Molecular mechanisms of mitotic spindle assembly and accurate

122 Pages·2013·3.18 MB·English

Molecular mechanisms of mitotic spindle assembly and accurate chromosome segregation

Yige Guo

Submitted in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
in the Graduate School of Arts and Sciences

COLUMBIA UNIVERSITY
2013
© 2013
Yige Guo
All rights reserved
Abstract
Molecular mechanisms of mitotic spindle assembly and accurate chromosome segregation
Yige Guo

During the cell cycle, duplicated DNA in S phase is segregated, in the form of
chromatids, into two daughter cells in mitosis. The accuracy of chromosome segregation is
essential as two daughter cells have the same genetic contents as the mother cell. Two major
mechanisms are utilized by the cell to ensure accurate chromosome segregation. First,
interactions between the dynamic microtubules and kinetochores, the proteinaceous structures
built on centromeres of mitotic chromosomes that act as the attachment site for microtubules,
serve as major forces to position each pair of chromosomes to the metaphase plate. Secondly, a
surveillance system, known as the mitotic checkpoint, put the anaphase onset on hold until each
pair of sister chromosomes are aligned at the metaphase plate and appropriately attached with
microtubule plus ends by kinetochores.
In the first part (Chapter 2) of this thesis, I illustrate the role of the auto-phosphorylation
of BubR1, a mitotic checkpoint protein, in kinetochore-microtubule attachment and the mitotic
checkpoint. Using a phospho-specific antibody against the auto-phosphorylation site identified
by mass spectrometry, I demonstrate that kinetochore-associated BubR1 phosphorylates itself in
human cells in vivo and that this phosphorylation is dependent on its binding partner, the
kinetochore-associated kinesin motor CENP-E. Studies using cells expressing a non-
phosphorylatable BubR1 mutant revealed that the CENP-E–dependent BubR1 phosphorylation
at unattached kinetochores is important for a full-strength mitotic checkpoint to prevent single
chromosome loss. Furthermore, replacing endogenous BubR1 with the non-phosphorylatable
BubR1 mutant or depletion of CENP-E, the BubR1 kinase activator, results in metaphase
chromosome misalignment and increased incidents of syntelic attachments. Using indirect
immunofluorescence, I have discovered a decreased level of Aurora B–mediated Ndc80
phosphorylation at the kinetochore of cells expressing the non-phosphorylatable BubR1 mutant,
which might contribute to the alignment defect. Moreover, expressing a phosphomimetic BubR1
mutant substantially reduces the incidence of polar chromosomes in CENP-E–depleted cells,
further supporting a signaling cascade function of CENP-E and BubR1 on the kinetochore. Thus,
the state of CENP-E–dependent BubR1 auto-phosphorylation in response to spindle microtubule
capture by CENP-E is important for kinetochore functions in achieving accurate chromosome
segregation.
In the second part (Chapter 3), my colleague and I demonstrate a novel mechanism of
mitotic spindle assembly in Xenopus egg extracts and mammalian cells. I show that the MRN
(Mre11, Rad50, and Nbs1) complex is required for metaphase chromosome alignment.
Consistent with the result of my colleague using Xenopus egg extracts, disruption of MRN
function by depleting Mre11 using an inducible shRNA system, or Mre11 inhibitor mirin,
triggers a metaphase delay and disrupts the RCC1-dependent Ran-GTP gradient. Addition of
mirin to mammalian cells reduces RCC1 association with mitotic chromosomes and changes the
confirmation of RCC1. Thus, the MRN-CtIP pathway contributes to Ran-dependent mitotic
spindle assembly by modulating RCC1 chromosome association.
In summary, my novel findings have revealed a pair of molecular mechanisms not known
previously, which are important to the mitosis field.
Contents

Chapter 1. Introduction: Regulation of Chromosome Alignment and the Mitotic Checkpoint in Mitosis . 1
Chromosome alignment and Error correction ....................................................................................... 2
Spindle formation ................................................................................................................................ 3
Kinetochore-microtubule attachment error correction ........................................................................... 5
The Mitotic checkpoint ...................................................................................................................... 12
Aneuploidy ........................................................................................................................................ 14
BubR1 ............................................................................................................................................... 16
CENP-E ............................................................................................................................................ 20
The MRN complex ............................................................................................................................ 21

Chapter 2. CENP-E–dependent BubR1 auto-phosphorylation enhances chromosome alignment and the
mitotic checkpoint ................................................................................................................................. 22
Introduction ....................................................................................................................................... 23
Methods ............................................................................................................................................ 26
Results .............................................................................................................................................. 28
Discussion ......................................................................................................................................... 52

