  a	  much	  more	  dynamically	  interacting	  complex,	  with	  the	  ability	  to	  be	  influenced	  by	  not	  just	  the	  known	  wall-­‐embedded/associated	  proteins	  and	  polysaccharides,	  but	  by	  their	  indirect	  effect	  on	  CSCs	  through	  microtubules	  and	  potentially	  more	  direct	  effects	  by	  MAPs.	  	   	   20 2.2	  Materials	  &	  Methods: 2.2.1	  Plant	  material	  &	  growth	  conditions	   To	  investigate	  the	  role	  of	  microtubule	  domains	  on	  CSC	  activity,	  previously	  generated	  lines	  of	  wild-­‐type	  (Col-­‐O	  ecotype)	  and	  mor1-­‐1	  seedlings	  expressing	  a	  CESA6-­‐YFP	  (Paredez	  et	  al.	  2006;	  Fujita	  et	  al.,	  2011)	  were	  grown	  and	  assessed	  under	  specific	  conditions.	  Both	  lines	  also	  carried	  the	  procuste1	  mutation,	  a	  null	  allele	  of	  the	  gene	  encoding	  CESA6	  (Fagard	  et	  al.	  2000).	  In	  the	  presence	  of	  endogenous	  CESA6,	  the	  CESA-­‐YFP	  is	  expressed	  but	  is	  not	  observed	  at	  the	  plasma	  membrane	  (Fujita	  et	  al	  2011).	    A	  solution	  made	  of	  3%	  hydrogen	  peroxide	  and	  50%	  ethanol	  in	  distilled	  water	  sterilized	  the	  seedlings	  prior	  to	  three	  rinses	  with	  distilled	  water.	  Sterilized	  seeds	  were	  planted	  on	  agar-­‐solidified	  Hoagland’s	  medium	  [90μM	  Fe-­‐EDTA,	  5mM	  Ca(NO3)2,	  1mM	  KH3P04,	  2mM	  KNO3,	  2mM	  	  MgSO4,	  46μM	  H2BO3,	  9.2μM	  MnCl2,	  0.77µM	  ZnSO4,	  0.32uM	  CuSO4,	  0.11μM	  MoO3,	  	  530μM	  myo-­‐Inositol	  (Sigma,	  Canada),	  50μM	  thiamine	  hydrochloride	  (Lancaster	  Synthesis),	  1.2%	  (w/v)	  Bacto	  Agar	  (BD	  Chemical)].	    Approximately	  5	  seeds	  were	  planted	  per	  round	  plate	  (Fisher	  Brand),	  sealed	  with	  microtape	  (Micropore,	  3M,	  USA),	  wrapped	  in	  foil	  to	  simulate	  etiolation,	  and	  vertically	  placed	  in	  wooden	  racks.	  After	  four	  days	  at	  4°C,	  plates	  were	  moved	  to	  21°C	  under	  continuous	  light	  (80-­‐100	  μmol.m-­‐2.s-­‐1)	  for	  two	  days,	  followed	  by	  one	  day	  at	  29°C.	  	  Seedlings	  were	  confirmed	  as	  wild	  type	  or	  mor1-­‐1	  based	  on	  their	  growth	  phenotypes	  at	  29°C.	  	  Seedlings	  remained	  under	  etiolated	  conditions	  throughout	  growth	  and	  imaging.   21 2.2.2	  Mutant	  backgrounds	  &	  reporter	  constructs In	  order	  to	  observe	  cellulose	  synthesis	  activity	  in	  vivo,	  reporter	  constructs	  in	  mutant	  lines	  with	  appropriate	  genetic	  backgrounds	  allowed	  for	  visualization	  of	  both	  the	  CSC	  and	  microtubules.	  To	  observe	  CesA6	  trajectories	  and	  velocities	  in	  etiolated	  hypocotyls,	  wild-­‐type	  and	  mor1-­‐1	  lines	  were	  previously	  crossed	  with	  prc1,	  complemented	  by	  YFP-­‐CESA6,	  and	  segregated	  out	  for	  these	  analyses.	  During	  assessment	  of	  microtubule	  domains,	  RFP-­‐TUB6	  visualized	  along	  the	  entire	  length	  of	  microtubules	  in	  both	  genotypes.	  Assessment	  of	  successful	  reporter	  constructs	  was	  verified	  by	  the	  presence	  of	  fluorescence	  during	  live	  cell	  imaging	  with	  near-­‐TIRF,	  specifically	  at	  the	  plasma	  membrane	  in	  concentrated,	  linearly	  arranged	  punctae	  for	  YFP-­‐CesA6.	    2.2.3.	  Drug	  treatment	  &	  hormone	  mounting	  solution Prior	  to	  imaging,	  seedlings	  designated	  for	  oryzalin	  treatment	  were	  incubated	  for	  a	  total	  of	  3	  hours	  each.	  The	  solution	  was	  previously	  made	  of	  either	  2µM	  oryzalin	  in	  0.1%	  DMSO	  or	  20µM	  oryzalin	  in	  0.1%	  DMSO	  and	  warmed	  to	  31°C	  prior	  to	  incubation.	  Seedling	  incubations	  and	  imaging	  were	  staggered	  one	  hour	  apart	  to	  guarantee	  equal	  exposure	  to	  oryzalin	  treatment.	  The	  effectiveness	  of	  the	  oryzalin	  treatment	  in	  disrupting	  microtubule	  polymers	  and	  organization	  was	  assessed	  during	  imaging.	    To	  preserve	  rapid	  growth	  conditions,	  a	  hormone	  solution	  was	  prepared	  as	  described	  in	  Vineyard	  et	  al.	  (2013)	  containing	  10μM	  gibberellic	  acid	  (GA4)	  and	  0.5μM	  indole	  acetic	  acid	  in	  distilled	  water.	  Stored	  at	  4°C	  prior	  to	  mounting	  the	  sample,	  the	  solution	  was	  heated	  to	  29°C	  and	  30μl	  was	  aliquoted	  to	  the	  centre	  of	  the	  imaging	  dish	  with	  the	  coverslip	  at	  the	   22 bottom	  (MatTek	  Co.	  USA)	  for	  each	  seedling.	  Seedlings	  were	  allowed	  approximately	  10	  minutes	  to	  acclimate	  before	  imaging	  began.  2.2.4	  Live-­‐cell	  imaging	   Assessing	  microtubule	  domain	  influence	  on	  CSC	  activity	  required	  simultaneous	  time-­‐lapse	  imaging	  of	  both	  YFP-­‐CesA6	  and	  RFP-­‐TUB6	  in	  both	  wild	  type	  and	  mor1-­‐1.	  	  For	  near-­‐TIRF	  analysis,	  our	  system	  consisted	  of	  a	  Zeiss	  Observer.Z1	  microscope,	  TIRF	  3.1,	  equipped	  with	  a	  Q	  Imaging	  Rolera	  EMCCD	  camera	  and	  a	  63X/1.46	  Oil	  Korr	  TIRF	  objective	  lens,	  controlled	  by	  Zen	  software	  (Zeiss).	  A	  488	  nm	  excitation	  laser	  and	  525/50	  nm	  emission	  filter	  were	  used	  for	  YFP	  imaging.	  	  A	  561	  nm	  excitation	  laser	  and	  601/25	  nm	  emission	  filter	  was	  used	  for	  RFP	  imaging.	  	  For	  appropriate	  imaging,	  the	  temperature	  of	  both	  the	  objective	  and	  stage	  was	  maintained	  at	  29°C.  Seedlings	  were	  mounted	  one	  at	  a	  time	  in	  the	  dark,	  into	  the	  mounting	  solution	  described	  above.	  Plates	  with	  the	  remainder	  of	  seedlings	  were	  stored	  in	  temperature	  controlled	  boxes	  until	  mounted	  into	  the	  microscope	  to	  maintain	  both	  light	  and	  temperature	  controls.	  Hoagland	  media	  plates	  were	  heated	  to	  29°C	  prior	  to	  imaging	  and	  dissected	  into	  rectangular	  blocks.	  In	  order	  to	  observe	  areas	  closest	  to	  the	  coverslip,	  vertical	  pressure	  was	  applied	  to	  the	  seedling	  from	  above.	  This	  was	  accomplished	  by	  stacking	  several	  agar	  blocks	  on	  top	  of	  the	  mounted	  seedling	  without	  exceeding	  the	  microscope	  stage	  dimensions.	  During	  the	  time	  of	  imaging,	  other	  seedlings	  were	  maintained	  in	  darkness	  at	  a	  minimum	  of	  29°C.	     23 Once	  placed	  onto	  the	  stage,	  561	  nm	  light	  was	  used	  to	  locate	  the	  seedling	  without	  adding	  excess	  reorienting	  light	  stimuli.	  Imaging	  with	  near-­‐TIRF	  requires	  changes	  in	  the	  incident	  angle	  of	  applied	  light;	  488	  nm	  light	  for	  YFP-­‐CesA6	  and	  561	  nm	  light	  for	  RFP-­‐TUB6.	  The	  angle	  required	  for	  imaging	  CSCs	  at	  the	  plasma	  membrane	  and	  the	  angle	  required	  for	  visualizing	  cortical	  microtubules	  should	  be	  within	  2-­‐6	  degrees	  of	  one	  another.	  Gain	  value	  for	  the	  EMCCD	  camera	  was	  set	  at	  2800	  with	  a	  laser	  strength	  of	  approximately	  32%.	  Angles	  ranged	  from	  65	  to	  85	  degrees	  from	  either	  side.	    Five-­‐minute	  time-­‐lapse	  videos	  of	  YFP-­‐CesA6	  and	  RFP-­‐TUB6	  in	  both	  wild	  type	  and	  mor1-­‐1	  were	  recorded	  in	  10-­‐second	  increments	  closest	  to	  the	  elongation	  zone	  of	  the	  hypocotyl.	  Data	  files	  were	  saved	  and	  stored	  as	  TIF	  files.	   2.2.5	  CSC	  velocity	  analysis Images	  were	  processed	  using	  Image	  J	  (Abramoff	  et	  al.	  2004).	  They	  were	  first	  converted	  to	  8-­‐Bit	  RGB	  and	  then	  split	  into	  their	  respective	  fluorescence	  channels	  to	  be	  edited	  separately.	  Images	  were	  brightness	  and	  contrast	  optimized,	  cropped,	  and	  aligned	  before	  re-­‐merging	  and	  color	  correcting.	  Images	  were	  separated	  once	  more	  into	  separate	  channels	  in	  order	  to	  produce	  single	  image	  projections	  of	  the	  videos	  for	  visualizing	  trajectories	  and	  linear	  movement.	    Average	  projections	  of	  both	  CesA	  trajectories	  and	  microtubules	  were	  produced.	  To	  assess	  microtubule	  association,	  linear	  trajectories	  were	  traced	  from	  the	  CesA	  image,	  superimposed	  onto	  the	  microtubule	  projection,	  and	  then	  used	  to	  obtain	  a	  kymograph	  of	  the	   24 particle	  trajectory	  using	  the	  Image	  J	  multikymograph	  plugin.	  From	  the	  kymograph,	  linear	  trajectories	  were	  traced	  and,	  from	  it,	  slopes	  were	  determined	  via	  additional	  macros	  that	  source	  data	  from	  the	  tsp	  (time	  series	  projection).	  From	  the	  kymograph	  slope,	  a	  value	  was	  obtained	  to	  convert	  to	  physical	  particle	  velocity.	  One-­‐way	  ANOVA	  followed	  by	  Tukey’s	  range	  test	  was	  performed	  using	  GraphPad	  Prism,	  version	  7.0a	  for	  Macintosh,	  GraphPad	  Software,	  La	  Jolla	  California	  USA.	  	  	  2.2.6	  CSC	  and	  microtubule	  coincidence	  analysis	  Sections	  of	  YFP-­‐CESA6	  and	  RFP-­‐TUB6	  projections	  with	  distinct	  patterning	  visible	  in	  both	  images	  were	  identically	  cropped.	  If	  necessary,	  the	  Subtract	  Background	  command	  can	  further	  isolate	  fluorescence	  from	  non-­‐signal.	  Using	  the	  threshold	  function	  of	  Image	  J,	  the	  signal	  to	  no	  signal	  ratio	  is	  determined	  by	  balancing	  the	  overlay	  with	  the	  original	  image’s	  CSC	  trajectory	  or	  potentially	  subtending	  microtubule.	  The	  Image	  Calculator	  function	  then	  can	  merge	  these	  two	  overlays	  and	  assess	  correspondence	  of	  fluorescence	  in	  each	  image	  in	  a	  single	  resulting	  merged	  image.	  