Roles of TACC related protein, mia1p in MTOCs and microtubule dynamics in schizosaccharomyces pombe 1

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ROLES OF TACC-RELATED PROTEIN, Mia1p IN MTOCs
AND MICROTUBULE DYNAMICS IN
SCHIZOSACCHAROMYCES POMBE









ZHENG LILING









A THESIS SUBMITTED
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
TEMASEK LIFE SCIENCES LABORATORY
NATIONAL UNIVERSITY OF SINGAPORE
2007

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Acknowledgements
I would like to express my heartfelt thanks to my supervisor Dr. Snezhka Oliferenko for
her excellent guidance, continuous support and encouragement, especially in the past two
years after my son was born. Without her understanding and support, this thesis would
not have been possible.

I thanks to my co-supervisor Prof. Mohan Balasubramanian who provided opportunity to
work as ARO in his lab and continually support me to be JRF in Dr Snezhka’s lab. Many
thanks to his valuable commemts on my thesis project.

I am also thankful to my thesis committee members Dr.Maki Murata Hori, Dr. Wang Yue
and Dr. Thirumaran Thanabalu for the time and effort they put in this project and their
helpful suggestions on this thesis project.

I thank all past and present members of the cell dynamics laboratory and the cell division
laboratory, Fungal Patho-biology for their help and discussions.

I am thankful to TLL facilities and staff for general support.

I thank Dr. Snezhka Oliferenko,Yuenchayo Ling and Aleksandar Vjestica for critical
reading of my thesis.


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I would like to thank Dr Phong Tran for his help with the spinning disk confocal
microscope.

I am thankful to Temasek Holdings, Singapore for their financial support through to my
work.

Finally, I would like to thank my family for their constant support and encouragement.

















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TABLE OF CONTENTS
page
Title page i
Acknowledgements ii
Table of contents iv
List of Figures xi
List of abbreviations xiii
Summary xiv

Publications xv

CHAPTER I: Introduction
1.1 A general introduction of microtubules
1.1.1 Properties of microtubules 1
1.1.2 Centrosome 3
1.1.3 Non-centrosomal microtubule 3
1.1.4 Schizosaccharomyces pombe (S. pombe) as a model for studying
microtubule dynamics 4
1.2 MTOC and microtubule cytoskeleton in S. pombe 5
1.2.1 Microtubule nucleating sites—MTOCs in S. pombe 5
1.2.1.1 Spindle pole body (SPB) 5
1.2.1.2 Non-centrosomal MTOCs 8
1.2.1.2.1 Interphase microtubule organizing centers (iMTOCs) 8
1.2.1.2.2 Equatorial microtubule organizing centers (eMTOC) 9
1.2.1.2.3 γ-TuRC satellites 10

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1.2.2 Components of MTOCs 10
1.2.2.1 The γ-TuRC 10
1.2.2.2 Mto1p and Mto2p 12
1.2.3 Organizing microtubule bundles 13
1.2.3.1 Minus-end-directed motor protein—Klp2p 14
1.2.3.2 Bundling protein—Ase1p 14
1.2.3.3 Model of maintaining ordered microtubules 15
1.2.4 Arrangement of microtubule cytoskeleton in S. pombe 18
1.2.4.1 Interphase microtubule cytoskeleton 18
1.2.4.1.1 Architecture and dynamics of interphase microtubule
cytoskeleton 18
1.2.4.1.2 Functions of interphase microtubule cytoskeleton 19

1.2.4.1.2.1 Maintaining cell morphology 19
1.2.4.1.2.2 Controlling central localization of nucleus 22
1.2.4.2 Mitotic microtubule cytoskeleton 22
1.2.4.2.1 Architecture of mitotic microtubule cytoskeleton 22
1.2.4.2.2 Functions of mitotic microtubule cytoskeleton 23
1.2.5 Self-organization of fission yeast microtubules 26
1.3 Mia1p as a TACC-related protein 27
1.3.1 TACCs in human 27
1.3.2 TACC in Drosophila 28
1.3.3 Current knowledge about Mia1p in S. pombe 30
1.4 Aim and objectives of this thesis 32

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1.5 Significance of this study 32

Chapter II Materials and Methods
2.1 Strains, reagents and genetic methods 33
2.1.1 Schizosaccharomyces pombe strains 33
2.1.2 Growth, media and conditions 34
2.1.3 Plasmids 34
2.1.4 Enzymes, antibodies and drugs used 35
2.2 Molecular methods and yeast methods 35
2.2.1 Recombinant DNA techniques 35
2.2.2 Transformation of E.coli by electroporation 36
2.2.3 LiAc transformation of S. pombe 36
2.2.4 Mia1p/Microtubule association experiments 37
2.2.5 Extraction of S. pombe genomic DNA 37
2.3 Construction of knock out mutants and epitope tagging of genes 38
2.3.1 Construction of knock out mutants 38
2.3.2 Construction of epitope tagging of genes 39

