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Genome-wide screen for new centrosome position regulators

Final Report Summary - CENTROLOC (Genome-wide screen for new centrosome position regulators.)

The microtubule cytoskeleton is a network of tubule like polymers organized through the cell cytoplasm to distribute correctly protein complexes and other cellular organelles. Its organization impacts therefore on overall cell architecture. Proper microtubule cytoskeleton structure and dynamic are critical during cell division to segregate chromosomes and in non-dividing cells for processes as migration, signaling, and cell polarization. Microtubules (MTs) in animal cells are organized from two main “microtubule organizing centers” (MTOCs) the centrosomes and the Golgi apparatus. The position and activity of the MTOCs should be therefore precisely regulated in order to establish an adequate MT network in each biological cell context.

The scientific objective of my research proposal was to identify new regulators of one of these MTOCs: the centrosome. Our global aim was to understand how the cell controls centrosome localization. Centrosomes are extremely well conserved across evolution. They are composed of a pair of centrioles surrounded by a cloud of pericentriolar material. Centrioles themselves are microtubule based cylindrical structures, of approximately 500nm length and 200nm wide in humans. The centrosomes/centrioles are the MTOCs that build the mitotic spindle, cilia and flagella, and as a consequence they are required for fundamental processes such as chromosome segregation, cell signalling and cell motility. Centrosome abnormalities have been linked to cancer progression and are thought to be at the root of genetic disorders as microcephaly and ciliopathies. Although proteomic analysis have identified approximately 200 proteins as part of the centrosome-proteasome, the role of most of these proteins in centrosome biology is still a mystery. In addition, other proteins not localizing to the centrosome may be involved in regulating fundamental centrosomal functions. We performed a genome wide analysis to discover new players of centrosome biology, focusing our attention on centrosome positioning. Our analysis tested the function of almost every single gene of the human genome by using a siRNA library to switch off the expression of the genes in the cells and subsequently analysing the consequences on centrosome position.

The centrosome, during interphase of actively dividing cells, is usually in close proximity to the nuclear surface. It is believed that this connection between the nucleus and the centrosome could be critical for several cellular processes. To screen for factors involved in this connection, we measured the distance from the centrosomes to the nuclear membrane systematically in our genomic approach (Figure 1).

With this strategy we uncovered 52 new regulators required for keeping the centrosome close to the nuclear surface. Among these candidates we selected one of them (CEP170) and characterized its molecular role in centrosome positioning. We revealed that CEP170 controls centrosome positioning by influencing the stability of the microtubule network surrounding the nucleus.

In addition we studied the molecular function of TRIM37, a gene identified in a previous screen as a mayor regulator of centrosome number. TRIM37 is an E3 ubiquitin ligase which loss of function mutations underlies a rare autosomal recessive multi-organ disorder of unknown molecular etiology: Mulibrey nanism (MUL). On the other hand TRIM37 overexpression is frequently found in breast cancer cells where it contributes to tumorigenesis. We discovered that TRIM37 restrict extra centrosome number formation by inhibiting two centriolar formation pathways.

Overall, the results produced in this study, once published, will increase the understanding of the scientific community on centrosome related diseases as cancer, microcephaly and ciliopathies. Furthermore the molecular mechanisms revealed that connect the E3 ligase TRIM37 with two centriole biogenesis pathways, might shed light on the molecular origin of the rare disorder Mulibrey nanism, and contribute therefore to envisage new therapeutic strategies.