Identification of centrosomal proteomes using COMPACT, a novel tool for purification of centrosomes
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 proteome in a panel of human cell lines, allowing for the identification of tissue-specific as well as differentially expressed centrosomal candidates. In addition to this, a quantitative mass spectrometry-based analysis of centrosome composition in different cell types revealed the absence of distal appendages in the T lymphocyte cell line, Jurkat, a novel and exciting finding. COMPACT was also shown to be effective at isolating centrosomes from primary tissue, revealing its potential for use as a tool to explore centrosome composition in various centrosome-associated diseases, including microcephaly and cancer. Finally, by comparing COMPACT across a wide range of cell lines, I was able to identify a number of novel centrosomal candidates, which were independently validated using various techniques. Loss-of-function studies in HEK 293T cells were performed to determine the function of a specific candidate, TRIM27. This study not only provides a novel method to isolate centrosomes in a spatially and temporally controlled manner, but also an effective tool for the identification of new or previously uncharacterised centrosomal proteins that may have significant roles in centrosome biology.
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