Using	  the	  measure	  command,	  the	  percent	  of	  signal	  of	  each	  image	  can	  be	  obtained.	  To	  obtain	  coincidence,	  the	  percent	  of	  signal	  in	  the	  tubulin	  image	  is	  multiplied	  by	  the	  percent	  of	  signal	  in	  the	  CSC	  trajectory	  image	  and	  divided	  by	  the	  CSC	  trajectory	  value.	   	   25 2.3	  Results:  2.3.1	  CSCs	  are	  mostly	  confined	  to	  microtubule	  domains Approximately	  equal	  proportions	  of	  CSCs,	  67.2%	  in	  wild	  type	  and	  66.7%	  in	  mor1-­‐1,	  were	  found	  to	  be	  coincident	  with	  subtending	  microtubule	  tracks	  in	  both	  ecotypes	  despite	  the	  microtubule	  perturbations	  in	  mor1-­‐1	  (Table	  2.1).	  	  By	  extension,	  a	  similar	  proportion	  of	  complexes,	  approximately	  33%,	  travel	  outside	  of	  microtubule	  domains	  in	  both	  wild	  type	  and	  mor1-­‐1.	    Table	  2.1	  Comparison	  of	  mean	  velocities	  of	  CSCs	  and	  percent	  association	  of	  CSCs	  with	  microtubule	  domains	  by	  confocal	  and	  near-­‐TIRF	  microscopy  Previous	  wild	  type	  data1 Previous	  mor1-­‐1	  data2 near-­‐TIRF	  wild	  type	  data near-­‐TIRF	  mor1-­‐1	  data Mean	  CSC	  Velocity	  (nm/min) 354	   410 346	  ±	  121 415	  ±	  118 %	  of	  CSCs	  	  in	  Microtubule	  Domain 62.8  48.2 67.2 66.7  %	  of	  CSCs	  not	  in	  Microtubule	  Domain 37.2 51.8 32.8 33.3 1	  Average	  wild	  type	  velocity	  referenced	  from	  Fujita	  et	  al	  2011. 2	  Average	  mor1-­‐1	  velocity	  referenced	  from	  Fujita	  et	  al	  2011	  	   	   26 2.3.2	  Microtubule	  domains	  restrict	  CSC	  velocity	  in	  wild	  type	  during	  rapid	  cell	  elongation Near-­‐TIRF	  analysis	  performed	  at	  29°C	  determined	  that	  the	  mean	  velocity	  of	  the	  overall	  population	  of	  CSCs	  in	  cells	  from	  the	  hypocotyl	  elongation	  zone	  was	  346	  ±	  121	  nm/min	  (Table	  2.1).	  This	  velocity	  is	  similar	  to	  the	  354	  ±	  73	  nm/min	  mean	  velocity	  reported	  by	  Fujita	  et	  al	  (2011).	  I	  next	  compared	  the	  velocity	  of	  CSCs	  tracking	  within	  and	  outside	  of	  microtubule	  domains.	  In	  wild	  type	  at	  29°C,	  CSCs	  moving	  inside	  of	  microtubule	  domains	  travel	  at	  a	  mean	  velocity	  of	  317	  ±	  110	  nm/min	  (Fig.	  2.1),	  while	  those	  outside	  of	  microtubule	  domains	  were	  observed	  to	  be	  traveling	  much	  more	  quickly,	  at	  a	  mean	  velocity	  of	  429	  ±	  109	  nm/min.	   	  	   	   27  	  Figure	  2.1|	  CSC	  velocity	  in	  microtubule	  domains	  and	  non-­‐microtubule	  domains. YFP-­‐CESA6	  tagged	  CSC	  trajectories	  were	  visualized	  in	  prc1	  dark-­‐grown	  hypocotyls	  at	  29°C	  as	  projections	  of	  movement	  using	  time-­‐lapse	  image	  sequences	  via	  near-­‐TIRF	  microscopy.	  (A)	  The	  histogram	  displays	  the	  overall	  distribution	  of	  CSC	  velocity	  within	  microtubule	  domains	  (blue)	  and	  in	  microtubule-­‐free	  space	  (red).	  CSCs	  confined	  to	  microtubule	  domains	  displayed	  a	  lower	  average	  velocity,	  317	  ±	  110	  nm/min	  (n	  =	  742	  particles),	  than	  those	  moving	  in	  microtubule-­‐free	  space,	  429	  ±	  112	  nm/min	  (n	  =	  254	  particles).	  Overall	  mean	  CSC	  velocity	  was	  346	  ±	  121	  nm/min,	  n	  =	  996	  particles	  taken	  from	  37	  cells	  across	  15	  seedlings.	  (B)	  The	  CSC	  trajectories	  (left)	  and	  velocities	  were	  analyzed	  in	  relation	  to	  RFP-­‐TUB6	  tagged	  microtubules	  (middle);	  overlap	  is	  clearly	  visible	  in	  the	  merged	  image	  (right).	  (C)	  Kymographs	  were	  produced	  for	  each	  trajectory.	  CSC	  velocity	  was	  calculated	  from	  the	  slope	  of	  kymographs.	     28 2.3.3	  Microtubules	  fail	  to	  properly	  restrict	  CSC	  velocity	  in	  mor1-­‐1 Near-­‐TIRF	  analysis	  found	  comparable	  results	  to	  previous	  data	  sets	  ,	  measuring	  the	  mean	  CSC	  velocity	  in	  mor1-­‐1	  at	  415	  ±	  118	  nm/min	  (Table	  2.1).	  This	  velocity	  is	  significantly	  elevated	  compared	  to	  wild-­‐type	  velocity	  levels.	  Comparison	  of	  CSC	  velocities	  within	  and	  outside	  microtubule	  domains	  revealed	  that	  the	  mor1-­‐1	  mutation	  had	  its	  greatest	  effect	  on	  CSCs	  tracking	  within	  microtubule	  domains.	  CSCs	  moving	  outside	  of	  microtubule	  domains	  had	  similar	  mean	  velocities	  in	  mor1-­‐1,	  450	  ±	  135	  nm/min,	  as	  wild	  type,	  429	  nm/min.	  By	  comparison,	  CSC	  mean	  velocities	  within	  microtubule	  domains	  were	  381	  ±	  109	  nm/min	  in	  mor1-­‐1	  compared	  to	  317	  nm/min	  in	  wild	  type,	  representing	  a	  20%	  increase	  (Fig.	  2.3).	  	      29   Figure	  2.2|	  CSC	  velocity	  in	  microtubule	  domains	  and	  non-­‐microtubule	  domains	  in	  mor1-­‐1 YFP-­‐CESA6	  tagged	  CSC	  trajectories	  were	  visualized	  in	  mor1-­‐1/prc1	  dark-­‐grown	  hypocotyls	  at	  29°C	  as	  projections	  of	  movement	  using	  time-­‐lapse	  image	  sequences	  via	  near-­‐TIRF	  microscopy. (A)	  The	  histogram	  displays	  the	  overall	  distribution	  of	  CSC	  velocity	  within	  microtubule	  domains	  (blue)	  and	  in	  microtubule-­‐free	  space	  (red).	  CSCs	  confined	  to	  microtubule	  domains	  displayed	  a	  lower	  average	  velocity,	  381	  ±	  109	  nm/min	  (n	  =	  781	  particles),	  than	  those	  moving	  in	  microtubule	  free	  space,	  450	  ±	  135	  nm/min	  (n	  =	  215	  particles).	  Overall	  mean	  CSC	  velocity	  was	  415	  ±	  121	  nm/min,	  n	  =	  996	  particles	  taken	  from	  48	  cells	  across	  26	  seedlings.	  (B)	  The	  CSC	  trajectories	  (left)	  and	  velocities	  were	  analyzed	  in	  relation	  to	  RFP-­‐TUB6-­‐tagged	  microtubules	  (middle);	  overlap	  is	  clearly	  visible	  in	  the	  merged	  image	  (right).	  (C)	  Kymographs	  were	  produced	  for	  each	  trajectory.	  CSC	  velocity	  was	  calculated	  from	  the	  slope	  of	  kymographs.	  	   30 	   Figure	  2.3|	  Comparison	  of	  CSC	  velocity	  in	  microtubule	  (MT)	  domains	  and	  non-­‐microtubule	  domains	  in	  wild	  type	  (WT)	  and	  mor1-­‐1	  by	  mean	  CSC	  velocity	  and	  frequency	  (%).	  	  	  (a)	  At	  29°C,	  CSCs	  travel	  at	  a	  mean	  velocity	  comparable	  to	  wild	  type	  in	  mor1-­‐1	  when	  outside	  of	  microtubule	  domains.	  A	  20%	  increase	  in	  CSC	  velocity	  is	  observed	  in	  mor1-­‐1	  seedlings	  (381	  ±	  109	  nm/min),	  relative	  to	  wild	  type	  controls	  (317	  +	  110	  nm/min)	  when	  aligned	  with	  microtubules.	  Asterisk	  indicates	  a	  significant	  value	  determined	  with	  one	  way	  ANOVA	  test;	  p-­‐value	  <	  0.0001.	  	  (B)	  Wild	  type	  Distribution	  of	  CSCs	  inside	  and	  outside	  of	  microtubule	  domains	  by	  frequency	  (%).	  (C)	  mor1-­‐1	  Distribution	  of	  CSCs	  inside	  and	  outside	  of	  microtubule	  domains	  by	  frequency	  (%).	  Asterisk	  indicates	  a	  significant	  difference	  determined	  with	  one	  way	  ANOVA	  test;	  p-­‐value	  <	  0.0001.	  	  Post-­‐hoc	  comparisons	  between	  assessments	  were	  made	  using	  the	  Tukey	  range	  test,	  p-­‐values	  <	  .0001.	  	   	   31 2.3.4	  Oryzalin	  disruption	  of	  cortical	  microtubule	  network	  does	  not	  impact	  mean	  CSC	  velocity	   Table	  2.2	  Comparison	  of	  mean	  velocities	  of	  CSCs	  and	  percent	  association	  of	  CSCs	  with	  microtubule	  domains	  by	  confocal	  and	  near-­‐TIRF	  microscopy	  in	  oryzalin	  treated	  seedlings  Wild	  Type	  Data Wild	  Type	  with	  2µM	  Oryzalin Wild	  Type	  with	  20µM	  	  Oryzalin Mean	  CSC	  Velocity	  (nm/min) 345	  ±	  121 365	  ±	  137 343	  ±	  122 %	  of	  CSCs	  in	  Microtubule	  Domain 67.2 52.1 34.9  %	  of	  CSCs	  not	  in	  Microtubule	  Domain 32.8  47.9 65.1   Oryzalin	  was	  applied	  at	  two	  concentrations,	  2	  and	  20μM.	  The	  2μM	  concentration	  was	  used	  to	  simulate	  the	  mor1-­‐1	  phenotype;	  microtubules	  were	  only	  partially	  depolymerized,	  became	  disorganized	  over	  time,	  and	  growth	  anisotropy	  was	  inhibited.	  Wild-­‐type	  CSC	  velocity	  in	  microtubule	  domains	  without	  oryzalin	  incubation	  was	  observed	  to	  be	  317	  nm/min	  (Fig.	  2.1).	  Seedlings	  incubated	  in	  2μM	  oryzalin	  did	  not	  differ	  from	  the	  control	  within	  microtubule	  domains	  (Fig.	  2.4),	  with	  a	  mean	  velocity	  within	  microtubule	  domains	  of	  329	  ±	  123	  nm/min	  (Fig.	  2.6).	  The	  proportion	  of	  CSCs	  outside	  microtubule	  domains	  was	  increased	  to	  47.9%	  of	  all	  CSCs	  and	  the	  velocity	  of	  CSCs	  outside	  of	  domains	  was	  also	  elevated	  compared	  to	  CSCs	  within	  domains	  at	  468	  ±	  123	  nm/min.	  Thus,	  at	  oryzalin	  concentrations	  that	  affect	  microtubule	  arrays	  to	  a	  similar	  extent	  as	  the	  mor1-­‐1	  mutant,	  there	  was	  no	  detectable	  change	  in	  CSC	  velocities	  either	  within	  or	  outside	  microtubule	  domains,	  but	  there	  was	  a	  displacement	  of	  CSCs	  from	  their	  microtubule	  association.	   32 	   	  Figure	  2.4|	  CSC	  velocity	  in	  microtubule	  domains	  and	  non-­‐microtubule	  domains	  in	  2	  μM	  oryzalin	  YFP-­‐CESA6	  tagged	  CSC	  trajectories	  were	  visualized	  in	  prc1	  dark-­‐grown	  hypocotyls	  treated	  for	  3	  hours	  prior	  to	  imaging	  and	  during	  imaging	  with	  a	  2μM	  concentration	  of	  oryzalin	  at	  29°C.	  They	  were	  observed	  as	  projections	  of	  movement	  using	  time-­‐lapse	  image	  sequences	  via	  near-­‐TIRF	  microscopy.