5.1  Conclusion.  

 

During   the   last   decades,   a   vast   amount   of   publications   have   unraveled   the   importance  of  mitochondrial  dynamics  during  cells’s  life  and  death.  Nowadays,  it   is   clear   that   the   control   of   mitochondrial   shape   through   the   balance   of   the   opposite   processes   of   fusion   and   fission   is   essential   in   mitochondrial   functions   such   as   respiration   and   cell   death.   However,   many   aspects   of   the   mechanisms   underlying  mitochondrial  dynamics  are  yet  unclear.  In  particular,  mitochondrial   fission  was  though  to  be  a  two-­‐component  system,  with  Fis1  being  the  receptor   for   the   translocation   of   Drp1   to   the   OMM.   The   discovery   of   Mff   and   MiDs   as   receptors   for   Drp1   in   mammals   challenged   the   relevance   of   Fis1   in   mitochondrial  fission.  In  this  project,  we  found  compelling  evidence  supporting   the  importance  of  Fis1  gene  in  mitochondrial  morphology  with  consequences  in   mitochondrial   functionality   and   cell   death.   Additionally,   we   report   the   unexpected   discovery   that   Fis1   is   alternatively   spliced   in   three   variants   expressed   in   several   tissues.   While   two   of   these   variants   play   a   role   in   mitochondrial  fission,  increased  mFis1.2  levels  cause  mitochondrial  elongation.  

By  mapping  the  relative  expression  of  mFis1.2  in  pathophysiological  conditions,   we   found   it   increased   during   macroautophagy   in   a   PKA-­‐dependent   manner   to   produce   mitochondrial   elongation.   Hence,   we   have   identifying   a   novel   mechanism   for   mitochondrial   morphology   changes   during   autophagy   that,  

together   with   the   already   known   mechanism   of   PKA-­‐dependent   inhibition   of   Drp1,  helps  to  enhance  respiration  in  conditions  of  nutrient  deprivation.  

 

5.2  Perspectives.  

 

Great  emphasis  has  been  placed  in  the  many  post-­‐translational  modification  that   Drp1   suffers   (phosphorylation,   ubiquitination   and   sumoylation)   and   how   they   modulate   mitochondrial   fission.   On   the   other   hand,   Drp1   receptors   have   been   treated   as   invariable   proteins,   ready   to   interact   with   Drp1   whenever   it   is   activated.   The   results   presented   in   this   work   highlight   the   relevance   of   the   modulation  of  Fis1  variant  expression,  especially  of  mFis1.2,  in  the  regulation  of   mitochondrial  morphology.  Likewise,  other  fission  factors  may  be  regulated  in  a   similar  manner.  For  example,  Mff  also  has  splice  variants  that  may  have  different   expression  according  to  internal  or  external  cues  as  we  showed  for  mouse  Fis1.  

It   is   interesting   to   note   that   only   a   small   deletion   in   the   amino-­‐terminus   of   mFsi1.2   induces   an   apposite   effect   on   mitochondrial   morphology,   given   to   the   coiled   coil   domain   in   this   region   a   regulatory   role   in   mitochondrial   dynamics.  

However,  whether  the  coiled  coil  arm  is  important  in  the  interaction  of  Fis1  with   itself   or   with   other   mitochondrial   shaping   proteins   still   has   to   be   investigated.  

Additionally,  human  Fis1  gene  is  also  able  to  produce  four  splice  variants,  some   with   differences   in   the   coiled   coil   arm,   however,   nothing   is   known   about   their   expression   pattern   and   regulation   nor   about   their   role   in   mitochondrial   morphology.  Hence,  it  is  possible  that  mitochondrial  shape  in  human  cells  may   also   be   regulated   by   Fis1   splice   variants.   To   this   regard,   it   is   of   paramount   importance   to   direct   studies   in   mouse   and   human   cells   aimed   to   address   the  

  121   hierarchical   interaction   between   the   different   Drp1   receptors   and   their   splice   variants   in   order   to   produce   a   specific   mitochondrial   shape   in   response   to   a   given   stimulus.   Most   likely,   a   detailed   mapping   of   the   mitochondrial   fission   transcriptome   and   interactome   will   contribute   to   a   clearer   view   of   how   mitochondrial  fragmentation  is  achieved.  

Finally,   mitochondria   have   a   central   role   in   cancer   and   neurodegenerative   diseases  and  data  in  this  work  clearly  show  that  mFis1  impinges  not  only  in  the   efficiency   of   mitochondrial   respiration   but   as   well   in   the   progression   of   cell   death.  Future  work  in  animal  models  should  clarify  whether  the  modulation  of   mFis1  variants  expression  could  have  an  impact  in  physiological  processes  with   pathological  consequences.  

     

 

 

Chapter  6  

determines  mitochondrial  network  architecture  and  mitochondrial  metabolism.  

