What is the cause of ring peeling in adf1 mutant cells? 98

circumferential   tension   imbalance   in   the   AMR   (Figure   3.8A).   This   idea   was   inspired   by   recent   work   performed   in   Drosophila   embryos,   examining   the   role   of   actin   dynamics   during   gastrulation   [151].   In   this   study,   the   authors   targeted   Profilin,   Cofilin,   and   Cyclase-­‐associated   protein   (all   of   which   are   involved   in   the   turnover   of   actin),   and   also   injected  cells  with  latrunculin  A  (actin  monomer  sequestering  drug)  and   phalloidin  (actin  disassembly  inhibitor),  in  order  to  probe  the  effects  of   reduced  actin  turnover.  The  authors  found  that  when  actin  turnover  was   reduced   the   balance   of   tension   along   the   apical   surface   was   lost,   and  

epithelial   cells   would   often   become   stretched   and   distorted,   and   subsequently  some  of  the  embryos  even  failed  to  form  a  ventral  furrow   during  apical  constriction  [151].  

When   trying   to   find   a   possible   explanation   for   our   peeling   phenotype,   we   realised   a   similar   model   would   also   provide   an   explanation  for  our  observations  in  S.  pombe:  If  reduced  turnover  of  actin   leads   to   tension   imbalance   around   the   ring,   then   some   regions   will   experience  a  higher  inwards  force  [134],  and  these  regions  may  undergo   peeling.  We  do  not  know  whether  a  non-­‐uniform  distribution  of  tension   is   a   common   feature   of   contractile   actomyosin   systems   when   actin   turnover   has   been   reduced.   The   importance   of   actin   turnover   for   the   generation  of  tension  and  contractility  has  only  been  realised  as  a  result   of  theoretical  studies  [1],  so  it  seems  likely  that  further  theoretical  work   would  be  the  best  way  to  determine  the  effect  of  reduced  actin  turnover   within  an  AMR,  and  whether  this  leads  to  tension  heterogeneity.  

It  has  also  been  hypothesised  that  ADF/Cofilin  proteins  are  able  to   regulate   actomyosin   assembly   and   contractility,   not   just   by   severing   and/or   depolymerising   actin   filaments,   but   by   also   competing   with   myosin   II   for   actin   binding   sites   [152].   The   authors   of   this   study   used   ADF/cofilin   from   a   range   of   organisms   (human,   chick,   Xenopus,   Drosophila,  acanthamoeba,  starfish  and  yeast  –  presumably  S.  cerevisiae)   to  perform  F-­‐actin  cosedimentation  along  with  myosin  S1  fragments.  By   doing  so,  they  found  that  the  molar  ratio  of  actin-­‐bound  myosin  S1  to  F-­‐ actin  decreased  as  the  concentration  of  ADF/cofilin  increased,  indicating   that  the  ADF/cofilin  is  able  to  competitively  inhibit  binding  of  myosin  II   to   F-­‐actin.   If   this   is   also   the   case   for   Adf1   in  S.  pombe,   then   this   would   further   support   our   model,   as   it   was   found   that   Adf1-­‐M2   and   Adf1-­‐M3   have   reduced   actin   binding   affinity,   which   would   create   more   actin   binding   sites   for   myosin   II   in   the   ring,   and   therefore   increase   ring   tension.  If  the  overall  ring  tension  is  higher,  in  addition  to  there  being  an   imbalance   of   tension   around   the   ring,   then   this   would   further   increase   the  propensity  for  ring  peeling  to  occur  in  adf1-­‐M2  and  adf1-­‐M3  cells.  

Additionally,   this   may   provide   a   potential   explanation   for   the   differences  in  phenotypes  observed  between  adf1-­‐M2/adf1-­‐M3  cells  and   adf1-­‐1   cells:   Adf1-­‐M2   and   Adf1-­‐M3   were   previously   biochemically   characterised  and  found  to  have  reduced  binding  to  actin  filaments  [66],   while  Adf1-­‐1  has  not  undergone  such  characterisation.  It  is  possible  that   Adf1-­‐1  displays  more  normal  binding  kinetics  to  actin  filaments,  and  only   struggles  with  severing/depolymerisation,  for  example.  If  this  is  the  case,   then   Adf1-­‐1   would   still   compete   with   myosin   II   for   actin   binding   sites,   unlike   Adf1-­‐M2   and   Adf1-­‐M3,   meaning   that   the   overall   ring   tension   would  not  be  increased,  which  would  then  affect  the  exact  behaviour  of   the  peeling  bundles  (Figure  3.1B,  Figure  3.1C,  Figure  3.2B).  

