Abstract
Protein functions are often revealed by their localization to specialized cellular sites. Recent reports demonstrated that swiprosin-1 is found together with actin and actin-binding proteins in the cytoskeleton fraction of human mast cells and NK-like cells. However, direct evidence of whether swiprosin-1 regulates actin dynamics is currently lacking. We found that swiprosin-1 localizes to microvilli-like membrane protrusions and lamellipodia and exhibits actin-binding activity. Overexpression of swiprosin-1 enhanced lamellipodia formation and cell spreading. In contrast, swiprosin-1 knockdown showed reduced cell spreading and migration. Swiprosin-1 induced actin bundling in the presence of Ca2+, and deletion of the EF-hand motifs partially reduced bundling activity. Swiprosin-1 dimerized in the presence of Ca2+ via its coiled-coil domain, and a lysine (Lys)-rich region in the coiled-coil domain was essential for regulation of actin bundling. Consistent with these observations, mutations of the EF-hand motifs and coiled-coil region significantly reduced cell spreading and lamellipodia formation. We provide new evidence of how swiprosin-1 influences cytoskeleton reorganization and cell spreading.
Highlights
Motile cells display dynamic movement by lamellipodia- or filopodia-based membrane extensions at the leading cell edge
Actin-filament bundles and networks in the lamellipodia of motile cells contain several actin cross-linking proteins, including fascin, fimbrin, filamin, and a-actinin [7]. Proteins such as fascin and fimbrin are suited for forming strong bundles because they are short, compact, and form monomeric cross-links between adjacent actin filaments
Cell Culture Jurkat T (ATCC CRL-1651) and Raji B (ATCC CCL-86) cells obtained from American Type Culture Collection (Manassas, VA) were grown in RPMI medium supplemented with 10% heatactivated fetal bovine serum (FBS), penicillin G (100 IU/ml), streptomycin (100 mg/ml), and L-glutamine (2 mM)
Summary
Motile cells display dynamic movement by lamellipodia- or filopodia-based membrane extensions at the leading cell edge. The lamellipodium is a sheet-like protrusion that contains an extensively branched network or meshwork of actin filaments [1,2]. Filopodia are rod-like extensions composed of long, unbranched, parallel bundles of actin [3]. Actin-filament bundles and networks in the lamellipodia of motile cells contain several actin cross-linking proteins, including fascin, fimbrin, filamin, and a-actinin [7]. Proteins such as fascin and fimbrin are suited for forming strong bundles because they are short, compact, and form monomeric cross-links between adjacent actin filaments. Crosslinking of actin filaments is critical for cell motility and is a fundamental process during filopodia protrusion and lamellipodia formation
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