A  novel  regulatory  mechanism  altered  in  obesity.  J  Biol  Chem  278:  17190-­‐17197  

  123   ligase  that  regulates  mitochondrial  fission.  EMBO  reports  10:  748-­‐754  

 

Chan  DC  (2006b)  Mitochondrial  fusion  and  fission  in  mammals.  Annual  review  of   cell  and  developmental  biology  22:  79-­‐99  

 

Chang   CR,   Blackstone   C   (2007)   Cyclic   AMP-­‐dependent   protein   kinase   phosphorylation   of   Drp1   regulates   its   GTPase   activity   and   mitochondrial   morphology.  The  Journal  of  biological  chemistry  282:  21583-­‐21587  

 

mediated   Drp1   phosphorylation   induced   elongated   mitochondrial   morphology   against  oxidative  stress.  PloS  one  7:  e49112   cytochrome  c  release  during  apoptosis  via  OPA1-­‐dependent  cristae  remodeling.  

Cell  126:  163-­‐175  

  125   SOD1G93A-­‐mediated  toxicity.  Cell  metabolism  8:  425-­‐436  

 

Dyall  SD,  Brown  MT,  Johnson  PJ  (2004)  Ancient  invasions:  from  endosymbionts   to  organelles.  Science  304:  253-­‐257  

 

Elgass   K,   Pakay   J,   Ryan   MT,   Palmer   CS   (2013)   Recent   advances   into   the   understanding  of  mitochondrial  fission.  Biochim  Biophys  Acta  1833:  150-­‐161    

Eura   Y,   Ishihara   N,   Yokota   S,   Mihara   K   (2003)   Two   mitofusin   proteins,   mammalian   homologues   of   FZO,   with   distinct   functions   are   both   required   for   mitochondrial  fusion.  Journal  of  biochemistry  134:  333-­‐344  

  controls  apoptotic  cristae  remodeling  independently  from  mitochondrial  fusion.  

Cell  126:  177-­‐189   protein  Mff  controls  mitochondrial  and  peroxisomal  fission  in  mammalian  cells.  

Mol  Biol  Cell  19:  2402-­‐2412   initiates  DRP1-­‐regulated  remodelling  of  mitochondrial  cristae  during  apoptosis.  

EMBO  J  24:  1546-­‐1556   membrane.  Journal  of  cellular  biochemistry  115:  632-­‐640  

  protein,  trigger  autophagy.  Biochim  Biophys  Acta  1777:  860-­‐866  

  127    

Gomes   LC,   Scorrano   L   (2011)   Mitochondrial   elongation   during   autophagy:   a   stereotypical  response  to  survive  in  difficult  times.  Autophagy  7:  1251-­‐1253    

Heo  JM,  Rutter  J  (2011)  Ubiquitin-­‐dependent  mitochondrial  protein  degradation.  

The  international  journal  of  biochemistry  &  cell  biology  43:  1422-­‐1426    

Herzig   S,   Raemy   E,   Montessuit   S,   Veuthey   JL,   Zamboni   N,   Westermann   B,   Kunji   ER,   Martinou   JC   (2012)   Identification   and   functional   expression   of   the   mitochondrial  pyruvate  carrier.  Science  337:  93-­‐96  

 

Hoppins   S,   Lackner   L,   Nunnari   J   (2007)   The   machines   that   divide   and   fuse   mitochondria.  Annual  review  of  biochemistry  76:  751-­‐780  

 

Ishihara  N,  Nomura  M,  Jofuku  A,  Kato  H,  Suzuki  SO,  Masuda  K,  Otera  H,  Nakanishi   Bap31  bridge  the  mitochondria-­‐ER  interface  to  establish  a  platform  for  apoptosis   induction.  The  EMBO  journal  30:  556-­‐568   mitochondrial   Fis1,   the   mitochondrial   fission-­‐stimulating   protein.  Biochemical   and  biophysical  research  communications  333:  650-­‐659  

  organelles  shows  that  mitochondrial  fusion  is  blocked  during  the  Bax  activation   phase  of  apoptosis.  J  Cell  Biol  164:  493-­‐499  

  129   Karbowski  M,  Norris  KL,  Cleland  MM,  Jeong  SY,  Youle  RJ  (2006)  Role  of  Bax  and   Bak  in  mitochondrial  morphogenesis.  Nature  443:  658-­‐662  

  (2006)  Hierarchical  regulation  of  mitochondrion-­‐dependent  apoptosis  by  BCL-­‐2   subfamilies.  Nat  Cell  Biol  8:  1348-­‐1358   fragmentation  in  neurodegeneration.  Nature  reviews  Neuroscience  9:  505-­‐518     Structural  basis  of  mitochondrial  tethering  by  mitofusin  complexes.  Science  305:  

858-­‐862    

Kroemer   G,   Galluzzi   L,   Brenner   C   (2007)   Mitochondrial   membrane   permeabilization  in  cell  death.  Physiological  reviews  87:  99-­‐163  

 

Lee   YJ,   Jeong   SY,   Karbowski   M,   Smith   CL,   Youle   RJ   (2004)   Roles   of   the   mammalian  mitochondrial  fission  and  fusion  mediators  Fis1,  Drp1,  and  Opa1  in   apoptosis.  Mol  Biol  Cell  15:  5001-­‐5011  

 