2.3.3 Generation of cells overexpressing Mia1p 39
2.4 Cell biology and microscopy 39
2.4.1 Immunofluorescence staining 39
2.4.2 Flow chamber experiments 41
2.4.3 Time-lapse fluorescence microscopy 41
2.4.4 Electron microscopy techniques 42

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2.4.5 Laser microsurgery 42

Chapter III: Results
3.1 Mia1p in assembly and maintenance of persisent iMTOCs at nuclear envelope 44
3.1.1 Lack of microtubule binding protein Mia1p results in fewer interphase
Microtubules 44
3.1.1.1 Mia1p is a microtubule binding protein 44
3.1.1.2 mia1Δ cells exhibit disordered and heterogeneous microtubule bundles 46
3.1.2 mia1Δ cells are defective in attachment of microtubules to the nucleation sites 48
3.1.2.1 Microtubules detached from the nucleation sites in mia1Δ cells 48
3.1.2.2 Microtubules remained attached to the SPB in alp14Δ cells 51
3.1.3 Mia1p is not required for microtubule nucleation 53
3.1.3.1 Microtubules could nucleate on the preexisting microtubules in
mia1Δ Cells
53
3.1.3.2 Microtubules could be nucleated from several sites around the nuclear
envelope in mia1Δ cells
56
3.1.4 Mia1p is required for maintenance of microtubule bundles 61
3.1.5 Mia1p is required for sustaining proper post-anaphase array dynamics 63
3.1.5.1 PAA dynamics is abnormal in mia1Δ cells 63
3.1.5.2 Mia1p is not required for the assembly of the eMTOC 66

3.1.6 There are no detectable γ-tubulin-rich iMTOC structures in mia1Δ cells 68

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3.1.7 Microtubules are required for emergence of iMTOC structures upon entry
into the new cell cycle 70
3.1.7.1 Why iMTOC structures could be absent from mia1Δ cells
70
3.1.7.2 Experimental confirmation of hypothesis 72
3.1.7.3 Model of establishment of the iMTOCs in fission yeast 75
3.2 The SPBs facilitate the NE division during closed mitosis in fission yeast 76
3.2.1 Cells overexpressing Mia1p arrest in mitosis with fully elongated spindles
and nonsegregated chromosomes
76
3.2.1.1 Mia1p-overexpressing cells arrest in mitosis 76
3.2.1.2 Mia1p-overexpressing cells exhibit fully elongated mitotic spindle
with non-segregated chromosomes
78
3.2.2 The SPBs are displaced from spindle poles and NE division fails in
Mia1p-overexpressing cells
80
3.2.3 Spindles in Mia1p-overexpressing cells are bipolar and require the
microtubule-crosslinking protein, Ase1p, for their assembly
85
3.2.4 The SPBs ensure the NE division by preventing abnormal deformation

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of the NE by mitotic spindle

89
3.2.5 Failure of chromosome segregation was not due to defects in kinetochore
attachment 91
3.2.6 Genetic ablation of the NE allows chromosome segregation in Mia1p-
overexpressing cells 93
3.3 Role of Mia1p in maintenance of microtubule polarity 98
3.3.1 Mia1p was required for symmetrical distribution of cortical tip proteins
at cell tips
98
3.3.2 Polarity of microtubules was altered in mia1Δ cells 100


Chapter IV Discussion
4.1 Comparison between mia1Δ cells and other γ-TuRC mutants 105
4.2 Role of Mia1p in microtubule attachment but not in microtubule nucleation 106
4.3 Microtubule attachment sites serve as MTOCs to establish microtubule
architecture 107
4.4 Establishment of the iMTOCs requires microtubule attachment to the NE 108
4.5 Relationship between Mia1p and Alp14p 109
4.5.1 Mia1p interacts with Alp14p but not Dis1p 109
4.5.2 Different aspect of Mia1p and Alp14p functions in microtubule

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attachment to the NE
110
4.5.3 Lack of Mia1p and Alp14p result in failure of iMTOCs establishment 110
4.6 De novo assembly of the MTOCs is not specific to S. pombe cells 111
4.7 Role of Mia1p in modulating mitotic microtubule dynamics 112
4.8 Conclusion and future perspectives 113
4.8.1 Conclusion 113

4.8.2 Future perspectives 114
4.8.2.1 Microtubules are nucleated by the iMTOCs and the SPB: are
they different or similar?
114
4.8.2.2 Distribution of the iMTOCs at the NE: random or specific? 115
4.8.2.3 What’re the partners of Mia1p in terms of anchoring microtubule
to the NE?
115
References 119

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LIST OF FIGURES
Figures page
Figure3.1.1.1: Microtubule-associated protein Mia1p binds microtubules and
is a component of S. pombe MTOCs.
45
Figure3.1.1.2: Cells lacking Mia1p exhibit disordered and heterogeneous
microtubule bundles.
47
Figure3.1.2.1: Mia1p is required for attachment of microtubules to the SPBs
and the NE.
49-50
Figure3.1.2.2: Although SPBs remain stationary in alp14Δ cells, microtubules
are attached to the SPBs.
52
Figure3.1.3.1: Microtubule could be nucleated on the preexisting microtubule
in mia1Δ cells.
54-55
Figure3.1.3.2: Microtubule could be nucleated form several sites around the NE
in mia1Δ cells.