(A)	  The	  histogram	  displays	  the	  overall	  distribution	  of	  CSC	  velocity	  within	  microtubule	  domains	  (blue)	  and	  in	  microtubule-­‐free	  space	  (red).	  CSCs	  confined	  to	  microtubule	  domains	  displayed	  a	  lower	  average	  velocity,	  329	  ±	  123	  nm/min	  (n	  =	  371	  particles),	  than	  those	  moving	  in	  microtubule-­‐free	  space,	  450	  ±	  135	  nm/min	  (n	  =	  127	  particles).	  Overall	  mean	  CSC	  velocity	  was	   365	  ±	  137	  nm/min,	  n	  =	  498	  particles	  taken	  from	  17	  cells	  across	  4	  seedlings.	  (B)	  The	  CSC	  trajectories	  (left)	  and	  velocities	  were	  analyzed	  in	  relation	  to	  RFP-­‐TUB6	  tagged	  microtubules	  (middle);	  overlap	  is	  clearly	  visible	  in	  the	  merged	  image	  (right).	    33  At	  the	  higher	  20μM	  concentration	  (Fig.	  2.5),	  microtubules	  showed	  extensive	  depolymerization	  but	  not	  all	  cells	  exhibited	  full	  microtubule	  depolymerization.	  Similar	  to	  2μM	  oryzalin-­‐treated	  lines,	  velocities	  within	  microtubule	  domains	  remained	  low	  at	  314	  ±	  98	  nm/min	  (Fig.	  2.6).	  In	  contrast	  to	  the	  2μM	  oryzalin	  treatment,	  20μM	  oryzalin	  caused	  a	  reduction	  in	  CSC	  velocity	  outside	  of	  microtubule	  domains	  (365	  ±	  132	  nm/min),	  correlated	  with	  27.2%	  increase	  in	  the	  proportion	  of	  CSCs	  tracking	  outside	  of	  microtubules	  (Table	  2.2).	  Trajectories	  of	  CSCs	  also	  appeared	  to	  have	  a	  wider	  variety	  of	  orientations,	  and	  were	  shorter	  in	  overall	  length	  compared	  to	  the	  2μM-­‐treated	  seedlings.	  	  	  	  	   	   34  	  Figure	  2.5|	  CSC	  velocity	  in	  microtubule	  domains	  and	  non-­‐microtubule	  domains	  in	  20μM	  oryzalin YFP-­‐CESA6	  tagged	  CSC	  trajectories	  were	  visualized	  in	  	  prc1	  dark-­‐grown	  hypocotyls	  treated	  for	  3	  hours	  prior	  to	  imaging	  and	  during	  imaging	  with	  a	  20μM	  concentration	  of	  oryzalin	  at	  29°C.	  They	  were	  observed	  as	  projections	  of	  movement	  using	  time-­‐lapse	  image	  sequences	  via	  near-­‐TIRF	  microscopy.	  (A)	  The	  histogram	  displays	  the	  overall	  distribution	  of	  CSC	  velocity	  within	  microtubule	  domains	  (blue)	  and	  in	  microtubule-­‐free	  space	  (red).	  CSCs	  confined	  to	  microtubule	  domains	  displayed	  a	  lower	  average	  velocity,	  314	  ±	  98	  nm/min	  (n	  =	  206	  particles),	  than	  those	  moving	  in	  microtubule	  free	  space,	  365	  ±	  132	  nm/min	  (n	  =	  289	  particles).	  	  Overall	  mean	  CSC	  velocity	  was	  365	  ±	  137	  nm/min,	  n	  =	  495	  particles	  taken	  from	  19	  cells	  across	  7	  seedlings.	  (B)	  The	  CSC	  trajectories	  (left)	  and	  velocities	  were	  analyzed	  in	  relation	  to	  RFP-­‐TUB6	  tagged	  microtubules	  (middle);	  overlap	  is	  clearly	  visible	  in	  the	  merged	  image	  (right).	  (C)	  Kymographs	  were	  produced	  for	  each	  trajectory.	  CSC	  velocity	  was	  calculated	  from	  the	  slope	  of	  kymographs.	  	   35  Figure	  2.6|	  Comparison	  of	  CSC	  velocity	  in	  microtubule	  domains	  and	  non-­‐microtubule	  domains	  in	  untreated,	  2μM	  oryzalin,	  and	  20μM	  treated	  seedlings	  mean	  CSC	  velocity	  and	  frequency	  (%).	  	  (a)	  At	  29°C	  in	  2μM	  oryzalin	  solutions,	  CSCs	  displaced	  at	  a	  mean	  velocity	  comparable	  to	  wild	  type	  in	  oryzalin-­‐treated	  seedlings	  both	  inside	  and	  outside	  of	  domains.	  At	  20μM	  oryzalin,	  CSC	  velocities	  inside	  domains	  remained	  similar	  to	  those	  in	  untreated	  seedlings,	  but	  a	  15%	  decrease	  (365	  ±	  137	  nm/min)	  from	  CSCs	  velocity	  in	  untreated	  seedlings	  (429	  ±	  112	  nm/min)	  was	  observed	  outside	  of	  microtubule	  domains.	  (B)	  Wild	  type	  +	  2μM	  oryzalin	  distribution	  of	  CSCs	  inside	  and	  outside	  of	  microtubule	  domains	  by	  frequency	  (%).	  (C)	  Wild	  type	  +	  20μM	  	  oryzalin	  distribution	  of	  CSCs	  inside	  and	  outside	  of	  microtubule	  domains	  by	  frequency	  (%).	  Asterisk	  indicates	  a	  significant	  difference	  determined	  with	  one	  way	  ANOVA	  test;	  p-­‐value	  <	  0.0001.	  	  Post-­‐hoc	  comparisons	  between	  assessments	  were	  made	  using	  the	  Tukey	  range	  test,	  p-­‐values	  <	  .0001.	  	   	   36 2.4	  Discussion:  The	  data	  obtained	  in	  the	  Fujita	  et	  al	  (2011)	  study	  suggested	  that	  the	  increased	  average	  velocity	  of	  CSCs	  in	  mor1-­‐1	  at	  restrictive	  temperature	  could	  be	  attributed	  to	  a	  higher	  number	  of	  CSCs	  tracking	  outside	  of	  microtubule	  domains.	  In	  that	  study,	  the	  use	  of	  spinning	  disk	  confocal	  microscopy	  limited	  the	  ability	  to	  distinguish	  CSCs	  within	  and	  outside	  microtubule	  domains.	  Previous	  research	  (Paredez	  et	  al.	  2006)	  has	  shown	  that	  CSCs	  can	  travel	  inside	  and	  outside	  of	  microtubule	  domains,	  making	  previous	  averages	  inconclusive.	  To	  date,	  no	  study	  has	  examined	  specifically	  how	  the	  proximity	  of	  CSCs	  to	  microtubules	  affects	  their	  activity.	  Near-­‐TIRF	  used	  in	  the	  current	  study,	  while	  in	  agreement	  with	  the	  overall	  velocity	  data	  from	  Fujita	  et	  al	  (2011),	  refuted	  the	  coincidence	  analyses	  and	  suggested	  instead	  that	  the	  major	  cause	  of	  increased	  CSC	  velocity	  in	  mor1-­‐1	  is	  the	  faster	  tracking	  of	  CSCs	  within	  microtubule	  domains.	  Evidence	  already	  supports	  a	  potentially	  direct	  linkage	  between	  the	  CSC	  and	  microtubules	  through	  linking	  proteins,	  such	  as	  Cellulose	  Synthase	  Interactive	  Protein	  1	  (CSI1)	  [Gu	  et	  al.	  2010,	  Li	  et	  al.	  2012].	  More	  direct	  or	  intimate	  relationships	  with	  other	  proteins,	  such	  as	  MOR1,	  may	  be	  discovered	  by	  analyses	  such	  as	  these.  Previous	  studies	  reporting	  CSC	  velocities	  place	  the	  average	  velocity	  for	  the	  CSC	  in	  wild	  type	  between	  300	  and	  350nm/min	  (Paredez	  et	  al.	  2006,	  Persson	  et	  al.	  2007).	  Unfortunately,	  with	  the	  exception	  of	  Fujita	  et	  al.	  (2011)	  and	  Fujita	  et	  al.	  (2013),	  most	  studies	  failed	  to	  perform	  temperature	  controls	  on	  their	  assessments	  so	  the	  values	  reported	  are	  not	  valid	  for	  comparison.	  Noting	  that	  Fujita	  et	  al.	  (2011)	  reported	  that	  small	  temperature	  shifts	  can	  generate	  massive	  differences	  in	  CSC	  velocity,	  it	  is	  critical	  that	  temperature	  be	  controlled	   37 and	  monitored	  during	  CSC	  velocity	  analysis.	  As	  has	  been	  shown	  (Paredez	  et	  al.	  2006,	  DeBolt	  et	  al.	  2007,	  Fujita	  et	  al.	  2011),	  the	  movement	  of	  the	  CSC	  is	  likely	  a	  kinetic/temperature	  dependent	  process	  that	  is	  reliant	  on	  its	  own	  synthetic	  output	  to	  move.	  Thus,	  analysis	  of	  CSC	  velocity	  should	  be	  carried	  out	  under	  strictly	  controlled	  conditions,	  just	  as	  with	  any	  enzyme/protein	  activity	  assay.	  Owing	  to	  the	  general	  lack	  of	  such	  controlled	  analysis	  in	  the	  literature	  for	  CSCs,	  the	  data	  presented	  here	  can	  only	  be	  compared	  to	  the	  limited	  studies	  incorporating	  temperature	  control	  and	  to	  subsequent	  results	  found	  in	  this	  thesis. Compared	  to	  the	  previous	  findings	  in	  Fujita	  et	  al.	  (2011),	  near-­‐TIRF	  analysis	  greatly	  improved	  the	  ability	  to	  distinguish	  CSCs	  tracking	  within	  and	  outside	  microtubule	  domains	  (Table	  2.3).	  In	  the	  Fujita	  et	  al.	  (2011)	  study,	  the	  coincidence	  analysis	  was	  restricted	  to	  regions	  devoid	  of	  background	  fluorescence	  from	  cytoplasmic	  contents,	  which	  greatly	  limited	  data	  acquisition.	  The	  ability	  of	  TIRF	  microscopy	  to	  attenuate	  the	  background	  fluorescence	  allowed	  for	  more	  accurate	  identification	  of	  the	  microtubules	  subtending	  the	  CSCs.	  The	  difference	  in	  results	  observed	  has	  transferred	  the	  likelihood	  of	  impact	  on	  cellulose	  synthesis	  in	  the	  mor1-­‐1	  mutant	  onto	  the	  malfunctioning	  MOR1	  protein	  or	  direct	  effects	  of	  altered	  polymer	  mass.  Ultimately,	  the	  greatest	  contribution	  to	  the	  overall	  increased	  CSC	  velocity	  measured	  in	  mor1-­‐1	  was	  the	  increase	  in	  CSC	  velocities	  within	  the	  microtubule	  domains	  of	  mor1-­‐1.	  Since	  there	  is	  a	  profound	  disruption	  of	  microtubule	  plus	  end	  dynamics	  in	  mor1-­‐1	  (Kawamura	  &	  Wasteneys,	  2008),	  one	  interpretation	  for	  the	  increased	  CSC	  velocity	  within	  these	  domains	  is	  that	  rapid	  microtubule	  dynamics,	  under	  the	  control	  of	  MOR1,	  is	  a	  requirement	  for	  the	  constraint	  of	  CSC	  velocity.	  This	  would	  result	  in	  production	  of	  cellulose	  of	  appropriate	   38 quality	  to	  maintain	  growth	  anisotropy.	  Alternatively,	  the	  MOR1	  protein	  might	  play	  a	  more	  direct	  role	  in	  constraining	  CSC	  movement	  through	  interactions	  with	  the	  CSC	  itself.	  Regardless	  of	  whether	  the	  influence	  of	  MOR1	  on	  CSCs	  is	  direct	  or	  indirect,	  the	  results	  do	  suggest	  that	  modulation	  of	  CSC	  velocity	  within	  microtubule	  domains	  at	  the	  plasma	  membrane	  helps	  to	  define	  wall	  crystallinity	  and,	  ultimately,	  growth	  anisotropy.	  