Estimación de cargas y balanceo del circuito 3

Based   on   our   working   model   of   ring   peeling   (Figure   3.8A),   our   second   prediction   was   that  adf1   mutant   cells   would   display   septation   defects,   due  to  the  hypothesised  imbalance  of  tension  around  the  ring.  To  test  for   this,  we  performed  CW  staining  on  WT,  adf1-­‐M2  and  adf1-­‐M3  cells,  and   quantified   the   different   septa   morphologies   that   we   observed,   performing   separate   analysis   for   fully-­‐septated   and   partially-­‐septated   cells.  For  the  fully  septated  cells,  we  saw  only  flat,  hemispherical,  or  wavy   septa   in   control   cells.   We   reasoned   that   the   wavy   septa   could   be   the   result   of   non-­‐uniform   septum   deposition,   and   therefore   an   indicator   of   non-­‐uniform  ring  tension.  By  quantifying  the  relative  proportion  of  each   morphology,   we   found   that   the   majority   of   WT   cells   displayed   either   hemispherical  or  flat  septa,  with  a  minority  showing  wavy  septa  (Figure   3.11A).   In  adf1-­‐M2   and  adf1-­‐M3   cells,   we   saw   a   greater   proportion   of   wavy   septa   compared   to   the   control   cells,   and   we   also   observed   a   number   of   cells   that   had   either   failed   septation   and   undergone   branching,   displayed   a   misplaced   hemispherical   septum,   or   cells   where   the   septum   was   particularly   bright   and   messy,   and   it   was   difficult   to   determine  a  specific  morphology  (Figure  3.11A).  

Next,   we   analysed   the   partially-­‐septated   cells,   by   looking   for   evidence   of   asymmetric   septum   deposition.   We   did   so   by   visually   examining  the  partially  septated  cells  in  our  images,  and  looking  for  cells   where  bright  CW  staining  (indicating  deposition  of  septum  material)  was   only   visible   partway   around   the   cell   circumference,   or   where   septation   was  almost  complete  and  the  remaining  hole  in  the  septum  was  radially   displaced  from  the  centre  of  the  division  plane  (Figure  3.11B  and  Figure  

0 0.2 0.4 0.6 Fraction adf1+ (N = 71) adf1-M2 (N = 246) adf1-M3 (N = 148) Not shown: 0 0.2 0.4 0.6 Fraction adf1+ (N = 71) adf1-M2 (N = 246) adf1-M3 (N = 148) Not shown: (A)$ (B)$ adf1+% adf1"M3% adf1"M2% CW(staining(

(low(intensity(">(Cell(wall,(high(intensity(">(Septum)(

Par?al(septa( Strain( Symmetric( Asymmetric(

adf1+% 37/38( 1/38((2.6(%)( adf1"M2% 63/102( 39/102((38(%)( adf1"M3% 73/89( 16/89((18(%)( adf1+% adf1"M2% adf1"M3% CW(staining( (C)$

Figure$13:$Effect$of$adf1$mutants$on$septa;on$

(A) Quan?fica?on(of(septum(morphology(in(fully(septated(WT,(adf1"M2(and(adf1"M3(cells,(from(CW(staining(of(fixed(cells.(

Septa(were(categorised(as(being(straight,(hemispherical((data(for(these(two(not(shown),(wavy((white),(branched(and( failed(septa?on((light(grey),(misplaced(hemispherical((dark(grey),(or(bright(and(improperly(organised((black).(

(B)  Representa?ve(images(of(symmetric(septa(in(WT(cells,(and(asymmetric(septa(in(adf1"M2(and(adf1"M3(cells,(from(CW(

staining(of(fixed(cells.(Images(have(been(segmented(into(low(and(high(intensity(regions,(to(represent(the(outer(cell(wall( and(the(division(septum,(respec?vely.(Table(shows(analysis(of(par?ally(septated(cells,(showing(the(propor?on(of(which( appeared(symmetric(or(asymmetric.(

(C)  Corresponding(non"segmented(images(for(those(shown(in((B),(showing(CW(staining(of(septa(in(WT,(adf1"M2,(and(adf1"

M3(cells.(

Scale(bars(in(single(?mepoint(images(are(2(μm.(

Figure  3.11:  Effect  of  adf1  mutants  on  septation.  

(A) Quantification  of  septum  morphology  in  fully  septated  WT,  adf1-­‐M2  and  adf1-­‐M3   cells,  from  CW  staining  of  fixed  cells.  Septa  were  categorised  as  being  straight,   hemispherical  (data  for  these  two  not  shown),  wavy  (white),  branched  and  failed   septation  (light  grey),  misplaced  hemispherical  (dark  grey),  or  bright  and   improperly  organised  (black).  

(B) Representative  images  of  symmetric  septa  in  WT  cells,  and  asymmetric  septa  in  

adf1-­‐M2  and  adf1-­‐M3  cells,  from  CW  staining  of  fixed  cells.  Images  have  been   segmented  into  low  and  high  intensity  regions,  to  represent  the  outer  cell  wall  and   the  division  septum,  respectively.  Table  shows  analysis  of  partially  septated  cells,   showing  the  proportion  of  which  appeared  symmetric  or  asymmetric.  

(C) Corresponding  non-­‐segmented  images  for  those  shown  in  (B),  showing  CW  staining   of  septa  in  WT,  adf1-­‐M2,  and  adf1-­‐M3  cells.  

3.11C).   In   WT   cells   we   only   observed   1/38   (2.6   %)   partially-­‐septated   cells   with   asymmetric   septum   deposition.   By   contrast,   in  adf1-­‐M3   cells   we   found   that   16/89   (18   %)   cells   displayed   asymmetric   septum   deposition,  whilst  in  adf1-­‐M2  cells  this  increased  to  39/102  (38  %)  of  the   partially  septated  cells  (Figure  3.11B).  

The   observation   that   more   adf1-­‐M2   cells   display   asymmetric   septum   deposition   than  adf1-­‐M3   cells   makes   sense,   as  adf1-­‐M2   is   the   more  severe  of  the  two  mutants,  so  it  would  presumably  have  the  greater   effect   on   ring   tension   during   AMR   contraction   [66].   Whilst   we   did   not   quantify   the   degree   of   asymmetry,   qualitative   observations   would   also   suggest   that   the   partial   septa   in  adf1-­‐M2   cells   were   more   asymmetric   than  those  in  adf1-­‐M3  cells,  especially  when  comparing  cells  at  the  later   stages  of  septation  (Figure  3.11B  and  Figure  3.11C).  However,  this  raises   the  questions  of  why  we  observe  slightly  more  aberrant  septa  in  adf1-­‐M3   cells  when  quantifying  the  fully  septated  cells  (Figure  3.11A).  Perhaps  it   is   the   case   that   a   greater   portion   of   the   septa   in  adf1-­‐M2   cells   are   so   asymmetric   that   they   are   unable   to   successfully   complete   septation   [134],   which   reduces   the   number   of   aberrant   septa   we   observe   when   quantifying   fully   septated   cells,   and   increases   the   portion   of   partially   septated  cells  that  are  observed  to  be  asymmetric.  

As   this   data   indicates   that   septum   synthesis   is   defective   in  adf1   mutant   cells,   this   also   supports   our   working   model   that   reduced   actin   turnover   during   AMR   contraction   leads   to   a   non-­‐uniform   tension   distribution  around  the  ring,  which  then  causes  ring  peeling  to  occur  at   regions  of  increased  tension  (Figure  3.8A).  

3.12. Attempting   to   recreate   and   rescue   the   ring   peeling