Effects of C-terminal Mutant of a Formin Protein on Actin Stress Fiber Formation and Cell Proliferation in Murine Fibroblasts

Induction of actin stress fibers may affect progression of the cell cycle and hence alter cell proliferation patterns. A C-terminal mutant (DC) of the formin family protein, formin homology domain containing-protein 1 (FHOD1), causes the appearance of thick actin stress fibers formation in murine fibroblasts, co-localizes with these stress fibers, and renders cells resistant to loss of stress fibers typically observed when cells are serum-starved. In contrast, FHOD1 wild type (WT) protein does not affect stress fiber architecture. Objective: To determine if cells over-expressing FHOD1 DC and FHOD1 WT present different propensities to proliferate. Methods: NIH-3T3 cells were plated on coverslips, transiently transfected with pCMV5 FHOD1 WT or pCMV5 FHOD1 DC (both were hemagglutinin[HA]-tagged). Twenty four hours after transfection, cells were cultured in the presence of 20nM bromodeoxyuridine (BrdU) for 8-24 hours. Cells were fixed in 3.7% paraformaldehyde, washed and exposed to immunofluorescence analysis after permeabilization/blocking, and incubation with primary antibodies against HA (rabbit) and BrdU (mouse), and appropriate anti-rabbit FITC- and anti-mouse Cy3-labelled secondary antibodies. Successfully transfected cells (FITC-labelled) were scored for BrdU incorporation (Cy3 labelled). The experiment was conducted four times (n=4) and a minimum of 160 cells were scored for each condition. The percentage of NIH 3T3 cells transfected with FHOD1 WT or FHOD1 DC that had incorporated BrdU was compared using a paired two-tailed Students t-test determined. Results: During the course of each experiment, a significantly larger percentage of cells over-expressing FHOD1 DC (37 + 12; mean + std. deviation) proliferated than cells over-expressing FHOD1 WT (27 + 10) (p=0.08). Conclusions: NIH3T3 cells over-expressing FHOD1 DC are characterized by the presence of thick actin stress fibers and are more likely to proliferate than cells over-expressing FHOD1 WT.