My	  finding	  that	  loss	  of	  anisotropy	  could	  be	  related	  to	  the	  change	  of	  velocity	  of	  CSCs	  within	  microtubule	  domains,	  in	  contrast	  to	  the	  previous	  assertions	  that	  domainless	  CSCs	  define	  the	  effect	  (Fujita	  et	  al.	  2011)	  led	  me	  to	  explore	  the	  domain	  effect	  further	  using	  oryzalin-­‐treated	  seedlings.  In	  oryzalin-­‐treated	  seedlings,	  CSC	  particle	  coincidence	  with	  microtubules	  decreased	  with	  increasing	  concentration	  while	  mean	  particle	  velocity	  in	  microtubule	  domains	  and	  outside	  of	  microtubule	  domains	  was	  unaltered.	  At	  higher	  concentrations	  of	  oryzalin,	  increased	  depolymerization	  of	  microtubules	  had	  a	  slightly	  more	  pronounced	  effect	  on	  CSC	  activity.	  Increased	  microtubule	  perturbation	  did	  not	  significantly	  affect	  CSC	  velocity	  within	  microtubule	  domains,	  when	  still	  present.	  The	  largest	  changes	  observed	  were	  the	  increase	  in	  the	  proportion	  of	  CSCs	  tracking	  outside	  of	  microtubule	  domains	  and	  a	  decrease	  in	  CSC	  velocity	  outside	  of	  the	  domains.	    Taken	  together,	  these	  velocity	  profiles	  indicate	  that	  it	  is	  not	  simply	  the	  depolymerization	  and	  disorganization	  of	  microtubules	  that	  affects	  cellulose	  synthesis;	  it	  is	  how	  they	  are	  disrupted.	  In	  view	  of	  the	  minimal	  effect	  witnessed	  on	  wild	  type	  CSC	  velocity	  by	  oryzalin,	  it	  can	  be	  assumed	  that	  the	  MOR1	  protein	  plays	  a	  much	  larger,	  potentially	  direct,	  role	  in	   39 modulating	  CSC	  activity	  than	  previously	  reported.	  Associations	  of	  the	  CSC	  with	  proteins	  such	  as	  KOR1	  and	  the	  addition	  of	  secreted	  xyloglucans	  could	  be	  affected	  due	  to	  the	  inability	  of	  microtubules	  in	  mor1-­‐1	  to	  be	  properly	  dynamic	  and	  organize/polymerize	  appropriately.	  At	  lower	  oryzalin	  concentrations,	  wild	  type	  microtubules	  were	  visibly	  comparable	  to	  mor1-­‐1	  and	  yet	  the	  associated	  CSCs	  did	  not	  display	  an	  increased	  velocity	  profile.	  Only	  after	  microtubule	  polymer	  mass	  was	  even	  more	  significantly	  reduced	  than	  mor1-­‐1	  with	  oryzalin	  was	  an	  effect	  observed.	  Nonetheless,	  CSCs	  continued	  to	  remain	  associated	  with	  microtubules	  and	  to	  be	  regulated	  appropriately	  even	  at	  the	  highest	  concentration	  when	  microtubule	  polymer	  mass	  was	  sparingly	  present.	  These	  data,	  in	  combination	  with	  the	  velocity	  profiles	  observed	  in	  mor1-­‐1,	  point	  to	  a	  more	  nuanced	  system	  of	  regulation	  of	  CSC	  velocity	  than	  has	  been	  previously	  suggested.	  	   	   40 Chapter	  3:	  Assessing	  the	  Nature	  of	  Microtubule	  Disruption	  in	  the	  Reduction	  of	  Growth	  Anisotropy  3.1	  Background: The	  indirect	  or	  direct	  manipulation	  of	  cellulose	  synthesis	  often	  causes	  aberrant	  growth	  phenotypes.	  Earlier	  studies	  between	  mor1-­‐1	  (Fujita	  et	  al.	  2011)	  and	  the	  CESA1	  mutant,	  anisotropy1	  D604N,	  any1	  (Fujita	  et	  al.	  2013),	  linked	  crystallinity	  and	  CSC	  velocity	  to	  establish	  proper	  anisotropy.	  any1’s	  aberrant	  and	  dwarf	  growth	  is	  ideal	  for	  studying	  anisotropy	  defects	  since	  the	  overall	  quantity	  of	  cellulose	  produced	  is	  unchanged,	  but	  relative	  to	  wild	  type,	  both	  CSC	  velocity	  and	  wall	  crystallinity	  are	  reduced	  (Fujita	  et	  al.	  2013).	  The	  point	  mutation	  in	  any1	  directly	  affects	  the	  catalytic	  domain	  of	  CESA1;	  a	  region	  containing	  many	  other	  allelic	  variants	  (Fujita	  et	  al.	  2013),	  but	  any1	  is	  distinct	  from	  other	  alleles	  by	  not	  having	  a	  lethal	  or	  conditional	  phenotype.	  This	  may	  be	  due	  to	  the	  location	  of	  the	  mutation,	  occurring	  within	  the	  catalytic	  domain	  between	  the	  second	  and	  third	  D	  in	  the	  DDDQxxRW	  motif,	  but	  distanced	  slightly	  from	  the	  species	  specific	  HVR.	  This	  disruption	  of	  catalytic	  activity	  interrupts	  proper	  anisotropy	  not	  through	  alterations	  in	  amount	  of	  cellulose,	  but	  for	  opposite	  crystallinity	  and	  CSC	  velocity	  to	  mor1-­‐1.	  This	  experiment	  was	  designed	  to	  assess	  how	  these	  two	  processes,	  CSC	  displacement	  velocity	  and	  wall	  crystallinity	  are	  related,	  by	  generating	  a	  double	  mor1-­‐1/any1	  mutant	  and	  by	  mimicking	  the	  disorganization	  and	  depolymerization	  of	  mor1-­‐1	  with	  oryzalin	  in	  any1	  seedlings.  There	  is	  no	  data	  on	  the	  effect	  of	  oryzalin	  on	  microfibril	  crystallinity,	  but	  differences	  in	  CSC	  velocity	  with	  the	  double	  mutant	  may	  distinguish	  the	  disrupted	  microtubules	  in	  each	  as	  separately	  or	  similarly	  acting.	  It	  is	  hoped	  that	  these	  simultaneous	  disruptions	  will	  expand	   41 on	  their	  relationship	  and	  hierarchical	  roles,	  if	  any.	  However,	  the	  means	  by	  which	  oryzalin	  and	  mor1-­‐1	  disrupt	  microtubules	  may	  impact	  their	  relationship	  with	  the	  any1	  mutant	  and	  must	  be	  carefully	  considered	  during	  analyses.	  Both	  oryzalin	  and	  the	  effects	  of	  mor1-­‐1	  may	  be	  more	  extensive	  than	  the	  limited	  effects	  seen	  individually,	  such	  as	  impacting	  transport	  or	  association	  with	  other	  proteins.	  With	  advancements	  in	  recent	  literature	  pointing	  out	  the	  ability	  for	  the	  CSC	  to	  determine	  actions	  of	  microtubules	  (Liu	  et	  al.	  2016),	  removing	  a	  majority	  of	  the	  microtubule	  support	  with	  oryzalin	  is	  unlikely	  to	  mimic	  mor1-­‐1,	  but	  may	  reveal	  aspects	  of	  the	  system	  that	  are	  more	  determinant	  for	  proper	  cellulose	  synthesis.	  Microtubules	  were	  found	  to	  influence	  CSC	  velocity	  when	  directly	  associated	  and	  the	  MOR1	  protein	  was	  postulated	  to	  play	  a	  more	  intimate	  role	  in	  modulating	  cellulose	  synthesis.	  I	  also	  explored	  other	  anisotropy	  and	  growth	  stunted	  mutants	  related	  to	  alterations	  of	  microtubule	  organization	  or	  polymerization.	    CLASP1	  is	  a	  MAP	  that	  has	  been	  implicated	  in	  microtubule	  organization	  and	  plant	  growth	  (Ambrose	  et	  al	  2007;	  Kirik	  et	  al	  2007).	  It	  was	  originally	  discovered	  as	  mor2,	  alongside	  mor1	  by	  Whittington	  et	  al	  (2001)	  in	  the	  same	  immunofluorescence-­‐based	  screen.	  This	  mutant,	  like	  the	  T-­‐DNA	  insertion	  line	  of	  clasp-­‐1,	  had	  hyperparallel	  microtubule	  organization,	  dwarf	  stature,	  and	  developmental	  defects	  indicated	  altered	  auxin	  levels	  or	  signaling.	  During	  a	  proteomic	  screen	  for	  tubulin-­‐binding	  proteins	  (Chuong	  et	  al.	  2004)	  it	  was	  rediscovered	  and	  mapped	  to	  the	  lower	  arm	  of	  chromosome	  2	  as	  CLASP.	  Sequencing	  indicated	  that	  some	  confusion	  between	  the	  two	  proteins	  may	  be	  due	  to	  original	  EMS	  experiments	  generating	  mutations	  in	  the	  protein’s	  promoter.	  Since	  the	  T-­‐DNA	  insertion	  in	  CLASP	  replicated	  all	  mor2	  mutant	  phenotypes,	  all	  further	  studies	  were	  conducted	  with	  the	  clasp-­‐1	  and	  clasp-­‐2	   42 mutants	  (Ambrose	  et	  al.	  2007).	  The	  protein,	  like	  MOR1,	  contains	  an	  N-­‐termial	  TOG	  domain,	  a	  large	  number	  of	  HEAT	  repeats,	  and	  also	  contains	  sequence	  homology	  to	  the	  human	  CLASP’s	  Cytoplasmic	  Linker	  Protein-­‐interacting	  domain	  (CLIP)	  [Inoue	  et	  al	  2000].	  This	  domain	  is	  considered	  It	  contains	  21	  exons	  and	  20	  introns	  and	  expression	  analyses	  show	  the	  functional	  protein	  to	  be	  present	  in	  all	  tissues.	  The	  clasp-­‐1	  mutant	  is	  a	  T-­‐DNA	  insertion	  mutant,	  in	  exon	  13,	  available	  via	  the	  SALK	  T-­‐DNA	  collection	  (Alonso	  et	  al.	  2003).	  The	  clasp-­‐1	  plant	  is	  dwarf	  with	  only	  mild	  loss	  of	  anisotropy,	  and	  microtubules	  tend	  to	  detach	  from	  the	  cortex	  in	  the	  clasp-­‐1	  mutant	  (Ambrose	  and	  Wasteneys	  2008).	  Preliminary	  cell	  wall	  analyses	  demonstrated	  that	  there	  is	  no	  alteration	  in	  cellulose	  microfibril	  orientations	  nor	  cell	  wall	  crystallinity	  (Miki	  Fujita,	  unpublished	  data).	  	  	  A	  recent	  study	  has	  shown	  that	  CLASP	  is	  downregulated	  in	  xyloglucan-­‐deficient	  mutants	  (Xiao	  et	  al.	  2016)	  to	  a	  similar	  enough	  degree	  as	  current	  research	  (Ruan	  unpublished	  data)	  that	  focused	  on	  disrupted	  growth	  due	  to	  large	  alterations	  in	  brassinosteroid	  signalling	  as	  a	  result	  of	  downregulated	  CLASP	  levels.	  Even	  though	  the	  downregulation	  of	  CLASP	  in	  these	  brassinosteroid	  mutants	  would	  have	  been	  considered	  insignificant	  in	  other	  backgrounds,	  this	  unpublished	  data	  focused	  on	  how	  small	  scale	  downregulation	  in	  this	  mutant	  can	  generate	  much	  larger	  impacts	  on	  growth	  than	  normally	  observed	  and	  for	  this	  reason	  identified	  CLASP	  as	  having	  potential	  for	  impacts	  on	  cellulose	  crystallinity	  through	  linkages	  with	  xyloglucan.	  I	  used	  clasp-­‐1	  mutant	  to	  examine	  the	  CSC	  velocity	  as	  an	  example	  of	  a	  MAP	  mutant	  with	  no	  obvious	  influence	  on	  cellulose	  synthesis.   