αII-Spectrin Is Critical for Cell Adhesion and Cell Cycle

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 ␣II-spectrin deficiency is associated with a defect in cell proliferation, which is related to a cell cycle arrest at the G 1 phase (first gap phase), as evaluated by DNA analysis and Rb phosphorylation. These observations coincided with elevated expression of the cyclin-dependent kinase inhibitor, p21 Cip . 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 ␣II-spectrin in cell cycle regulation and actin organization.First identified at the intracellular surface of the erythrocyte plasma membrane, the spectrin-based skeleton is considered as a nearly ubiquitous and complex spectrin-actin network in metazoan cells (1).Spectrins are giant extended flexible molecules composed of two subunits (␣ and ␤) that intertwine to form ␣␤ heterodimers. Spectrin is normally considered to exist as tetramers resulting from self-association of ␣␤ dimers. Spectrin tetramers constitute the filaments of the lattice, the nodes of which are cross-linked by actin filaments. This spectrin-based skeleton is bound to various transmembrane proteins either directly, or more frequently through two connecting proteins, ankyrin and protein 4.1. In mammals, the spectrin family currently includes seven genes encoding for two ␣-subunits (␣I and ␣II), four "conventional" ␤-subunits (␤I to ␤IV) and one ␤ heavy subunit (␤V), as well as multiple alternatively spliced variants, each of these species presenting its specific cellular expression pattern. For example, whereas ␣I-spectrin is essentially expressed in the mature erythrocyte, ␣II-spectrin is the most common form in nucleated cells.The functions clearly determined up to date for the spectrin network emerge from human mutations associated with diseases as well as from animal models. Numerous studies on red cells, particularly those in hereditary hemolytic anemia, have clearly established its importance for supporting cell shape and for maintaining cell membrane integrity and stability (2, 3). In nucleated cells, 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 direct interaction between TRCP4 channel and spectrin is involved in the regulation of the channel surface expression and activation. One consistent feature observed when spectrin or its binding partner ankyr...

Cell organization, growth, and neural and cardiac development require αII-spectrin

“…These include a role in the formation and maintenance of specialized plasma membrane domains defining apicalbasolateral and planar polarity in epithelial cells, muscle and neurons (Bennett and Baines, 2001); in the structural support of the plasma membrane and the maintenance of cell shape (Gallagher and Jarolim, 2000;Kizhatil et al, 2007); as a scaffold upon which calcium-mediated and tyrosine kinase-phosphatase signal transduction pathways converge (Nicolas et al, 2002;Nedrelow et al, 2003); as a tumor-suppressor protein involved in TGF-b-SMAD regulation (Tang et al, 2003); as a cargo selection mechanism in the secretory and endocytic pathways (De Matteis and Morrow, 2000); as a regulator of macropinocytosis (Xu et al, 2000); as a tether linking trafficking vesicles to microtubule motors (Holleran et al, 2001;Muresan et al, 2001); as a nuclear scaffold organizer (McMahon et al, 1999;Tse et al, 2001); and most recently, as a potential mechano-sensing ligand-binding switch (Stabach et al, 2009). Deletion of aII-spectrin in Drosophila melanogaster and Caenorhabditis elegans leads to late embryonic-early larval stage lethality (Moorthy et al, 2000;Dubreuil, 2006;Hammarlund et al, 2007), and recent knockdown studies of aII-spectrin in cultured cells have demonstrated growth and adhesion defects (Metral et al, 2009). However, the role of aII-spectrin in vertebrate development remains unexplored.…”

Intercellular adhesion molecule-4 and CD36 are implicated in the abnormal adhesiveness of sickle cell SAD mouse erythrocytes to endothelium

“…Alternatively, ICAM-4 molecules on SAD RBC could be gathered as clusters, enabling stronger binding to aVβ3 integrin clusters on endothelial cells. 31,32 Likewise, an increased clustering of CD36 on SAD RBC, as observed by electronic microscopy in platelet membranes, 33 may strengthen the interaction between erythrocyte CD36 and endothelial thrombospondin.…”

Novel role for the Lu/BCAM–spectrin interaction in actin cytoskeleton reorganization

“…We and others have demonstrated that the interaction of Lu/BCAM with erythroid spectrin negatively regulated its adhesive receptor function in normal and spectrin-deficient HS RBCs [15,18]. In non-erythroid adherent cells, partial DII-spectrin depletion was associated with loss of cell spreading, defective adhesion and decrease and irregularity of focal adhesion points [32].…”

“…The role of DII-spectrin in actin organization has been recently demonstrated, as DII-spectrin knocked-down melanoma WM-266 cells exhibited modifications of the actin cytoskeleton such as a loss of stress fibers [32]. DII-Spectrin, via its SH3 domain, has been also implicated in initiating Rac activation in the specialized E3 integrin clusters that initiate cell adhesion and spreading [35].…”