Student Theses and Dissertations

Date of Award

2019

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Kapoor Laboratory

Abstract

Cell division in eukaryotes requires the assembly and maintenance of a structure, the bipolar spindle, self-organized from microtubules and their associated proteins. A multitude of components have been identified to be involved in this organization, and solving the combinatorial of the conditions that lead to the specific set of configurations present in vivo remains an open question. This thesis presents reconstitutions from purified components used to study aspects of microtubule self-organization: first, the microtubule-nucleation-related augmin octameric complex was assessed at a single-molecule interaction level with microtubules; second, the cross-linker PRC1's frictional response resisting motion between two microtubule filaments was analyzed to determine the dependence of the frictional force on the binding conditions of the cross-linker. For the augmin complexes studied sub-second interaction times were observed, yielding diffusive tracks on the lattice of microtubules. Microtubule bundles driven to slide showed GFP-PRC1 accumulation with a near-contant frictional force recorded. From the data, a theoretical model was produced linking the accumulation of GFP-PRC1 to the time evolution of the force trace. The observations on the reconstituted augmin complex establish constrains (diffusive and short lived) on an entity proposed to anchor newly-nucleated microtubules to pre-existing microtubules at a given branching angle. The observations on PRC1 mechanics lead to the hypothesis of a changing mechanical behavior of the cross-linker to its exchange kinetics, potentially modulated by external factors.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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