Degradation of Calponin 2 is Required for Cytokinesis

Abstract

Calponin 2 is an actin filament-associated protein and plays a role in regulating cytoskeletal functions such as cell adhesion, migration, phagocytosis and cell division. We previously showed that the level of calponin 2 is inversely correlated with the rate of cell proliferation. However, the mechanism by which calponin 2 is down-regulated during cytokinesis is unknown. In the present study, we investigate the levels of endogenous calponin 2 in NIH/3T3 cells and HEK293 cells decreased prior to cell division characterized by an absence in the F-actin-based contractile ring. Nocodazole-synchronized NIH/3T3 cells showed decreased calponin 2, and MG132, a ubiquitin proteasomal inhibitor (UPS), increased the overall calponin 2 level in the cells post nocodazole treatment. The results suggest that M phase cells have a downregulation of calponin 2, which is likely mediated through UPS. Furthermore, GFP-fusion calponin 2 was constructed for dynamic studies in live cells. Control experiments using NIH/3T3 cells and HEK293 cells showed that non-fusion and GFP-fusion calponin 2 had minimal difference in cytoskeleton association and effects on cell proliferation. A smooth muscle-originated cell line SM3 lacking endogenous calponin was transiently transfected with GFP-calponin 2 expression plasmid and imaged using fluorescent microscopy at a series of time points. The results showed that 1) the level of calponin 2 decreased prior to cell division, especially at the contractile ring; 2) the overall level of calponin 2 expression had an inverted correlation to the duration of cytokinesis; and 3) cells had high level ectopic expression of calponin 2 could not complete cytokinesis, which was followed by cell death. The data suggest that degradation of calponin 2 is a factor determining the rate and fate of cytokinesis, which may be targeted to control cell proliferation during development, wound healing, inflammation, pathological remodeling, and tumor growth and metastasis.