Spindle assembly checkpoint-independent functions of Bub1 and Bub3 in fission yeast mitosis [Elektronische Ressource] / vorgelegt von Hanna Windecker

Hanna Heller

Sujets

• Biologie

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2010
• Nombre de lectures: 20
• Langue: English
• Poids de l'ouvrage: 12 Mo

Extrait

Spindle assembly checkpoint-independent
functions of Bub1 and Bub3 in fission yeast
mitosis





Dissertation
der Mathematisch-Naturwissenschaftlichen Fakultät
der EBERHARD KARLS UNIVERSITÄT TÜBINGEN
zur Erlangung des Grades eines
Doktors der Naturwissenschaften
(Dr. rer. nat.)




vorgelegt von
Hanna Windecker
aus Mainz






Tübingen
2010























Tag der mündlichen Qualifikation: 16.12.2010
Dekan: Prof. Dr. Wolfgang Rosenstiel
1. Berichterstatter: Prof. Dr. Thilo Stehle
2. Berichterstatter: Prof. Dr. Klaus Harter ACKNOWLEDGEMENTS

I am thankful to everybody who supported me during the last years, be it in the institute or outside,
including many people not mentioned below.

First and foremost, I would like to thank Dr. Silke Hauf for letting me do my PhD thesis in her lab. I
am grateful for her guidance, generous support and continuous encouragement as well as her
patience when things went not that well.

I would like to thank my PhD examiners Prof. Dr. Klaus Harter, Prof. Dr. Thilo Stehle, Dr. Silke
Hauf and PD Dr. Ulrike Zentgraf. I am thankful to Prof. Dr. Thilo Stehle and Prof. Dr. Klaus Harter
for agreeing to be my PhD supervisors. Furthermore, special thanks go to them and the other
members of my PhD advisory committee, Dr. Dmitri Ivanov and Dr. Elisa Izaurralde, for listening to
my annual progress report and discussing my results as well as for their helpful advice and
suggestions.

I am very thankful to the whole lab, current and previous lab members, for the nice working
atmosphere and also the enjoyable activities outside the lab. I thank André Koch, Ashapurno
Biswas, Julia Kamenz, Maria Langegger, Stephanie Heinrich and Yu-Hua Huang for great support
and fruitful discussions. Especially, I would like to thank Eva Illgen for excellent technical help and
keeping the lab shipshape. In addition, I thank the undergraduate research assistants Julia Binder,
Janet Peper, Melanie Maerklin-Stradinger and Paula Quecke for technical support.

Furthermore, I thank the Ivanov lab, in particular Dmitri, for discussions about my data. In addition,
I am grateful to Stephen Huisman and Virgilio Failla for helping me with microscopy and data
analysis, even though they assisted me with experiments not shown in this thesis, but I learned a
lot from them and appreciate their help.

I would like to thank my friends in Tübingen and the surrounding area, thanks to whom I really felt
at home here, as well as friends far away for moral support.

I am grateful to my family for continuous support and encouragement during the whole time of my
PhD. I especially thank my parents for always believing in me and my abilities.

Special thanks go to Christine Vogler for proofreading the manuscript, and to all dear friends and
family members who offered to proofread my thesis and to type for me when I was able to use only
my left hand. I really appreciate these generous offers!


I TABLE OF CONTENTS

ACKNOWLEDGEMENTS .....................................................................................................I  
LIST  OF  FIGURES..............VI  
ABBREVIATIONS ............................................................................................................VIII  
SUMMARY........................X  
ZUSAMMENFASSUNG .....................................................................................................XI  
1   Introduction ......................... 1  
1.1   The  cell  cycle................................................................1  
1.2   Fission  yeast  as  a  model  organism........................2  
1.3   Mitosis ...................................................................................................................................3  
1.3.1   Th e  mitotic  spindle  in  fission  yeast................5  
1.4   The  spindle  assembly  checkpoint ..........................................................................................6  
1.4.1   The  ‘Mad2-­‐template  model’...........................7  
1.4.2   The  spindle  assembly  checkpoint  kinase  Mps1  (Mph1) .................................................8  
1.4.3   Activation  of  the  spindle  assembly  checkpoint............................10  
1.4.4   Bub1  and  Bub3 .............................................................................11  
1.4.4.1   Bub1.......................................................11  
1.4.4.2   Bub1  kinase  activity ...............................................................13  
1.4.4.3   Bub1  has  functions  outside  the  SAC ......................................................................14  
1.4.4.4   Bub1  acts  upstream  of  shugoshin..........16  
1.4.4.5   Bub3 .......................................................................................................................19  
1.5   The  fission  yeast  kinesin -­‐8  family  members  Klp5  and  Klp6.................22  
1.5.1   Synthetic  lethal  interactors  of   klp5 ..............................................................................24  
1.5.1.1   Synthetic  interactions............................24  
1.5.1.2   The  anaphase -­‐promoting  complex/cyclosome  (APC/C)........25  
1.5.1.3   The   Dam1/DAS H  complex ......................................................................................26  
1.5.1.4   Dis1  and  Alp14 .......................................................................................................26  
1.5.1.5   The  Ras  pathway  in  fission  yeast...........26  
1.5.1.6   α-­‐tubulin ................................................................................................................27  
1.5.1.7   γ-­‐tubulin.................27  
1.6   Aim  of  this  study28  
2   Results ................................ ................................ ................................ ............................... 29  
II 2.1   Contributions ......................................................................................................................29  
2.2   Bub1  shares  a  function  with  the  kinesin -­‐8  family  proteins  Klp5  and  Klp6 ..........................29  
2.2.1   A  so  far  uncharacterized  function  of  Bub1  is  required  for  growth  of  kinesin -­‐8-­‐deleted  
cells   29  
2.2.2   Phenotype  of  klp5 Δ  bub1 Δ  mutants ............................................................................32  
2.2.3   Proteins  that  could  act  tog ether  with  Bub1  to  exert  the  unknown  function ...............34  
2.2.3.1   klp5  synthetic  lethal  screen ...................................................................................34  
2.2.3.2   Known  synthetic  lethal  interactors  of   klp5 Δ..........................36  
2.2.4   Bub1  separation -­‐of -­‐functi on  mutants ..........................................................................40  
2.2.4.1   Genetic  screen  for   bub1  separation -­‐of -­‐function  mutants.....40  
2.2.4.2   Spindle  assembly  checkpoint  activity  of   bub1  mutants.........40  
2.3   Bub1  and  Bub3  share  function(s)  outside  the  spindle  assembly  checkpoint ......................46  
2.3.1   Deletion  of   bub3  causes  synthetic  lethality  with   klp5 Δ...............................................46  
2.3.2   Bub3  is  not  essential  for  the  mitotic  delay  in  the  presence  of  chromosome  attachment  
errors  in  fission  yeast ................................................................................48  
2.3.3   Sgo2  localization  in   bub1 -­‐ΔGLEBS  and  bub3 Δ  cells ......................................................51  
2.3.4   Chromosome  segregation  and  spindle  defects  in  the   klp5 Δ  bub3 Δ