Zyxin is not colocalized with vasodilator-stimulated phosphoprotein (VASP) at lamellipodial tips and exhibits different dynamics to vinculin, paxillin, and VASP in focal adhesions.
Actin polymerization is accompanied by the formation of protein complexes that link extracellular signals to sites of actin assembly such as membrane ruffles and focal adhesions. One candidate recently implicated in these processes is the LIM domain protein zyxin, which can bind both Ena/vasodilator-stimulated phosphoprotein (VASP) proteins and the actin filament cross-linking protein alpha-actinin. To characterize the localization and dynamics of zyxin in detail, we generated both monoclonal antibodies and a green fluorescent protein (GFP)-fusion construct. The antibodies colocalized with ectopically expressed GFP-VASP at focal adhesions and along stress fibers, but failed to label lamellipodial and filopodial tips, which also recruit Ena/VASP proteins. Likewise, neither microinjected, fluorescently labeled zyxin antibodies nor ectopically expressed GFP-zyxin were recruited to these latter sites in live cells, whereas both probes incorporated into focal adhesions and stress fibers. Comparing the dynamics of zyxin with that of the focal adhesion protein vinculin revealed that both proteins incorporated simultaneously into newly formed adhesions. However, during spontaneous or induced focal adhesion disassembly, zyxin delocalization preceded that of either vinculin or paxillin. Together, these data identify zyxin as an early target for signals leading to adhesion disassembly, but exclude its role in recruiting Ena/VASP proteins to the tips of lamellipodia and filopodia.
Rottner
K
K
Department of Cell Biology, Gesellschaft für Biotechnologische Forschung, D-38124 Braunschweig, Germany.
Krause
M
M
Gimona
M
M
Small
J V
JV
Wehland
J
J
D000185:Actinin / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D005347:Fibroblasts; D022001:Focal Adhesions / Q000378:metabolism*; D006023:Glycoproteins; D006367:HeLa Cells; D006801:Humans; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D011554:Pseudopodia / Q000378:metabolism*; D051381:Rats; D016596:Vinculin / Q000378:metabolism*; D060589:Zyxin; D000098662:Vasodilator-Stimulated Phosphoprotein
D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D006023:Glycoproteins; D008667:Metalloproteins; D008840:Microfilament Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; C496062:Pxn protein, mouse; C496063:Pxn protein, rat; C092842:ZYX protein, human; C498285:Zyx protein, mouse; D060589:Zyxin; D000098662:Vasodilator-Stimulated Phosphoprotein; D000185:Actinin; D016596:Vinculin