αII-Spectrin Is Critical for Cell Adhesion and Cell Cycle

Sylvain Metral,Didier Dhermy,Beata Machnicka,Marie-Christine Lecomte,Yves Colin,Sylvain Bigot

doi:10.1074/jbc.m801324200

Abstract

Spectrins are ubiquitous scaffolding components of the membrane skeleton that organize and stabilize microdomains on both the plasma membrane and the intracellular organelles. By way of their numerous interactions with diverse protein families, they are implicated in various cellular functions. Using small interfering RNA strategy in the WM-266 cell line derived from human melanoma, we found that alphaII-spectrin deficiency is associated with a defect in cell proliferation, which is related to a cell cycle arrest at the G1 phase (first gap phase), as evaluated by DNA analysis and Rb phosphorylation. These observations coincided with elevated expression of the cyclin-dependent kinase inhibitor, p21Cip. Concomitantly, spectrin loss impaired cell adhesion and spreading. These cell adhesion defects were associated with modifications of the actin cytoskeleton, such as loss of stress fibers, alterations of focal adhesions, and modified expression of some integrins. Our results provide novel insights into spectrin functions by demonstrating the involvement of alphaII-spectrin in cell cycle regulation and actin organization.

Highlights

Spectrins are giant extended flexible molecules composed of two subunits (␣ and ␤) that intertwine to form ␣␤ heterodimers
We studied the relative contributions of ␣II-spectrin to cell behavior using a small interfering RNA2 knock-down approach in a cellular model
Effective after 48 h Transfection—To select optimal and specific small interfering RNA (siRNA) targeting ␣II-spectrin, six siRNA duplexes were tested on spectrin expression in the WM-266 melanoma cells during 4 days after transfection

Summary

Introduction

Spectrins are giant extended flexible molecules composed of two subunits (␣ and ␤) that intertwine to form ␣␤ heterodimers. The spectrin-based skeleton has been shown to participate in the stabilization or activation of several specialized membrane proteins, as recently reported for the TRCP channels [4]. The consequences of loss of function of ␤-spectrins and ankyrins are progressively better explained, the cellular consequences of ␣-spectrin defects are less well established, except in the context of red blood cells: mutations in the gene coding for ␣I-spectrin mainly expressed in mature red blood cells from mammals, are associated with severe hemolytic anemia and in some cases with a short survival [11, 12]. We studied the relative contributions of ␣II-spectrin to cell behavior using a small interfering RNA (siRNA) knock-down approach in a cellular model. For the first time we show that depletion of ␣II-spectrin is associated with a loss of cell adhesion as well as with an arrest of cell proliferation

Results

Discussion

Conclusion