Chapter 3. The MRN-CtIP pathway is required for metaphase chromosome alignment ......................... 60
Introduction ....................................................................................................................................... 61
Methods ............................................................................................................................................ 63
Results .............................................................................................................................................. 67
Discussions ....................................................................................................................................... 82

Chapter 4. Discussions and Future Directions ....................................................................................... 84
The BubR1’s kinase activity requires a co-activator CENP-E ............................................................. 85
CENP-E is a microtubule sensor independent of tension to regulate Aurora B-mediated
phosphorylation prior to end-on attachment ....................................................................................... 96
CENP-E – BubR1 regulates phosphatase recruitment ....................................................................... 100

Reference ............................................................................................................................................ 103

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List of Figures
Figure 1.1. Initial interaction between kinetochores and microtubules. ..................................................... 4
Figure 1.2. Correct and incorrect microtubule kinetochore attachment. .................................................... 6
Figure 1.3. A model for establishing proper stable kinetochore-microtubule attachment. ........................ 10
Figure 1.4. Illustration of different domains shows similarity between BubR1 and Bub1. ....................... 16
Figure 2.1. BubR1 phosphorylates itself. ............................................................................................... 31
Figure 2.2. BubR1 auto-phosphorylation at the kinetochore is sensitive to spindle microtubule attachment.
.............................................................................................................................................................. 32
Figure 2.3. BubR1 auto-phosphorylation at the kinetochore is CENP-E dependent. ................................ 33
Figure 2.4. BubR1 auto-phosphorylation is essential for accurate chromosome segregation. ................... 36
Figure 2.5. BubR1 kinase has other substrates that are important for kinetochore function. .................... 37
Figure 2.6. BubR1 auto-phosphorylation is required for efficient kinetochore targeting of Mad2 and a
prolonged mitosis induced by nocodazole. ............................................................................................. 39
Figure 2.7. The nonphosphorylated form of BubR1 reduces the levels of Mad1 association with
unattached kinetochores induced by nocodazole treatment. .................................................................... 40
Figure 2.8. BubR1 auto-phosphorylation is necessary for metaphase chromosome alignment. ................ 43
Figure 2.9. The nonphosphorylated form of BubR1 reduces Aurora B–mediated Ndc80 phosphorylation
at kinetochores. ..................................................................................................................................... 46
Figure 2.10. CENP-E depletion, but not BubR1 depletion, in human cells causes a decrease of Aurora B–
mediated Ndc80 phosphorylation at the kinetochore. ............................................................................. 47
Figure 2.11. The polar chromosome phenotype in CENP-E–depleted cells can be rescued by expression of
a phosphomimetic BubR1 mutant. ......................................................................................................... 51
Figure 2.12. A model for the role of CENP-E–dependent BubR1 auto-phosphorylation at the kinetochore.
.............................................................................................................................................................. 59
Figure 3.1. The MRN Complex Is Essential for Metaphase Chromosome Alignment in Xenopus Egg
Extracts (These experiements were performed by Dr. Lorene Rozier) .................................................... 69
Figure 3.2. Inhibition of MRN Results in Prolonged Metaphase in Mammalian Cells (These experiements
were performed by Dr. Lorene Rozier) .................................................................................................. 72
Figure 3.3. Reducing the expression level of MRE11 causes a metaphase delay in mammalian cultured
cells. ...................................................................................................................................................... 74
Figure 3.4. MRN inhibition disrupts the Ran-GTP gradient during metaphase. ....................................... 76
Figure 3.5. MRN inhibition results in an RCC1 conformational change ................................................. 78
Figure 3.6. MRN inhibition results in a reduction of RCC1 binding to chromatin. .................................. 79
Figure 3.7. MRN functions to stabilize RCC1 interaction with chromosomes during mitosis. ................. 81
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.............................................................................................................................................................. 89
Figure 4.1. Sequence analysis reveals the four key amino acids on BubR1 kinase domain crucial for the
catalytic activity. ................................................................................................................................... 89
Figure 4.2. The “GFSGS” motif may substitute the degenerated P-loop and facilitate ATP binding upon
CENP-E binding to BubR1 and the induction of confirmation change. ................................................... 91
Figure 4.3. Zebrafish BubR1 does not possess the crucial Asp in the “HRD” motif in the catalytic loop. 92
Figure 4.4. Phylogenetic analysis of BubR1, Bub1 and CENP-E among different species. ..................... 94
Figure 4.5. Phosphorylation of T608 is not dependent on Aurora B, Mps1, Cdk and Plk1. ..................... 95
Figure 4.5. A model for the role of CENP-E–dependent BubR1 auto-phosphorylation to facilitate initial
microtubule capture. .............................................................................................................................. 97
Figure 4.6. The CENP-E-dependent BubR1 auto-phosphorylation at the kinetochore is sensitive to lateral
microtubule binding prior to end-on attachment. .................................................................................... 99
Figure 4.7. CENP-E-dependent BubR1 auto-phosphorylation at T608 residue controls the access of Cdk1
to T620, which is essential for Plk1 binding and phosphorylation of the KARD domain. ...................... 101