43 Other	  associated	  proteins,	  KATANIN	  (McClinton	  et	  al.	  2001)	  and	  RIC1	  (Fu	  et	  al.	  2005),	  show	  changes	  in	  microtubule	  organization	  and/or	  polymer	  mass	  related	  to	  their	  respective	  disrupted	  and	  enhanced	  anisotropies.	  A	  mutant	  variant	  of	  the	  p60	  subunit	  of	  KATANIN,	  botero1,	  and	  an	  overexpression	  line	  of	  RIC1,	  RIC1-­‐OX,	  were	  studied	  previously	  alongside	  mor1-­‐1	  to	  determine	  if	  altered	  wall	  crystallinity	  was	  a	  trend	  amongst	  microtubule	  perturbations	  (Fujita	  et	  al.	  2011).	  KATANIN	  is	  known	  to	  be	  a	  heterodimeric	  ATPase	  with	  two	  subunits;	  the	  catalytic	  p60	  subunit	  and	  the	  p80	  subunit,	  which	  determines	  proper	  localization.	  During	  a	  screen	  for	  mutants	  with	  reduced	  hypocotyl	  cellular	  elongation,	  botero1,	  a	  point	  mutation	  generated	  via	  EMS	  in	  the	  p60	  subunit	  of	  KATANIN,	  was	  discovered	  (Bichet	  et	  al	  2001).	  In	  this	  genotype,	  microtubules	  lose	  parallel	  order	  as	  in	  mor1-­‐1	  but	  the	  polymer	  mass	  is	  not	  reduced.	  The	  mutated	  KATANIN	  subunit	  fails	  to	  sever	  microtubules,	  normally	  causing	  catastrophe	  in	  selective	  regions.	  According	  to	  Fujita	  et	  al	  (2011),	  crystallinity	  in	  bot1	  mutants	  was	  not	  significantly	  reduced.	  Based	  on	  the	  correlation	  observed	  between	  crystallinity	  and	  CSC	  velocity	  in	  both	  mor1-­‐1	  and	  any1,	  we	  hypothesized	  that	  the	  CSC	  velocity	  in	  bot1	  should	  be	  similar	  to	  that	  of	  wild	  type.	    Unlike	  the	  loss-­‐of-­‐anisotropy	  mutants	  mor1-­‐1	  and	  bot1,	  RIC1-­‐OX	  plants	  displayed	  enhanced	  anisotropy,	  with	  longer	  and	  narrower	  organs	  in	  comparison	  to	  wild	  type	  (Fu	  et	  al.	  2005).	  Through	  a	  bait	  screen	  with	  a	  constitutively	  active	  ROP1	  mutant,	  a	  mutant	  in	  the	  Rho	  GTPase	  (ROP)	  signaling	  pathway,	  a	  clone	  was	  identified	  that,	  through	  further	  sequence	  analyses,	  lead	  to	  the	  discovery	  of	  another	  CRIB-­‐motif	  containing	  protein,	  Rop-­‐interactive	  CRIB-­‐containing	  protein1	  (RIC1)	  [Wu	  et	  al.	  2001].	  This	  CRIB	  motif	  was	  shown	  to	  be	  essential	  for	  proper	  interactions	  between	  the	  RIC1	  protein	  and	  ROPs.	  Further	   44 investigations	  into	  the	  overexpression	  of	  RIC1	  showed	  enhancement	  of	  growth	  phenotypes,	  possibly	  related	  to	  the	  increased	  transverse	  alignment	  and	  bundling	  of	  microtubules	  (Fu	  et	  al.	  2005).	  Compared	  to	  previously	  tested	  mutants,	  these	  phenotypes	  appear	  most	  likely	  to	  be	  indicators	  of	  proper	  or	  maximized	  adaptations	  for	  elongation.	  In	  Fujita	  et	  al.	  (2011),	  the	  crystallinity	  of	  inflorescence	  stems	  in	  RIC1-­‐OX	  lines	  showed	  a	  significant	  decrease	  in	  wall	  crystallinity	  compared	  to	  wild	  type	  at	  21°C	  and	  29°C.	  If	  wild-­‐type	  adaptations	  are	  to	  reduce	  crystallinity	  of	  the	  wall	  for	  proper	  elongation	  via	  microfibril	  separation,	  then	  we	  predicted	  that	  CSC	  velocity	  should	  be	  elevated	  or	  comparable	  to	  adaptations	  by	  wild	  type	  in	  RIC1-­‐OX	  seedlings.	  	  	   	   45 3.2	  Materials	  &	  Methods: 3.2.1.	  Plant	  material	  &	  growth	  conditions Previously	  isolated	  lineages	  of	  any1	  (Fujita	  et	  al.	  2013)	  ,	  clasp-­‐1	  (Ambrose	  et	  al.	  2007),	  bot1,	  provided	  by	  Dr.	  Herman	  Höfte	  (Centre	  de	  Versailles-­‐Grignon,	  INRA,	  France),and	  RIC1-­‐OX,	  provided	  by	  Dr.	  Zhen-­‐Biao	  Yang	  (Center	  for	  Plant	  Cell	  Biology,	  University	  of	  California	  at	  Riverside,	  CA)	  were	  grown	  and	  assessed	  under	  specific	  conditions.	  Procedure	  otherwise	  follows	  chapter	  2,	  section	  2.2.1	  except	  that	  no	  RFP	  reporter	  was	  available	  for	  visualizing	  microtubules.	  	  I	  generated	  the	  any1/mor1-­‐1	  double	  mutant	  via	  manual	  crossing	  and	  isolated	  them	  for	  imaging.	  F1s	  displayed	  WT	  phenotypes,	  while	  F2s	  were	  the	  expected	  9:3:3:1	  ratio	  of	  WT	  :	  any1	  :	  mor1-­‐1	  :	  any1/mor1-­‐1.	  Seedlings	  grown	  at	  21°C	  appeared	  most	  like	  any1	  seedlings,	  with	  mor1-­‐1	  phenotypes	  mostly	  suppressed	  by	  bulged	  and	  disrupted	  roots.	  Seedlings	  grown	  at	  29°C	  proceeded	  toward	  lethality	  immediately	  following	  germination.	  By	  following	  the	  standard	  procedure	  set	  in	  this	  document	  for	  growing	  and	  imaging	  seedlings	  (Section	  2.2.1),	  etiolated	  hypocotyls	  of	  the	  double	  mutant	  were	  discernable	  from	  single	  mutants.	  Grown	  for	  4	  days	  at	  4°C,	  2	  days	  at	  21°C,	  and	  1	  day	  at	  29°C,	  any1/mor1-­‐1	  seedlings	  were	  more	  dwarf	  than	  either	  single	  mutant.	  However,	  this	  was	  not	  always	  a	  clear	  distinction,	  so	  seedlings	  were	  re-­‐plated	  post-­‐imaging	  and	  assessed	  for	  seedling	  lethality	  following	  return	  to	  growth	  conditions.	  Images	  for	  seedlings	  that	  were	  able	  to	  grow	  normally	  per	  any1	  or	  mor1-­‐1	  phenotypes	  at	  21°C	  after	  re-­‐plating	  were	  discarded	  from	  use.	     46 3.2.2	  Mutant	  backgrounds	  &	  reporter	  constructs Procedure	  is	  as	  described	  previously	  in	  chapter	  2,	  section	  2.2.2.	    3.2.3.	  Hormone	  treatment	  &	  mounting	  solution Procedure	  is	  as	  described	  previously	  in	  chapter	  2,	  section	  2.2.3.  3.2.4	  Live-­‐cell	  imaging	   Procedure	  is	  as	  described	  previously	  in	  chapter	  2,	  section	  2.2.4.	  No	  RFP	  reporter	  was	  available	  for	  visualizing	  microtubules.  3.2.5	  Image	  J	  analysis Procedure	  is	  as	  described	  previously	  in	  chapter	  2,	  section	  2.2.5.	  No	  RFP	  reporter	  was	  available	  for	  visualizing	  microtubules.	  	   	   47 3.3	  Results: 3.3.1	  Disruption	  of	  cortical	  microtubule	  network	  by	  oryzalin	  exacerbates	  any1	  CSC	  velocity	  phenotype	  The	  observed	  CSC	  velocity	  in	  any1	  seedlings	  was	  elevated,	  268	  ±	  70	  nm/min	  (Fig.	  3.1),	  compared	  to	  previous	  measurements,	  229	  nm/min	  (Fujita	  et	  al.	  2011),	  but	  statistically	  still	  reduced	  compared	  to	  wild	  type	  (Fig	  2.1).	  Velocity	  was	  measured	  as	  an	  average	  of	  CSCs	  within	  and	  outside	  of	  microtubule	  domains.	  Unlike	  wild-­‐type	  plants,	  any1	  mutants	  displayed	  an	  enhancement	  of	  their	  already	  reduced	  CSC	  velocity.	  At	  the	  lower	  2μM	  concentration	  (Fig.	  3.2),	  any1	  mutants	  showed	  a	  significantly	  reduced	  mean	  velocity	  at	  179	  ±	  74	  nm/min	  compared	  to	  untreated	  any1	  seedlings	  at	  268	  nm/min	  (Fig.	  3.3).	  	  	   	   48   Figure	  3.1|	  CSC	  velocity	  in	  any1	  mutant	  with	  YFP-­‐CESA6	  in	  prc1 YFP-­‐CESA6	  tagged	  CSC	  trajectories	  were	  visualized	  in	  any1/prc1	  dark-­‐grown	  hypocotyls	  at	  29°C	  as	  projections	  of	  movement	  using	  time-­‐lapse	  image	  sequences	  via	  near-­‐TIRF	  microscopy.(A)	  The	  histogram	  displays	  the	  overall	  distribution	  of	  CSC	  velocity.	  CSCs	  displayed	  a	  velocity	  of	  268	  ±	  70	  nm/min	  (n	  =	  500	  particles	  from	  14	  cells	  in	  4	  seedlings).	  (B)	  YFP-­‐Projection	  image	  indicating	  cellular	  morphology.	  (C)	  Kymographs	  were	  produced	  for	  each	  trajectory.	  CSC	  velocity	  was	  calculated	  from	  the	  slope	  of	  kymographs.  49  	  	  Figure	  3.2|	  CSC	  velocity	  in	  the	  any1	  mutant	  with	  2μM	  oryzalin	  with	  YFP-­‐CESA6	  in	  prc1 YFP-­‐CESA6	  tagged	  CSC	  trajectories	  in	  any1/prc1	  dark-­‐grown	  hypocotyls	  treated	  with	  a	  2μM	  concentration	  solution	  of	  oryzalin	  at	  29°C	  were	  visualized	  as	  projections	  of	  movement	  using	  time-­‐lapse	  image	  sequences	  via	  near-­‐TIRF	  microscopy.	  (A)	  The	  histogram	  displays	  the	  overall	  distribution	  of	  CSC	  velocity.	  (B)	  Cell	  morphology	  was	  more	  isotropic	  than	  the	  any1	  mutant	  and	  CSCs	  displayed	  a	  velocity	  of	  179	  ±	  74	  nm/min	  (n	  =	  501	  particles	  from	  9	  cells	  in	  7	  seedlings).	  (C)	  Kymographs	  were	  produced	  for	  each	  trajectory.	  CSC	  velocity	  was	  calculated	  from	  the	  slope	  of	  kymographs.	  	   50  Figure	  3.3|	  CSC	  velocity	  in	  the	  any1	  mutant	  with	  20μM	  oryzalin	  with	  YFP-­‐CESA6	  in	  prc1 YFP-­‐CESA6-­‐tagged	  CSC	  trajectories	  in	  any1/prc1	  dark-­‐grown	  hypocotyls	  treated	  with	  a	  20μM	  concentration	  solution	  of	  oryzalin	  at	  29°C	  were	  visualized	  as	  projections	  of	  movement	  using	  time-­‐lapse	  image	  sequences	  via	  near-­‐TIRF	  microscopy.	  (A)	  The	  histogram	  displays	  the	  overall	  distribution	  of	  CSC	  velocity.	  (B)	  Cell	  morphology	  was	  more	  isotropic	  than	  the	  any1	  mutant	  and	  CSCs	  displayed	  a	  velocity	  of	  140	  ±	  85	  nm/min	  (n	  =	  500	  particles	  from	  11	  cells	  in	  4	  seedlings).	  (C)	  Kymographs	  were	  produced	  for	  each	  trajectory.	  CSC	  velocity	  was	  calculated	  from	  the	  slope	  of	  kymographs.	    51 An	  even	  further	  reduction	  in	  CSC	  velocity	  was	  observed	  at	  140	  140	  ±	  85	  nm/min	  in	  seedlings	  treated	  with	  20μM	  concentrations	  of	  oryzalin	  (Fig	  3.3).	  