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Title: Structural studies of PCM scaffold assembly in Drosophila melanogaster
Author: Feng, Zhe
ISNI:       0000 0004 6353 136X
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2015
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Centrosomes are the major microtubule (MT) organising centres in most animal cells, and they play important roles in many cellular processes. Centrosomes comprise a pair of centrioles surrounded by a matrix of proteins known as the pericentriolar material (PCM). Centrosomes assemble around the mother centriole when the PCM is recruited to nucleate and organise the MTs. For decades, the PCM has been viewed as an amorphous cloud of proteins. Although there are hundreds of PCM proteins, a small number of these are thought to assemble an underlying PCM scaffold that extends around the mitotic centriole and subsequently recruits other mitotic PCM proteins. In flies, Centrosomin (Cnn) and Spindle-defective protein 2 (Spd-2) are such essential proteins, and Asterless (Asl) helps recruit these proteins to the mother centriole. However, it is unclear how these proteins assemble into a scaffold at the centrosome, and none of these core PCM assembly proteins have been characterised at a structural level. In this thesis, I have identified and recombinantly expressed functional domains of Cnn, Spd-2 and Asl in order to characterise them in vitro. In collaboration with colleagues in the laboratory, the biological significance of these domains has been investigated in vivo. I define a Phospho-Regulated Multimerisation (PReM) domain within Cnn that is phosphorylated by Polo kinase in vitro, that dimerises via a leucine zipper, and that can form higher order oligomers when phospho-mimicking mutations are incorporated into the protein in vitro. In vivo, forms of Cnn that cannot be phosphorylated in the PReM domain cannot efficiently assemble a Cnn scaffold, while phospho-mimicking forms can spontaneously assemble into scaffold structures. I show that the Centrosomin-Motif-2 (CM2) domain of Cnn is also required to promote Cnn scaffold assembly. The crystal structure of CM2 reveals an unusual asymmetric dimer that further assembles into a tetramer in crystallo. Strikingly, Cnn-CM2 demonstrates a zinc-binding activity that stabilises the dimer, and preliminary in vivo studies indicate that the tetramer interface is essential for Cnn scaffold assembly in vivo. Finally, I show that Cnn-CM2 exhibits a weak interaction with the first of three ASPM, Spd-2, Hydin (ASH) domains in Spd-2, and that this domain facilitates Spd-2 expansion into the PCM region. The structure of two of the Spd-2 ASH domains has been investigated using X-ray crystallography and Nuclear Magnetic Resonance spectroscopy, revealing an Immunoglobulin (Ig)-like protein fold. These studies provide the first atomic insights into the molecular mechanism underlying mitotic PCM assembly, and reveal how the assembly of a Cnn scaffold may be controlled to ensure robust centrosome assembly in vivo.
Supervisor: Raff, Jordan ; Lea, Susan Sponsor: Sir William Dunn School of Pathology
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available