The Nucleoporin Nup98 Regulates Microtubule Dynamics During SpindleAssembly Open Access

Cross, Marie K (2009)

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Abstract During interphase, the nuclear pore complex regulates trafficking between the nucleus and the cytoplasm. However, during mitosis, the nuclear pore complex is disassembled and increasingly, nucleoporins have been found to have alternate mitotic functions in mitotic checkpoints and in assembly of the mitotic spindle. Using Xenopus extract spindle assembly assays, we found that the nucleoporin Nup98 functions in mitotic spindle assembly through regulation of microtubule dynamics. When added to spindle assembly assays, the C-terminal domain of Nup98 stimulates uncontrolled growth of microtubules. Addition of inhibitory antibodies or depletion of Nup98 from extract leads to formation of stable monopolar spindles. Importantly, addition of purified Nup98 C-terminus to depleted extract rescued bipolar spindle formation. This activity does not require the known interaction between Nup98 and Nup96, one member of a nucleoporin complex associated with both the kinetochore and spindle. We have mapped the relevant region of Nup98 to a portion of the C-terminal domain lacking a previously characterized function. Both interphase and mitotic phosphorylations occur within this region of Nup98, and mutation of selected mitotic phosphorylation sites significantly alters the excess microtubule phenotype. Furthermore, we have identified a molecular interaction between the C-terminal domain of Nup98 and the microtubule depolymerizing kinesin MCAK. These data support a model in which Nup98 acts through MCAK to regulate the microtubule dynamics required for spindle bipolarity.

Table of Contents

Table of Contents Chapter 1 Introduction 1

Introduction to Thesis 1

The Nuclear Pore Complex 3

Structure 3 Nucleoporins 4 Dynamics of the pore 8

Nuclear Transport 11

Transport Receptors 11 Transport Signals 12 The RanGTPase System 14 mRNA Export 18 Models of Translocation 19 The Mitotic Spindle 23 Microtubules 24 Centrosomes and Kinetochores 30 Centrosome-driven Spindle Assembly 32 Chromatin-driven Spindle Assembly 33 Spindle Assembly Factors 35 Factors Involved in Microtubule-Kinetochore 41


Nucleoporins In Mitosis 45 Nup107 complex and ELYS 46 Nup358/RanBP2 50 Rae1 52 Nup153 and TPR 54 Nup98 55

Nup98 Domains and Post-translational 55

Modifications Nup98 Function at the Pore 56 Subcellular Localization 57 Nup98 and Viral Infection 61 Nup98 Knockout Mouse 62 Nup98 in Leukemia 62 Xenopus Egg Extract 63 Scope of the Dissertation 67

Chapter 2 Nup98 regulates bipolar spindle assembly 70

through the depolymerizing kinesin, MCAK.

Abstract 71 Introduction 72 Results 76 Addition of purified C-terminal domain to 76

CSF extract disrupts spindle assembly .

Nup98 and alternate spindle assembly 83 pathways .

Nup98 antibodies inhibit formation of 89

bipolar spindles but do not affect Ran asters or Preformed spindles.

Endogenous Nup98 is required for bipolar 93

spindle assembly in egg extract.

A fraction of Nup98 is associated with 97

centrosomes. Mitotic phosphorylations influence the 101

function of Nup98 in spindle assembly.

Nup98 C-terminal domain interacts with the 105

microtubule depolymerizing kinesin, MCAK

Discussion 112 Experimental Procedures 119 Acknowledgements 126

Chapter 3 Microtubule Flux 127

Investigating Flux 128 Fluorescent Speckle Microscopy Requirements 129 FSM Analysis 130 FSM and Nup98 131 Chapter 4 Conclusions 136 Future Directions 139 Binding Partners 140 Phosphorylation 141 Final Thoughts 143 Nup98 and Rae1 During Mitosis 143 Implications for Nup98 in Cancer 144 Model for Nup98 During Mitosis 145 Bibliography146

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