Vitiello, E; (2014) DLC-2, a CDC42 gap involved in attachment of microtubules at the cell cortex and at the kinetochores. Doctoral thesis , UCL (University College London).

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Abstract

Maintenance of epithelial tissue integrity requires correct organization of the polarity plane. This is promoted by assembly of cell-cell junctions, defining distinctive apical and basolateral domains. The establishment of polarity is a key point in tissue organization since it controls the plane in which cells divide. When a cell replicates, it duplicates DNA and aligns them before partitioning them evenly between the two daughter cells. Although results from different laboratories indicate a role of cell-cell junctions in the orientation of the mitotic spindle, it is poorly understood if there is a cross-talk between the mitotic checkpoint and the attachment status of the microtubules at the cell cortex. Previous works suggest that the small Rho GTPase Cdc42 regulates spindle orientation and chromosome capture. Cdc42 is also a crucial regulator of cell-cell junction assembly and dynamics. Cell junctions need to remodel during cell division to adapt to the changing cell shape, and allow the condensed chromosomes to be properly aligned. Therefore, we hypothesized that specific regulators of Cdc42 guide the interplay between cell junctions and the mitotic machinery. Using a siRNA approach, we identified a GAP for Cdc42, DLC2, that associates with cell-cell contacts and mitotic spindles, and regulates junctional integrity and chromosome attachment during mitosis. Upon depletion of DLC2 in epithelial cells, the normally continuous immunofluorescence staining of junctional markers was disrupted in mitotic cells, and chromosomes are misaligned. Following DLC2 depleted cells on live, it results clear that these cells are arrested in metaphase for longer timing, with some chromosomes unattached. The orientation of mitotic spindles relative to the substrate was not affected, suggesting that the defect was not due to a loss of polarity. Depletion of DLC2 led to increased levels of GTP-bound Cdc42, indicating that deregulation of Cdc42 contributed to the observed phenotypes. Indeed, partial depletion of Cdc42 by RNA interference rescued the phenotype induced by depletion of DLC2. We also found that DLC2 associates with the plus-end motor Kif1B, a kinesin-3 family member, and that depletion of Kif1B resulted in a similar phenotype as depletion of DLC2. Based on our observations, we speculate that DLC2 regulates Cdc42 to guide association of microtubules with the cell cortex and the kinetochores in metaphase and to regulate forces they exert at these sites.

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