Leinninger   GM,   Backus   C,   Sastry   AM,   Yi   YB,   Wang   CW,   Feldman   EL   (2006)   Mitochondria  in  DRG  neurons  undergo  hyperglycemic  mediated  injury  through   Bim,  Bax  and  the  fission  protein  Drp1.  Neurobiology  of  disease  23:  11-­‐22   mitochondrial  function.  Biochim  Biophys  Acta  1762:  140-­‐147  

 

  131   generated  by  Cre-­‐  and  Flp-­‐mediated  recombination.  Nature  genetics  18:  136-­‐141    

Michalak   EM,   Villunger   A,   Adams   JM,   Strasser   A   (2008)   In   several   cell   types   tumour   suppressor   p53   induces   apoptosis   largely   via   Puma   but   Noxa   can   contribute.  Cell  death  and  differentiation  15:  1019-­‐1029  

  stimulates  Bax  oligomerization.  Cell  142:  889-­‐901  

  Inter-­‐mitochondrial  complementation:  Mitochondria-­‐specific  system  preventing   mice  from  expression  of  disease  phenotypes  by  mutant  mtDNA.  Nature  medicine   required  for  Parkin-­‐induced  mitochondrial  clustering  but  not  mitophagy;  VDAC1   is  dispensable  for  both.  Autophagy  6:  1090-­‐1106  

 

Narendra  D,  Tanaka  A,  Suen  DF,  Youle  RJ  (2008)  Parkin  is  recruited  selectively  to   impaired  mitochondria  and  promotes  their  autophagy.  J  Cell  Biol  183:  795-­‐803    

Nargund   AM,   Pellegrino   MW,   Fiorese   CJ,   Baker   BM,   Haynes   CM   (2012)  

dysregulated   mitochondrial   fission   and   neurodegeneration.   Acta   neuropathologica  123:  189-­‐203  

  133   during   ceramide-­‐induced   cardiomyocyte   early   apoptosis.   Cardiovascular   research  77:  387-­‐397   formation   protects   mitochondria   from   autophagosomal   degradation   during   nutrient  starvation.  Proceedings  of  the  National  Academy  of  Sciences  of  the  United   oxidative  phosphorylation  protein  complexes.  Methods  in  enzymology  260:  190-­‐

202    

Schrader   M   (2006)   Shared   components   of   mitochondrial   and   peroxisomal   division.  Biochim  Biophys  Acta  1763:  531-­‐541  

 

Scorrano   L   (2013)   Keeping   mitochondria   in   shape:   a   matter   of   life   and   death.  

European  journal  of  clinical  investigation  43:  886-­‐893     regulates   mitochondrial   morphology   and   fission   through   self-­‐interaction.  

Experimental  cell  research  314:  3494-­‐3507  

 Shen   Q,   Yamano   K,   Head   BP,   Kawajiri   S,   Cheung   JT,   Wang   C,   Cho   JH,   Hattori   N,   dynamin-­‐related  protein  controls  the  distribution  of  mitochondria.  The  Journal  of   cell  biology  143:  351-­‐358  

 

Stojanovski   D,   Koutsopoulos   OS,   Okamoto   K,   Ryan   MT   (2004)   Levels   of   human   Fis1  at  the  mitochondrial  outer  membrane  regulate  mitochondrial  morphology.  J   Cell  Sci  117:  1201-­‐1210  

  135   recognition.  The  Journal  of  biological  chemistry  276:  17484-­‐17496  

  survival  in  cortical  neurons.  Experimental  neurology  218:  274-­‐285  

 

Wakabayashi  J,  Zhang  Z,  Wakabayashi  N,  Tamura  Y,  Fukaya  M,  Kensler  TW,  Iijima   M,  Sesaki  H  (2009)  The  dynamin-­‐related  GTPase  Drp1  is  required  for  embryonic   and  brain  development  in  mice.  J  Cell  Biol  186:  805-­‐816  

 

Walker  JE  (1998)  ATP  Synthesis  by  Rotary  Catalysis  (Nobel  lecture).  Angewandte   Chemie  International  Edition  37:  2308–2319  

 

Wang  K,  Long  B,  Jiao  JQ,  Wang  JX,  Liu  JP,  Li  Q,  Li  PF  (2012)  miR-­‐484  regulates   mitochondrial  network  through  targeting  Fis1.  Nature  communications  3:  781     hFis1  regulates  mitochondrial  fission  in  mammalian  cells  through  an  interaction   with  the  dynamin-­‐like  protein  DLP1.  Mol  Cell  Biol  23:  5409-­‐5420   species  in  hyperglycemic  conditions  requires  dynamic  change  of  mitochondrial   morphology.  Proc  Natl  Acad  Sci  U  S  A  103:  2653-­‐2658  

 

  137   reticulocyte  model.  Methods  in  enzymology  452:  227-­‐245  

 

Zhang   J,   Randall   MS,   Loyd   MR,   Dorsey   FC,   Kundu   M,   Cleveland   JL,   Ney   PA   (2009b)   Mitochondrial   clearance   is   regulated   by   Atg7-­‐dependent   and   -­‐

independent  mechanisms  during  reticulocyte  maturation.  Blood  114:  157-­‐164     machinery.  Cell  death  and  differentiation  16:  1419-­‐1425  

 

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