Identification of centrosomal proteomes using COMPACT, a novel tool for purification of centrosomes
type: CreativeWork
description: Centrosomes are small cytoplasmic organelles that play a critical role in multiple cellular processes; from mitotic spindle assembly and cell division, to cell migration, invasion and polarity. The human centrosome consists of two cylindrical structures known as centrioles, decorated at their distal ends by appendage structures, and surrounded by a proteinaceous matrix known as the pericentriolar material. Despite a number of advances in the field, focussing particularly on centrosome structure and assembly in normal and diseased cells, relatively little is known about the spatial and temporal dynamics of centrosome composition, and how this may vary between different cell, tissue, or disease types. Indeed, only a handful of studies have analysed the proteomic composition of the centrosome. In the first part of this study, I established a novel method for centrosome isolation that was developed in our laboratory. This approach, known as COMPACT (Centrosome Purification by Affinity Capture), involves a single-step affinity purification of centrosomes by a short peptide corresponding to a 33 amino acid C-terminal fragment of the centrosomal protein CCDC61. I have shown that the method is able to isolate centrosomes from a variety of cell types, and that analysis of purified centrosomes using mass spectrometry recovers the majority of known core centrosomal proteins. Importantly, I demonstrated that COMPACT purifies centrosomal components with higher efficiency and specificity than the traditional sucrose sedimentation-based centrosome isolation technique. In the second part of this study, I performed experiments to gain mechanistic insight into how COMPACT works. I generated cell lines lacking the centrosomal proteins, NIN or CEP128, and showed these proteins (located primarily at the subdistal appendages) to be directly involved in the peptide’s ability to purify centrosomes. In the third part of this study, I performed a comprehensive analysis of the centrosome prot…
abstract: Centrosomes are small cytoplasmic organelles that play a critical role in multiple cellular processes; from mitotic spindle assembly and cell division, to cell migration, invasion and polarity. The human centrosome consists of two cylindrical structures known as centrioles, decorated at their distal ends by appendage structures, and surrounded by a proteinaceous matrix known as the pericentriolar material. Despite a number of advances in the field, focussing particularly on centrosome structure and assembly in normal and diseased cells, relatively little is known about the spatial and temporal dynamics of centrosome composition, and how this may vary between different cell, tissue, or disease types. Indeed, only a handful of studies have analysed the proteomic composition of the centrosome. In the first part of this study, I established a novel method for centrosome isolation that was developed in our laboratory. This approach, known as COMPACT (Centrosome Purification by Affinity Capture), involves a single-step affinity purification of centrosomes by a short peptide corresponding to a 33 amino acid C-terminal fragment of the centrosomal protein CCDC61. I have shown that the method is able to isolate centrosomes from a variety of cell types, and that analysis of purified centrosomes using mass spectrometry recovers the majority of known core centrosomal proteins. Importantly, I demonstrated that COMPACT purifies centrosomal components with higher efficiency and specificity than the traditional sucrose sedimentation-based centrosome isolation technique. In the second part of this study, I performed experiments to gain mechanistic insight into how COMPACT works. I generated cell lines lacking the centrosomal proteins, NIN or CEP128, and showed these proteins (located primarily at the subdistal appendages) to be directly involved in the peptide’s ability to purify centrosomes. In the third part of this study, I performed a comprehensive analysis of the centrosome prot…
author: Carden, Sarah
language: eng
keywords: Centrosome, purification, proteomics
license: https://www.repository.cam.ac.uk/bitstreams/16e40be9-573a-4e6d-a318-953dc957cc62/download
license: https://www.rioxx.net/licenses/all-rights-reserved/
date published: 2021-08-20
date modified: 2026-07-24T01:33:33Z
provider: Cambridge University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Cambridge

PropertyValue
property ID: DOI
value: 10.17863/CAM.78647