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Abbreviation list
APC/C : Anaphase promoting complex/Cyclosome
Bub: Budding Uninhibited by Benzimidazole
BubR1: Bub-Related 1
CENP-E: Centromere protein-E
CPC: Chromosomal passenger complex
GEF: Guanine Nucleotide exchange factor
CtIP: C-terminal binding protein interacting protein
Hec1: Highly Expressed in Cancer protein 1
KD: Kinase dead
KT: Kinetochore
Mad: Mitotic-Arrest Deficient
MCC: Mitotic checkpoint complex
MEF: Mouse embryonic fibroblast
MRN complex: Mre11, Rad50, and Nbs1
MT: Microtubule

RCC1: Regulator of chromosome condensation 1

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Acknowledgements
First and foremost I want to thank my advisor Dr. Yinghui Mao. It has been an honor to
be his first Ph.D. student. He has always been very supportive to my study during these five
years and I have learnt a lot both inside and outside of the academia from him. He always tried to
help whenever I encountered any problems in my study, and his expertise in this field usually
offered great solutions to address those problems. He is a live example of an excellent scientific
researcher and a mentor, and acted as my role model during my study and training.
The members of the Mao lab have also made great contributions to my study. All of the
current and past members are excellent colleague, who are really nice to work with.
Collaborations among lab members made my project to progress much more efficiently. I
specifically want to acknowledge some lab members who have directly contributed to my thesis
work. Sana Ahmad used to be our lab technician when I joined the lab. She offered so much help
to me and I actually learnt most of my bench techniques from her. She was always very patient
whenever I have any questions regarding to the experiment methods, and helped me a lot with
troubleshooting. Her excellent skills of lab management made my experiment in the lab going on
smoothly. Dr. Jiayin Zhang, one of our former post-docs also contributed to the BubR1 auto-
phosphorylation study. She was actually the one who discovered the auto-phosphorylation site
through Mass Spectrometry and I was lucky enough to take over this project from her. I also
want to thank Dr. Lorene Rozier, the other post-doc when I joined the lab, who I later
collaborated with on the MRN project. She discovered some very interesting phenotypes in
Xenopus egg extracts and I followed up later with mammalian cells. Lorene is a very interesting
person to work with and she has extensive interests like camping, photographing and skiing.
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I also appreciate the time and ideas my thesis committee members have contributed to my
Ph.D. work. The committee, consists of great scientists in the cell biology field, Dr. Richard
Vallee, Dr. Gregg Gundersen, Dr. Ronald Liem and Dr. Geri Kreitzer together with my advisor
have provided many pieces of valuable advice to my research. Besides, they also generously
shared some resource in their lab. Dr. Susumu Antoku from the Gundersen lab taught me virus
transfection, which was a key experiment in my later project.
In my later study on the MRN project, I want to thank Dr. Theresa Swayne and Dr. Adam
White in the Shared Resource of Columbia University for their help with the FRET experiment.
No one in the Mao lab has any previous experience with FRET and this experiment is known to
be hard to perform. Adam and Theresa are definitely experts in the imaging field and I was able
to acquire some really nice images and analyzed the data without much difficulty.
I also want to thank Ms. Zaia Sivo, the program coordinator of my graduate program,
who provided a lot of help and advice to me when I was trying to find a lab to rotate in the first
year of my Ph.D. study. She did a great job to keep everyone in the graduate program on track
and made my Ph.D. study trouble-free and so efficient.
Five years was quite a period of time, and Ph.D. study can sometimes be very stressful,
especially when experiments did not work. But I feel my time at Columbia and New York City
enjoyable in large part, and I have made a lot of friends here. I was also an active member of
several student organizations, and I thank them for providing such a good platform for me to
build up my social network.
Lastly, I want to thank my family for all their support and love. As the only child of my
family, I especially want to show gratitude to my parents in China. Without their support, it
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