58-60
Figure3.1.4: Mia1p is required for maintenance of microtubule bundles. 62
Figure3.1.5.1: Mia1p-GFP is required for PAA organization. 65
Figure3.1.5.2: Mia1p is not essential for the assembly of the eMTOC. 67

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Figure3.1.6: Components of the γ-tubulin complex are not found as prominent
structures around the NE.
69
Figure3.1.7.1: A model of the iMTOC formation in S. pombe cells 71
Figure3.1.7.2: Microtubules are required for emergence of iMTOC structures
upon entery into the new cell cycle.
74

Figure3.2.1.1: Fission yeast cells over-expressing Mia1p arrest in mitosis with
fully extended mitotic spindles.
77
Figure3.2.1.2: Cells overexpressing Mia1p exhibit fully elongated mitotic spindle
with non-segregated chromosomes
79
Figur3.2.2: Cells overexpressing Mia1p causes the SPBs to be displaced from the
mitotic spindle poles and NE division failure.
82-84 Figure3.2.3: Spindle structures in Mia1p-overexpressing cells are bipolar
and
anti-parallel.
87-88
Figure 3.2.5: The majority of Mia1p-overexpressing cells exhibit Mad2p-GFP
on the NE.
92

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Sup-Figure 3.2.6: DNA segregation occurs in cells overexpressing Mia1p when
the NE is fragmented.
95-97
Figure 3.3.1: Symmetrical cell tip localization of the cell end marker requires
Mia1p.
99
Figure 3.3.2: Orientation of microtubules is altered in mia1Δ cells. 102-104
Sup-Figure 4.5.3: Localization of Mto1p-GFP in alp14Δ cells. 118





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ABBREVIATIONS
TACC the transforming acidic coiled-coil related protein
MTOC microtubule organizing center
iMTOC interphase microtubule organizing center
eMTOC equatorial microtubule organizing center
PAA post anaphase array
NE: nuclear envelope
MT microtubule
SPB spindle pole body
γ-TuRC γ-tubulin ring complex
Mia1 microtubule associated protein 1
DAPI 4’6,-diamidino-2-phenylindole
MBC methyl benzimidazol-2-yl-carbamate
DNA deoxyribonucleic acid
GFP green fluorescent protein

GTP guanosine triphosphate
GDP guanosine diphosphate
PCR polymerase chain reaction
ts: temperature sensitive
EMM Edinburgh minimal medium



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SUMMARY
Microtubule arrays of distinct geometries are important for cell function.
Microtubule-organizing centers (MTOCs) concentrate microtubule nucleation,
attachment and bundling factors and thus restrict formation of microtubule arrays in
spatial and temporal manner. It is essential to understand molecular mechanisms
underlying MTOC emergence and maintenance. In this thesis, I use a combination of cell
imaging, cell manipulation and genetics experiments in the fission yeast
Schizosaccharomyces pombe, to show that the Transforming Acidic Coiled Coil protein,
Mia1p, functions in sustaining proper MTOC and microtubule dynamics, both in
interphase and mitosis. Briefly, I show that Mia1p is required for microtubule attachment
to the nucleation sites. Furthermore, when overexpressed, Mia1p organized ectopic
spindle pole like structures that interfered with normal mitotic spindle assembly. These
studies led us to identify a role for the spindle pole bodies in segregation of the nuclear
envelope during closed mitosis.

Key words: S. pombe, microtubule, MTOC, iMTOCs, eMTOC, Mia1p, TACC

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Publications

First author publications:

Liling Zheng

, Cindi Schwartz

, Liangmeng Wee

, and Snezhana Oliferenko (2006). The
Fission Yeast Transforming Acidic Coiled Coil–related Protein Mia1p/Alp7p Is Required
for Formation and Maintenance of Persistent Microtubule-organizing Centers at the
Nuclear Envelope. Mol Bio Cell 17(5): 2212-2222.

Liling Zheng

, Cindi Schwartz

, Valentin Magidson, Alexey Khodjakov, Snezhana
Oliferenko (2007). The Spindle Pole Bodies Facilitate Nuclear Envelope Division during
Closed Mitosis in Fission Yeast. PloS Biology 5(7):1530-1542