Our  model  of  tension  imbalance  does  not  answer  the  question  of   how   a   peeled   bundle   subsequently   moves   across   the   ring   after   it   has   peeled   off:   Does   the   peeling   bundle   itself   contract,   or   is   it   reeled   in   through   its   attachment   points   (which   also   move   around   the   ring)?   Our   observation   of   a   bundle   that   peels   off   from   a   part   of   the   ring   with   a   noticeable  kink  would  support  the  second  idea,  as  the  kink  is  also  present   in   the   peeling   bundle,   which   suggests   that   the   central   region   of   the   bundle   is   not   contracting   or   under   tension   (Figure   3.1F).   This   implies   that   peeling   bundles   are   reeled   in   at   their   attachment   points,   with   the   shortening   of   the   peeled   bundle   pulling   the   attachment   points   along   passively.  Quantification  of  the  shortening  rate  of  peeling  bundles  shows   that  there  does  not  appear  to  be  a  difference  between  the  unbroken  peels   and   the   reeling-­‐in   of   the   snapped   bundles   (Figure   3.3D),   which   further   suggests  that  the  tension  stored  within  a  peeled  bundle  is  negligible.       In  this  case,  it  would  seem  likely  that  Myo2,  which  remains  in  the   ring   during   peeling   events,   would   be   the   motor   responsible   for   the   reeling-­‐in  at  the  attachment  points,  while  Myp2  and  Myo51  may  mostly   play   a   role   in   crosslinking   the   peeling   bundle.   However,   this   does   not   explain  why  the  absence  of  either  Myp2  or  Myo51  causes  ring  peeling  to   disappear  (Figure  3.5B,  Figure  3.5H),  as  the  other  protein  would  still  be   present   and   able   to   crosslink   the   peeling   bundle.   This   is   especially   puzzling  for  the  myo51  deletion,  as  Myo51  is  only  thought  to  play  a  minor  

role  in  AMR  contraction  [71,100,106],  and  there  are  estimated  to  be  5×   fewer  molecules  of  Myo51  in  the  ring  than  there  are  of  Myp2,  at  least  in   WT  cells  [64,71].    

Nonetheless,   the   requirement   that   all   three   myosins   are   present   does   explain   why   peeling   does   not   start   until   the   final   myosin,   Myp2,   arrives  in  the  ring  (Figure  3.4A).  The  back-­‐and-­‐forth  wave-­‐train  nature  of   ring   peeling   can   also   be   explained   by   the   observation   that   most   of   the   Myp2  is  pulled  off  the  ring  on  the  peeling  bundle  (Figure  3.5D),  and  ends   up   located   on   the   opposite   side   of   the   ring.   There,   it   would   generate   tension  heterogeneity,  and  subsequently  initiate  the  next  peeling  event  at   the   site   of   maximum   Myp2   density,   possibly   by   crosslinking   the   inner   side   of   the   AMR,   causing   a   fracture   to   form   between   different   layers   of   the  ring  [100,103].  

Based  on  this,  and  our  observation  that  peeling  bundles  shorten  at   a   constant   rate   (Figure   3.3D),   this   would   suggest   that   the   time   interval   between  successive  peeling  events  gets  shorter  as  the  AMR  contracts.  We   attempted   to   see   if   this   was   the   case,   however   our   results   were   inconclusive   (Figure   3.4D).   It   is   possible   that   for   the   first   few   peeling   events  the  ring  has  not  contracted  enough  in  order  to  noticeably  shorten   the   interval   between   these   events,   and   we   did   not   observe   many   cells   undergoing   four   peeling   events,   so   it   was   difficult   to   draw   conclusions   about   what   happens   when   the   ring   is   at   a   smaller   size,   particularly   for   adf1-­‐M3  cells  (Figure  3.4D).