Similar	  to	  wild-­‐type	  seedlings	  treated	  with	  oryzalin,	  CSC	  trajectories	  appeared	  impaired,	  showing	  movement	  of	  particles	  to	  be	  less	  ordered	  and	  shortened	  in	  overall	  trajectory	  length	  with	  increasing	  oryzalin	  concentrations.	  	   3.3.2	  The	  mor1-­‐1/any1	  double	  mutant	  displays	  reduced	  seedling	  viability	  and	  CSC	  velocity Both	  mor1-­‐1	  and	  any1	  display	  reduced	  growth	  when	  grown	  in	  the	  dark	  at	  29°C,	  and,	  when	  combined,	  have	  exacerbated	  growth	  phenotypes,	  making	  isolation	  of	  the	  mutant	  difficult	  due	  to	  lethality	  and	  impaired	  visualization	  of	  mor1-­‐1	  root-­‐skewing	  phenotypes	  (Whittington	  et	  al.	  2001).	  The	  double	  mutant	  seedlings	  at	  21°C	  display	  an	  overall	  any1-­‐like	  phenotype;	  reduced	  anisotropic	  growth	  and	  globular	  trichomes.	  However,	  even	  when	  grown	  at	  21°C,	  the	  double	  mutant	  failed	  to	  bolt,	  building	  large	  rosettes	  over	  several	  months	  until	  death	  (Fig.	  3.4).	  Attempts	  at	  modulating	  the	  photoperiod,	  light	  intensity,	  and	  temperature	  failed	  to	  provide	  the	  conditions	  necessary	  for	  seedling	  viability.	    52 	  Figure	  3.4|	  The	  any1/mor1-­‐1	  double	  mutant The	  any1/mor1-­‐1	  double	  mutant	  displayed	  severe	  growth	  phenotypes.	  Seedlings	  often	  failed	  to	  germinate,	  but	  with	  reduced	  temperature,	  reduced	  exposure	  to	  light,	  and	  short	  day	  settings,	  rosettes	  were	  able	  to	  form	  after	  	  months	  of	  growth.	  Seen	  here	  is	  full	  growth	  at	  3	  months	  time.	  No	  bolting	  occurred	  in	  any	  seedlings	  and	  none	  were	  viable	  long	  after	  reaching	  a	  maximum	  rosette	  size.	  	  	  	  At	  mor1-­‐1’s	  restrictive	  temperature,	  29°C,	  seedlings	  displayed	  additive	  phenotypes	  and	  progress	  toward	  lethality	  was	  enhanced.	  Seedlings	  kept	  at	  29°C	  from	  the	  start	  failed	  to	  grow	  past	  germination	  and	  seedling	  lethality	  was	  consistent.	  Failure	  to	  produce	  true	  leaves	  and	  progress	  past	  the	  cotyledon	  stage	  under	  continued	  exposure	  to	  restrictive	  temperature	  abolished	  visibility	  of	  any1	  phenotypes.	  Distinguishing	  the	  double	  mutant	  from	  single	  mutants	  was	  possible,	  however.	  After	  two	  days	  of	  growth	  at	  21°C,	  followed	  by	  one	  at	  29°C,	  etiolated	  hypocotyls	  of	  the	  double	  mutants	  had	  reduced	  growth	  compared	  to	  both	  any1	  and	  mor1-­‐1	  single	  mutants	  and	  upon	  re-­‐plating	  after	  imaging	  they	  failed	  to	  grow	  past	  the	  cotyledon	  stage.	  	  This	  allowed	  for	  post-­‐imaging	  identification	  of	  double	  mutants.	  	   53 Seedlings	  raised	  under	  the	  standard	  live-­‐imaging	  conditions	  detailed	  above	  were	  viable	  for	  imaging.	  CSC	  trajectories	  appeared	  to	  lack	  any	  consistent	  orientation	  and	  reduced	  in	  overall	  length.	  Cells	  were	  qualitatively	  more	  isotropically	  expanded	  than	  those	  of	  single	  mutants.	  When	  CSC	  velocity	  was	  measured	  in	  the	  double	  mutant	  at	  mor1-­‐1’s	  restrictive	  temperature,	  the	  mean	  CSC	  velocity	  was	  found	  to	  be	  233	  ±	  102	  nm/min	  (Fig.	  3.5).	  	  This	  reduction	  of	  CSC	  velocity	  in	  comparison	  to	  the	  any1	  single	  mutant	  (268	  nm/min)	  is	  not	  significant,	  but	  is	  still	  significant	  compared	  to	  wild	  type.	  	  	   	   54 	  Figure	  3.5|	  CSC	  velocity	  in	  the	  any1/mor1-­‐1	  double	  mutant	  with	  YFP-­‐CESA6	  in	  prc1 YFP-­‐CESA6	  tagged	  CSC	  trajectories	  in	  any1/mor1-­‐1/prc1	  dark-­‐grown	  hypocotyls	  at	  29°C	  were	  visualized	  as	  projections	  of	  movement	  using	  time-­‐lapse	  image	  sequences	  via	  near-­‐TIRF	  microscopy.	  (A)	  The	  histogram	  displays	  the	  overall	  distribution	  of	  CSC	  velocity.	  (B)	  Cell	  morphology	  was	  more	  isotropic	  than	  the	  any1	  mutant	  and	  CSCs	  displayed	  a	  velocity	  of	  233	  ±	  102	  nm/min	  (n	  =	  499	  particles	  from	  12	  cells	  in	  6	  seedlings).	  (C)	  Kymographs	  were	  produced	  for	  each	  trajectory.	  CSC	  velocity	  was	  calculated	  from	  the	  slope	  of	  kymographs.	  	     55 	  Figure	  3.6|	  Comparison	  of	  CSC	  velocity	  in	  wild	  type,	  untreated	  any1,	  any1/mor1-­‐1,	  any1	  +	  2μM	  oryzalin,	  any1	  +	  20μM	  oryzalin	  treated	  seedlings	  mean	  CSC	  velocity	  and	  frequency	  (%).	  	  	  (A)	  At	  29°C	  any1	  seedlings	  showed	  a	  23%	  reduction	  in	  CSC	  velocity	  (268	  ±	  70	  nm/min)	  from	  wild	  type	  (346	  ±	  121	  nm/min).	  The	  any1/mor1-­‐1	  double	  mutant	  showed	  a	  33%	  decrease	  (233	  ±	  102	  nm/min)	  in	  velocity	  relative	  to	  wild	  type.	  In	  2μM	  oryzalin	  solutions,	  CSCs	  in	  any1	  travel	  at	  an	  even	  further	  48%	  reduced	  velocity	  from	  wild	  type	  (179	  ±	  74	  nm/min).	  At	  20μM,	  the	  observed	  140	  	  ±	  85	  nm/min	  in	  any1	  continued	  the	  reduction	  in	  velocity	  to	  60%	  of	  wild	  type	  values.	  (B)	  any1	  distribution	  of	  CSCs	  by	  frequency	  (%).	  (C)	  any1/mor1-­‐1	  distribution	  of	  CSCs	  by	  frequency	  (%).	  (D)	  any1	  +	  2μM	  oryzalin	  distribution	  of	  CSCs	  by	  frequency	  (%).	  (E)	  any1	  +	  20μM	  oryzalin	  distribution	  of	  CSCs	  by	  frequency	  (%).	  Asterisks	  indicate	  significant	  difference	  relative	  to	  wild	  type	  determined	  with	  one	  way	  ANOVA	  test;	  p-­‐values	  <	  .0001.	  Post-­‐hoc	  comparisons	  between	  assessments	  were	  made	  using	  the	  Tukey	  range	  test	  and	  significant	  values	  compared	  to	  wild	  type	  and	  any1	  are	  indicated	  by	  double	  asterisks;	  p-­‐values	  <	  .0001.	  	   56 3.3.3	  Hypocotyl	  CSC	  velocities	  are	  elevated	  in	  clasp-­‐1	  mutant	  background	  	  	  CSC	  velocity	  in	  the	  clasp-­‐1	  mutant	  was	  significantly	  elevated	  compared	  to	  wild	  type	  at	  405	  ±	  119	  nm/min	  (Fig.	  3.7).	  The	  velocity	  profile	  was	  taken	  as	  an	  average	  value	  of	  both	  inside	  and	  outside	  of	  microtubule	  domains.	  Cellular	  morphology	  appeared	  unaltered	  and	  CSC	  trajectories	  were	  visibly	  comparable	  to	  wild	  type.	  	  	   	   57 	  	  Figure	  3.7|	  CSC	  velocity	  in	  clasp-­‐1	  with	  YFP-­‐CESA6	  in	  prc1 YFP-­‐CESA6	  tagged	  CSC	  trajectories	  in	  clasp-­‐1/prc1	  dark-­‐grown	  hypocotyls	  at	  29°C	  were	  visualized	  as	  projections	  of	  movement	  using	  time-­‐lapse	  image	  sequences	  via	  near-­‐TIRF	  microscopy.	  (A)	  The	  histogram	  displays	  the	  overall	  distribution	  of	  CSC	  velocity.	  (B)	  Cell	  morphology	  was	  similar	  to	  wild	  type	  and	  CSCs	  displayed	  a	  velocity	  of	  405	  ±	  119	  nm/min	  (n	  =	  499	  particles	  from	  29	  cells	  in	  8	  seedlings).	  (C)	  Kymographs	  were	  produced	  for	  each	  trajectory.	  CSC	  velocity	  was	  calculated	  from	  the	  slope	  of	  kymographs.	  	   	   58 3.3.4	  botero1	  displays	  unaltered	  mean	  CSC	  velocity	  compared	  to	  wild	  type	  values Microtubules	  in	  botero1	  are	  known	  to	  be	  disorganized	  and	  longer	  than	  wild	  type.	  Velocities	  of	  CSCs	  in	  botero1	  were	  349	  ±	  95	  nm/min,	  unchanged	  compared	  to	  wild	  type	  and	  maintained	  a	  normal	  distribution	  (Fig.	  3.8).	  Cellular	  morphology	  appeared	  slightly	  isotropic	  but	  trajectories	  appeared	  mostly	  wild	  type,	  both	  in	  length	  of	  trajectory	  and	  orientation.	  	   	   59 	   Figure	  3.8|	  CSC	  velocity	  in	  bot1	  with	  YFP-­‐CESA6	  in	  prc1	  	  YFP-­‐CESA6	  tagged	  CSC	  trajectories	  in	  bot1/prc1	  dark-­‐grown	  hypocotyls	  at	  29°C	  were	  visualized	  as	  projections	  of	  movement	  using	  time-­‐lapse	  image	  sequences	  via	  near-­‐TIRF	  microscopy.	  	  A)	  The	  histogram	  displays	  the	  overall	  distribution	  of	  CSC	  velocity.	  (B)	  Cell	  morphology	  was	  similar	  to	  wild	  type	  and	  CSCs	  displayed	  a	  velocity	  of	  349	  ±	  95	  nm/min	  (n	  =	  500	  particles	  from	  13	  cells	  in	  4	  seedlings).	  (C)	  Kymographs	  were	  produced	  for	  each	  trajectory.	  CSC	  velocity	  was	  calculated	  from	  the	  slope	  of	  kymographs.	  	   60 3.3.5	  CSCs	  in	  RIC1-­‐OX	  exacerbate	  wild	  type	  response	  to	  rapid	  elongation	  by	  increasing	  velocity	  The	  overexpression	  of	  RIC1	  lead	  to	  a	  significant	  increase	  in	  overall	  complex	  velocity	  at	  434	  ±	  132	  nm/min	  (Fig.	  3.9).	  CSC	  velocity	  in	  the	  RIC1-­‐OX	  line	  exceeded	  the	  wild-­‐type	  increase	  in	  CSC	  velocity	  that	  occurs	  under	  rapid	  expansion	  at	  higher	  temperatures.	  Cellular	  morphology	  also	  appeared	  to	  have	  enhanced	  anisotropy	  with	  narrower	  and	  longer	  cells,	  as	  previously	  reported	  (Fujita	  et	  al.	  2011).	  CSC	  trajectories	  were	  visibly	  similar	  to	  those	  observed	  in	  wild	  type	  but	  appeared	  to	  be	  more	  consistently	  transverse	  to	  the	  growth	  axis	   61 and	  shorter	  in	  length.  Figure	  3.9|	  CSC	  velocity	  in	  RIC1-­‐OX.with	  YFP-­‐CESA6	  in	  prc1	  	  YFP-­‐CESA6	  tagged	  CSC	  trajectories	  in	  RIC1-­‐OX/prc1	  dark-­‐grown	  hypocotyls	  at	  29°C	  were	  visualized	  as	  projections	  of	  movement	  using	  time-­‐lapse	  image	  sequences	  via	  near-­‐TIRF	  microscopy.	  (A)	  The	  histogram	  displays	  the	  overall	  distribution	  of	  CSC	  velocity.	  (B)	  Cell	  morphology	  was	  similar	  to	  wild	  type	  and	  CSCs	  displayed	  a	  velocity	  of	  434	  ±	   62 135	  nm/min	  (n	  =	  500	  particles	  from	  19	  cells	  in	  9	  seedlings).	  (C)	  Kymographs	  were	  produced	  for	  each	  trajectory.	  CSC	  velocity	  was	  calculated	  from	  the	  slope	  of	  kymographs.	    Figure	  3.1A|	  Comparison	  of	  CSC	  velocity	  in	  wild	  type,	  clasp-­‐1,	  bot1,	  and	  RIC1-­‐OX	  seedlings	  mean	  CSC	  velocity	  and	  frequency	  (%).	  	   (A)	  At	  29°C	  clasp-­‐1	  seedlings	  showed	  a	  17%	  increase	  in	  CSC	  velocity	  (405	  ±	  119	  nm/min)	  from	  wild	  type	  (346	  ±	  121	  nm/min).	  bot1	  seedlings	  showed	  no	  significant	  change	  (349	  ±	  95	  nm/min)	  in	  velocity	  relative	  to	  wild	  type.	  	  In	  RIC1-­‐OX	  seedlings,	  CSCs	  traveled	  at	  an	  even	  more	  increased	  25%	  compared	  (434	  ±	  135	  nm/min)	  to	  wild	  type	  velocity.	  (B)	  clasp-­‐1	  distribution	  of	  CSCs	  by	  frequency	  (%).	  (C)	  bot1	  distribution	  of	  CSCs	  by	  frequency	  (%).	  (D)	  RIC1-­‐OX	  distribution	  of	  CSCs	  by	  frequency	  (%).	  Asterisks	  indicate	  significant	  differences	  relative	  to	  wild	  type	  determined	  with	  one	  way	  ANOVA	  test;	  p-­‐values	  <	  .0001.	  Post-­‐hoc	  comparisons	  between	  assessments	  were	  made	  using	  the	  Tukey	  range	  test;	  p-­‐values	  <	  .0001.	  	   	   63 3.4	  Discussion: Previously,	  Fujita	  et	  al	  (2013)	  determined	  that	  wall	  crystallinity	  and	  CSC	  velocity	  are	  both	  reduced	  in	  any1	  mutants	  but	  total	  cellulose	  produced	  is	  unaffected.	  This	  indicates	  that	  CSC	  velocity	  is	  to	  a	  certain	  extent	  dependent	  on	  the	  physical	  characteristics	  of	  the	  CesA	  subunits	  to	  properly	  crystallize	  glucan	  chains	  into	  microfibrils.	  In	  this	  study,	  I	  found	  that	  CSC	  velocity	  was	  reduced	  further	  in	  any1	  mutants	  after	  treatment	  with	  oryzalin.	  The	  additive	  nature	  of	  this	  phenotype	  suggests	  that	  the	  reduction	  in	  CSC	  velocity	  brought	  on	  by	  oryzalin’s	  inhibition	  of	  microtubule	  polymerization	  is	  independent	  of	  the	  CSC’s	  catalytic	  activity. The	  observed	  decrease	  in	  CSC	  velocity	  in	  the	  any1/mor1-­‐1	  double	  mutant	  (relative	  to	  the	  any1	  single	  mutant)	  was	  not	  found	  to	  be	  significant	  but	  was	  in	  contrast	  to	  the	  predicted	  increase	  of	  CSC	  velocity	  associated	  with	  mor1-­‐1.	  This	  suggests	  that	  the	  relationship	  between	  microtubule	  dynamics	  and	  CSC	  activity	  is	  more	  complex	  than	  is	  discernible	  by	  these	  data	  alone.  Alterations	  in	  expression	  of	  specific	  MAPs	  and	  associated	  proteins	  investigated	  in	  this	  thesis	  mostly	  exhibit	  altered	  anisotropy	  as	  a	  result	  of	  changes	  in	  organization	  and/or	  polymerization	  of	  the	  microtubules.	  However,	  in	  the	  case	  of	  CLASP,	  it	  is	  more	  highly	  expressed	  in	  root	  and	  young	  meristematic	  tissues	  (Ambrose	  et	  al.	  2007).	  Unlike	  the	  other	  mutants	  explored	  in	  this	  thesis,	  clasp-­‐1	  mutants	  do	  not	  solely	  display	  defects	  in	  anisotropy.	  While	  plants	  display	  stunted	  growth,	  seedlings	  fail	  more	  consistently	  at	  cellular	  division	  than	  elongation.	  The	  increase	  in	  CSC	  velocity	  in	  clasp-­‐1	  is	  significant,	  but	  it	  does	  not	  continue	  the	  assertion	  that	  crystallinity	  is	  correlated	  to	  CSC	  velocity.	  The	  correlations	  observed,	  however,	  are	  in	  mutant	  seedlings	  with	  defects	  in	  anisotropy.	  In	  clasp-­‐1,	   64 anisotropy	  defects	  stemming	  from	  failed	  cellular	  division	  may	  disconnect	  the	  observed	  correlations	  seen	  in	  anisotropy	  mutants	  mor1-­‐1	  and	  any1,	  explaining	  the	  increased	  CSC	  velocity	  and	  unchanged	  crystallinity	  in	  clasp-­‐1	  (Fujita	  unpublished	  data).  RIC1	  and	  KATANIN	  are	  both	  highly	  expressed	  in	  hypocotyls	  during	  elongation	  and	  assist	  in	  some	  microtubule-­‐mediated	  responses	  for	  determining	  anisotropy.	  While	  both	  affect	  the	  organization	  of	  microtubules	  in	  the	  cell,	  they	  do	  so	  in	  distinct	  ways.	  This	  difference	  may	  be	  the	  impactful	  discrepancy	  in	  the	  two	  lines.	  In	  botero1,	  CSC	  mean	  velocity	  is	  unchanged	  compared	  to	  wild-­‐type	  values,	  but	  this	  is	  taken	  as	  an	  average	  and	  exhibits	  the	  same	  potential	  for	  hidden	  information	  as	  those	  performed	  previously	  (Fujita	  et	  al.	  2011,	  Fujita	  et	  al.	  2013).	  Considering	  the	  previous	  results	  concerning	  mor1-­‐1,	  which	  also	  alters	  microtubule	  organization	  as	  well	  as	  polymerization,	  some	  parallels	  can	  be	  drawn	  to	  understand	  the	  connection	  between	  which	  states	  of	  microtubule	  disarray	  are	  most	  effective	  at	  disrupting	  cellulose	  synthesis.	  In	  mor1-­‐1	  as	  well	  as	  bot1,	  we	  see	  a	  continuation	  of	  the	  trend	  correlating	  CSC	  velocity	  and	  crystallinity.	  It	  may	  be	  that	  failed	  anisotropy	  is	  a	  reflection	  of	  the	  inability	  of	  the	  cell	  to	  properly	  regulate	  CSC	  velocity	  and	  crystallinity,	  which	  increases	  its	  CSC	  velocity	  but	  decreases	  its	  wall	  crystallinity.	  While	  both	  bot1	  and	  mor1-­‐1	  fail	  to	  grow	  anisotropically,	  their	  velocity	  profiles	  are	  dissimilar	  as	  averages	  of	  inside	  and	  outside	  domain	  values,	  indicating	  that	  the	  relationship	  of	  CSC	  velocity	  with	  anisotropic	  growth	  is	  not	  dependent	  on	  the	  organization	  of	  microtubules	  alone.	  	  RIC1-­‐OX	  seedlings	  stray	  from	  this	  correlation	  of	  CSC	  velocity	  and	  crystallinity.	  CSC	  mean	  velocity	  in	  RIC1-­‐OX	  seedlings	  has	  an	  overall	  net	  increase,	  exhibiting	  the	  highest	  average	   65 velocity	  profile	  of	  any	  reported	  CSC.	  bot1	  and	  mor1-­‐1,	  both	  loss-­‐of-­‐function	  mutations,	  exhibit	  markedly	  more	  similar	  cellular	  phenotypes	  than	  either	  of	  them	  share	  with	  RIC1-­‐OX,	  a	  gain-­‐of-­‐function	  mutation.	  Microtubules	  in	  the	  mor1-­‐1	  and	  bot1	  mutants	  are	  in	  disarray	  and	  disorganized	  versus	  being	  hyper	  organized	  into	  transverse	  arrays	  in	  RIC1-­‐OX.	  It	  has	  yet	  to	  be	  determined	  via	  domain	  analysis	  whether	  RIC1-­‐OX	  and	  bot1	  may	  more	  directly	  influence	  CSC	  velocity	  or	  if	  their	  effects	  are	  based	  more	  on	  the	  physical	  disruption	  of	  microtubule	  tracks	  and	  associated	  proteins.	  When	  both	  organization	  and	  degree	  of	  microtubule	  polymerization	  are	  affected,	  the	  overall	  result	  is	  the	  same	  in	  both	  genotypes;	  CSC	  velocity	  increases.	  Even	  though	  RIC1-­‐OX	  and	  mor1-­‐1	  are	  opposites	  on	  the	  spectrum	  of	  anisotropy,	  their	  mean	  velocities	  of	  all	  CSCs	  respond	  appropriately	  as	  wild	  type	  does	  with	  a	  further	  net	  increase.	  This	  appears	  to	  link	  the	  degree	  of	  microtubule	  polymerization,	  positively	  or	  negatively,	  with	  an	  increase	  in	  CSC	  velocity.	  As	  a	  stand	  alone,	  RIC1-­‐OX	  seedlings	  closely	  mirrored	  and	  even	  enhanced	  the	  adaptive	  response	  observed	  in	  wild	  type	  to	  rapid	  elongation	  by	  reducing	  wall	  crystallinity	  (Fujita	  et	  al.	  2011)	  and	  increasing	  CSC	  velocity.	  This	  may	  confirm	  the	  ideal	  qualifications	  necessary	  for	  proper	  anisotropy	  as	  partially	  dependent	  upon	  appropriately	  amorphous	  cellulose	  being	  produced	  by	  complexes	  moving	  more	  quickly;	  a	  process	  determined	  largely	  by	  specific	  interactions	  with	  the	  CSC	  by	  associated	  proteins.  Thus,	  altered	  availability	  of	  MAPs	  and	  other	  necessary	  interacting	  proteins	  required	  for	  cellulose	  synthesis	  is	  a	  growing	  explanation	  for	  the	  altered	  velocity	  profiles	  amongst	  MAP	  mutants.	  The	  more	  dynamic	  plus	  ends	  of	  microtubules	  are	  oftentimes	  associated	  with	  MAPs	  performing	  various	  functions	  and	  their	  unavailability	  or	  inability	  to	  access	  key	   66 regulatory	  aspects	  of	  cellulose	  synthesis	  may	  be	  the	  cause	  for	  failed	  anisotropy.	  In	  mutants	  with	  microtubule	  disorganization	  as	  the	  main	  phenotype,	  such	  as	  bot1,	  the	  plus	  end	  dynamics	  are	  not	  altered	  or	  disrupted,	  only	  mislocalized,	  and	  associated	  proteins	  are	  free	  to	  act	  normally.	  In	  mutants	  such	  as	  mor1-­‐1,	  or	  the	  overexpression	  of	  RIC1,	  the	  change	  in	  polymer	  mass	  (Fu	  et	  al.	  2005,	  Fujita	  et	  al.	  2011)	  may	  directly	  influence	  the	  known	  direct	  interactions	  with	  CSCs,	  relating	  the	  changes	  in	  velocity	  or	  appropriate	  responses	  in	  crystallinity	  to	  other	  known	  and	  unknown	  CSC-­‐associated	  proteins,	  such	  as	  CSI1	  and	  KOR1.	  	   	   67 Chapter	  4:	  Summary	  &	  Future	  Directions  The	  use	  of	  improved	  optical	  plane	  selection	  and	  precise	  environmental	  controls	  via	  near-­‐TIRF	  microscopy	  provided	  enough	  evidence	  to	  question	  the	  interpretation	  of	  previous	  studies’	  on	  CSC	  movement.	  