Formation of stable microspikes containing actin and the 55 kDa actin bundling protein, fascin, is a consequence of cell adhesion to thrombospondin-1: implications for the anti-adhesive activities of thrombospondin-1.
The organisation of the actin cytoskeleton was examined in H9c2 and human intestinal smooth muscle cells adherent on fibronectin or thrombospondin-1. Whereas cells adherent on fibronectin adopted a polygonal shape and rapidly assembled prominent stress fibres and focal contacts, cells adherent on thrombospondin-1 assumed a more irregular morphology with large lamellae containing radial actin microspikes. Focal contacts were not detected in cells adherent on thrombospondin-1, as determined by indirect immunofluorescence staining for vinculin and other focal contact components. Instead, the radial microspikes stained positively for the actin-bundling protein, 55 kDa/fascin, and myosins. In cells adherent on fibronectin, 55 kDa/fascin immunoreactivity was diffuse and tended to be concentrated in the perinuclear region. In long-term adherent cells cultured in serum-containing medium, 55 kDa/fascin was detected in membrane ruffles, in stress fibres and in the perinuclear region. The microspikes formed within 40 minutes of plating cells on thrombospondin-1 and remained present when cells were treated with sodium orthovandate and hydrogen peroxide to increase intracellular phosphotyrosine levels. Indeed, although vanadate-treated cells tended to retract, the microspikes became more prominent and showed an increased intensity of staining for fascin. Under these conditions, a proportion of the microspikes did not appear to be in contact with the substratum: these spikes stained weakly for focal adhesion kinase, talin and vinculin. Cells treated with genistein also spread and formed fascin-containing microspikes which tended to be more slender than those of control cells. In contrast, cells adherent on fibronectin displayed a complex rearrangement of the actin cytoskeleton and a transient enrichment of 55 kDa/fascin-containing structures at the cell surface when treated with sodium orthovanadate and hydrogen peroxide. These observations indicate that cell interactions with fibronectin or thrombospondin-1 send distinct organisational signals to the actin cytoskeleton and may offer a mechanistic framework for further investigations of the anti-adhesive properties of thrombospondin-1.
Adams
J C
JC
MRC Laboratory for Molecular Cell Biology, Department of Biology, University College London, UK.
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism; D048429:Cell Size; D003599:Cytoskeleton / Q000737:chemistry / Q000648:ultrastructure*; D005353:Fibronectins / Q000378:metabolism; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism*; D009130:Muscle, Smooth / Q000187:drug effects / Q000648:ultrastructure; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D016608:Talin / Q000378:metabolism; D019699:Thrombospondins; D014638:Vanadates / Q000494:pharmacology; D016596:Vinculin / Q000378:metabolism
D000199:Actins; D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D005353:Fibronectins; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D016608:Talin; D019699:Thrombospondins; D016596:Vinculin; C020324:fascin; D014638:Vanadates; D006861:Hydrogen Peroxide; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; C496052:Ptk2 protein, rat