title: Calcium ions and tyrosine phosphorylation interact coordinately with actin to regulate cytoprotective responses to stretching.
abstract: The actin-dependent sensory and response elements of stromal cells that are involved in mechanical signal transduction are poorly understood. To study mechanotransduction we have described previously a collagen-magnetic bead model in which application of well-defined forces to integrins induces an immediate (< 1 second) calcium influx. In this report we used the model to determine the role of calcium ions and tyrosine-phosphorylation in the regulation of force-mediated actin assembly and the resulting change in membrane rigidity. Collagen-beads were bound to cells through the focal adhesion-associated proteins talin, vinculin, alpha 2-integrin and beta-actin, indicating that force application was mediated through cytoskeletal elements. When force (2 N/m2) was applied to collagen beads, confocal microscopy showed a marked vertical extension of the cell which was counteracted by an actin-mediated retraction. Immunoblotting showed that force application induced F-actin accumulation at the bead-membrane complex but vinculin, talin and alpha 2-integrin remained unchanged. Atomic force microscopy showed that membrane rigidity increased 6-fold in the vicinity of beads which had been exposed to force. Force also induced tyrosine phosphorylation of several cytoplasmic proteins including paxillin. The force-induced actin accumulation was blocked in cells loaded with BAPTA/AM or in cells preincubated with genistein, an inhibitor of tyrosine phosphorylation. Repeated force application progressively inhibited the amplitude of force-induced calcium ion flux. As force-induced actin reorganization was dependent on calcium and tyrosine phosphorylation, and as progressive increases of filamentous actin in the submembrane cortex were correlated with increased membrane rigidity and dampened calcium influx, we suggest that cortical actin regulates stretch-activated cation permeable channel activity and provides a desensitization mechanism for cells exposed to repeated long-term mechanical stimuli. The actin response may be cytoprotective since it counteracts the initial force-mediated membrane extension and potentially strengthens cytoskeletal integrity at force-transfer points.
authors.affiliation: MRC Group in Periodontal Physiology, University of Toronto, Ontario, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Glogauer
authors.forename: P
authors.initials: P
authors.lastname: Arora
authors.forename: G
authors.initials: G
authors.lastname: Yao
authors.forename: I
authors.initials: I
authors.lastname: Sokholov
authors.forename: J
authors.initials: J
authors.lastname: Ferrier
authors.forename: C A
authors.initials: CA
authors.lastname: McCulloch
mesh_terms: D000199:Actins / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002462:Cell Membrane / Q000502:physiology*; D048429:Cell Size / Q000502:physiology; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003599:Cytoskeleton / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology; D005881:Gingiva / Q000166:cytology; D006801:Humans; D008954:Models, Biological; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010812:Physical Stimulation; D015398:Signal Transduction; D013314:Stress, Mechanical
chemical_list: D000199:Actins; D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D002118:Calcium