In cell culture, entry into mitosis of many adherent mammalian cells is accompanied by substantial changes in cellular architecture. Flat, spread-out interphase cells detach from the extracellular matrix and become more spherical. These changes in cell shape are mediated by rearrangements in the actin cytoskeleton, a dynamic network of actin filaments that are organized by actin-binding proteins. Filamin A (FLNa) is a 280 kD actin-binding protein that crosslinks actin filaments into parallel bundles or three-dimensional orthogonal networks. We previously identified FLNa as an in vitro substrate of cyclin-dependent kinase 1 (Cdk1), a kinase that regulates entry into mitosis, and hypothesized that Cdk1 phosphorylation of FLNa regulates mitotic actin remodelling. Using mass spectrometry and a p-FLNa antibody, we show that FLNa is phosphorylated in vivo in HeLa cells on multiple Cdk1 sites, including serines 1084, 1459 and 1533. All three sites match the phosphorylation consensus sequence of Cdk1. We further show that p-FLNa is almost fully dephosphorylated by anaphase, consistent with it being a cell cycle-regulated substrate. Using a phospho-specific antibody, we find that p-FLNa has decreased cortical actin localization compared to total FLNa in mitotic cells. To investigate the functional role of mitotic FLNa phosphorylation, we mutated serines 1084, 1459 and 1533 to nonphosphorylatable alanine and expressed this FLNa mutant (FLNa-S1084A, S1459A, S1533A, referred to as “FLNa-AAA GFP”) in FLNa-deficient human M2 melanoma cells. FLNa-AAA GFP-expressing cells have enhanced FLNa-AAA GFP localization at sites of contact between daughter cells and this correlates with defects in cell division and impaired cell migration. Therefore, mitotic delocalization of cortical FLNa is critical for successful cell division and interphase cell behaviour.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/31732 |
| Date | January 2014 |
| Creators | Szeto, Sandy |
| Contributors | Lee, Jonathan |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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