Gathering minus ends

: in vitro study of the influence of NuMA on microtubule dynamics and dynein-dependent transport

Abstract

During cell division, various cell components need to move in order to effectively separate equally the chromosomic material. Some of the major actors of this process are the microtubules, which form the spindle in order to attach to the chromosomes and pull them apart. How motors form the spindle is not fully understood, one of the major aspects being spindle pole focusing. The NuMA protein has been shown to be an essential player in multiple aspects of cell division including spindle pole focusing. In the absence of NuMA, the defects triggered are the same that in the absence of dynein or dynactin. The dynein-dynactin complex is known to require an adaptor protein for processivity and cargo binding, and although dynein-dynactin is known to play a role in minus end focusing, the adaptor protein associated to this function remains unknown. NuMA can bind dynein in vitro and NuMA localizes to microtubule minus ends independently of dynein in cells. The goal of this thesis is to understand better the role of NuMA in spindle pole focusing, more specifically asking whether NuMA could act as a dynein-dynactin adaptor, activating dynein processivity and binding microtubule minus ends as a cargo.
The first part of this project focuses on the interactions of NuMA with microtubules by studying the specific binding interaction and the impact that NuMA has on microtubule dynamics. The second part of this project addresses the ability of NuMA to activate dynein processivity and explores the capacity of the dynein-dynactin-NuMA complex to move microtubules relatively to one another, and therefore to bring minus ends together. The third part of this project is a collaborative work and studies the effect of an inhibition-lifting mutation on the ability of the dynein adaptor Spindly to activate dynein processivity. Purified proteins are observed in TIRF microscopy to study the behaviour of microtubules in the presence of these proteins. The goal is to recreate an in vitro system to understand the molecular mechanism that governs spindle focusing.

Date of Award	1 May 2021
Original language	English
Awarding Institution	King's College London
Supervisor	Thomas Surrey (Supervisor)