The	  most	  noted	  and	  direct	  example	  stems	  from	  the	  lack	  of	  environmental	  controls	  on	  studied	  systems.	  Considering	  both	  microtubule	  dynamicity	  and	  CSC	  motility	  are	  kinetic	  processes,	  earlier	  literature	  discussing	  the	  relationship	  of	  anisotropy	  to	  CSC	  velocity	  must	  be	  seriously	  reevaluated.	  The	  data	  that	  exists	  for	  various	  cellulose	  synthesis	  mutants,	  MAP	  mutants,	  and	  other	  relevant	  wall	  proteins	  and	  polysaccharides	  shown	  in	  these	  thesis	  projects	  and	  previous	  literature	  has	  standardized	  environmental	  conditions	  for	  growth	  as	  necessary	  to	  properly	  compare.	  Similarly,	  comparisons	  have	  been	  made	  between	  various	  CESA	  isoforms	  despite	  the	  reduced	  support	  for	  functional	  redundancy.	  To	  fully	  understand	  the	  impact	  on	  cellulose	  synthesis	  in	  mutants	  such	  as	  those	  researched	  here,	  velocity	  profiles	  of	  each	  genotype	  or	  treatment	  should	  be	  generated	  under	  monitored	  conditions,	  and	  specific	  CESA	  comparisons	  should	  be	  maintained.	  	  It	  remains	  to	  be	  determined	  how	  the	  alteration	  of	  microtubule	  dynamics	  or	  the	  defect	  in	  the	  MOR1	  protein	  itself	  in	  mor1-­‐1	  could	  affect	  the	  CSC	  velocity	  in	  microtubule	  domains.	  It	  is	  also	  unsure	  if	  the	  correlation	  of	  velocity	  to	  crystallinity	  previously	  proposed	  holds	  true	  amidst	  increasing	  microtubule	  domain	  data.	  The	  trend	  correlating	  changes	  in	  CSC	  velocity	  to	  crystallinity	  in	  mor1-­‐1	  and	  any1	  loss-­‐of-­‐function	  mutants	  (Fujita	  et	  al	  2011,	  Fujita	  et	  al	  2013),	  without	  microtubule	  domain	  data	  in	  lines	  such	  as	  any1,	  is	  rendered	  inconclusive.	  Since	  bot1	  and	  RIC1-­‐OX	  seedlings	  had	  their	  crystallinity	  measured	  in	  inflorescence	  stems,	   68 there	  is	  some	  uncertainty	  in	  the	  comparisons	  between	  all	  the	  genotypes.	  Data	  does	  not	  exist	  on	  the	  immediate	  effect	  of	  crystallinity	  on	  oryzalin-­‐treated	  seedlings,	  and	  similarly	  upsets	  proper	  comparisons	  between	  the	  seedlings.	  Tests	  to	  assess	  crystallinity	  in	  all	  of	  these	  situations	  more	  accurately	  would	  provide	  more	  concrete	  definitions	  of	  the	  velocity	  and	  crystallinity	  relationship	  than	  we	  have	  currently	  (Fig.	  4.1).	  The	  any1,	  bot1,	  and	  mor1-­‐1	  mutants	  display	  a	  direct	  correlation	  between	  their	  crystallinity	  and	  velocity,	  while	  RIC1-­‐OX	  exhibits	  an	  inverse	  correlation	  during	  rapid	  elongation	  and	  the	  velocity	  alteration	  in	  clasp-­‐1	  may	  be	  related	  to	  processes	  other	  than	  growth	  anisotropy.  Figure	  4.1|	  CSC	  Velocity	  versus	  percent	  change	  in	  crystallinity	  from	  wild	  type.	   Data	  points	  representing	  each	  genotype	  follow	  the	  notation	  (%	  change	  of	  crystallinity	  from	  wild	  type,	  mean	  CSC	  velocity);	  WT	  (reduced	  from	  WT	  21°C,	  346	  nm/min),	  WT	  at	  21°C	  (0,	  84	  nm/min),	  any1	  (-­‐4.7,	  268	  nm/min),	  RIC1-­‐OX3	  (4,	  434	  nm/min),	  bot14	  (-­‐1.5,	  349	  nm/min),	  clasp-­‐15	  (unchanged	  from	  WT	  29°C	  1,	  405	  nm/min),	  mor1-­‐1	  (unchanged	  from	  WT	  21°C,	  415	  nm/min),	  mor1-­‐1	  at	  21°C	  (-­‐2,	  71	  nm/min).	  Green	  coloration	  denotes	  enhanced	  or	  regular	  anisotropy	  has	  been	  observed.	  Red	  coloration	  denotes	  mutations	  that	  affect	  anisotropy	  negatively.	  Shaded	  colors	  indicate	  velocity	  data	  from	  seedlings	  at	  21°C2. iFrom	  Fujita	  unpublished,	  2From	  Fujita	  et	  al.	  2011,	  3From	  Wu	  et	  al.	  2001,4From	  Bichet	  et	  al.	  2001,	  5From	  Ambrose	  et	  al.	  2007.  69  It	  would	  be	  of	  interest	  to	  see	  if	  the	  functional	  MOR1	  and	  partially	  functional	  mor1-­‐1	  proteins	  colocalize	  with	  CSCs	  or	  how	  they	  act	  differently	  in	  their	  microtubule	  associations;	  a	  difficult	  process	  due	  to	  the	  large	  number	  of	  exons	  found	  in	  the	  gene.	  By	  comparing	  the	  interactions	  of	  MOR1	  and	  mor1-­‐1	  with	  CSCs,	  it	  may	  be	  possible	  to	  observe	  the	  timing	  and	  length	  of	  the	  interaction,	  if	  any,	  as	  well	  as	  to	  see	  if	  other	  aspects	  of	  CSC	  dynamics,	  such	  as	  overall	  lifetime	  at	  the	  plasma	  membrane	  or	  turnover	  rate,	  are	  strongly	  affected.	  Double	  mutants	  of	  MOR1	  with	  proteins	  such	  as	  KORRIGAN1	  and	  the	  CC	  proteins	  could	  provide	  valuable	  information	  on	  how	  intimately	  MOR1	  is	  involved	  in	  CSC	  localization	  to	  microtubules	  or	  in	  modifying	  its	  velocity.	  Mutants	  such	  as	  botero1,	  which	  show	  no	  significant	  changes	  in	  velocity	  or	  crystallinity,	  but	  do	  show	  altered	  microtubule	  organization,	  could	  impact	  mor1-­‐1	  localization	  in	  a	  double	  mutant	  and	  effect	  CSC	  velocity	  even	  more	  drastically.	  Since	  the	  data	  here	  do	  not	  confirm	  or	  deny	  MOR1’s	  involvement	  with	  the	  CSC,	  experiments	  such	  as	  these	  are	  necessary	  to	  define	  the	  role	  MOR1	  has	  in	  modifying	  CSC	  velocity.	  	    Other	  data	  found	  here,	  such	  as	  the	  velocity	  profile	  for	  the	  clasp-­‐1	  mutant,	  as	  well	  as	  previously	  reported	  data,	  like	  the	  reduced	  CSC	  velocity	  profiles	  of	  xyloglucan-­‐deficient	  double	  mutant	  xxt1/xxt2	  (Xiao	  et	  al.	  2016),	  have	  placed	  emphasis	  on	  some	  less	  direct	  mechanisms	  for	  modulating	  cellulose	  synthesis.	  It	  was	  also	  found	  here	  that	  microtubule	  stability	  was	  impacted,	  implying	  a	  feedback	  loop	  that	  could	  incorporate	  microtubules	  not	  only	  in	  the	  transports	  of	  polysaccharides	  to	  the	  wall,	  but	  in	  mechanical	  signaling	  for	  proper	  wall	  synthesis.	  Even	  though	  CLASP1	  nor	  xyloglucan	  are	  specific	  to	  elongating	  cells	  in	  the	   70 hypocotyl,	  both	  impact	  CSC	  velocity	  in	  that	  tissue;	  a	  result	  possibly	  stemming	  from	  the	  mechanical	  feedback	  noted	  in	  xxt1/xxt2	  mutants	  and	  failed	  division	  in	  clasp-­‐1	  mutants.	  	  Interactions	  with	  other	  proteins,	  and	  thus	  investigation	  into	  the	  modification	  of	  xyloglucan	  in	  genotypes	  with	  altered	  microtubules,	  is	  key	  for	  understanding	  if	  crystallinity	  is	  regulated	  directly	  by	  more	  than	  just	  the	  activity	  of	  the	  CESAs.	  By	  performing	  such	  a	  cross	  in	  this	  thesis	  with	  mor1-­‐1	  and	  any1,	  based	  solely	  on	  their	  opposite	  velocity	  and	  crystallinity	  profiles,	  we	  discovered	  that	  the	  effects	  on	  CSC	  velocity	  are	  not	  additive.	  It	  was	  considered	  possible	  that	  their	  opposite	  natures	  could	  enhance	  either	  phenotype	  or	  have	  no	  effect.	  It	  appears,	  therefore,	  that	  there	  are	  numerous	  pathways	  for	  separately	  modulating	  cellulose	  synthesis	  and	  crystallization,	  but	  domain	  data	  must	  be	  attributed	  appropriately	  to	  actually	  understand	  the	  impact	  of	  the	  manipulation	  before	  any	  concrete	  evaluations	  can	  be	  made.	    In	  both	  bot1	  and	  RIC1-­‐OX,	  hidden	  domain	  data	  may	  reveal	  discrepancies	  from	  expectations.	  Since	  microtubule	  disorganization	  had	  little	  effect	  on	  CSC	  association	  with	  microtubules	  in	  the	  mor1-­‐1	  mutant,	  it	  is	  possible	  that	  this	  may	  be	  similarly	  unaffected	  in	  bot1.	  Confounding	  results	  may	  also	  be	  present	  in	  the	  RIC-­‐OX	  dataset.	  While	  the	  RIC1-­‐OX	  construct	  appears	  to	  enhance	  the	  wild	  type	  anisotropy	  response,	  it	  remains	  unknown	  whether	  the	  increase	  in	  organization	  and	  polymer	  mass	  will	  have	  as	  little	  of	  effect	  on	  localization	  of	  the	  CSC	  as	  it	  does	  in	  mor1-­‐1.	  Until	  it	  has	  been	  properly	  explored,	  it	  is	  uncertain	  if	  we	  can	  attribute	  the	  enhanced	  anisotropy	  to	  a	  protein-­‐specific	  interaction	  or	  the	  theorized	  increased	  availability	  of	  associated	  proteins	  with	  the	  CSC.	  More	  experiments	  investigating	  domain	  data	  and	  their	  in	  vivo	  dynamics	  in	  relation	  to	  the	  CSC	  are	  necessary.	     71 When	  considered	  alongside	  data	  such	  as	  that	  found	  in	  the	  clasp-­‐1	  mutant	  or	  the	  xyloglucan-­‐deficient	  xxt1/xxt2	  mutants,	  the	  diversity	  of	  interactions	  that	  impact	  the	  CSC	  during	  rapid	  elongation	  is	  extensive.	  The	  CSC	  can	  be	  modulated	  via	  its	  own	  constituents	  (any1),	  direct	  interactions	  (POM2),	  not	  yet	  fully	  defined	  MAP	  interactions	  (MOR1,	  BOT1,	  CLASP,	  RIC1),	  other	  microtubule-­‐related	  perturbations	  (transport,	  degree	  of	  dynamicity),	  or	  a	  number	  of	  biomechanical	  cellular	  properties	  (membrane	  lipidic	  composition,	  mechanical	  stresses,	  etc).	  Some	  of	  these	  results	  can	  be	  distinguished	  simply	  by	  assessing	  the	  influence	  of	  microtubule	  association	  on	  CSC	  velocity.	  Distinct	  changes	  within	  or	  outside	  of	  domains	  could	  indicate	  a	  more	  direct	  linkage	  between	  the	  protein	  and	  the	  CSC/associated	  proteins,	  or	  other	  wall	  polysaccharides.	  	  	  	   	   72 References	   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