Tyrosine phosphorylation and cytoskeletal tension regulate the release of fibroblast adhesions

We have investigated the mechanisms by which fibroblasts release their adhesions to the extracellular matrix substrata using a permeabilized cell system in which the adhesions remain relatively stable. A large number of different molecules were assayed for their effect on focal adhesion stability using immunofluorescence with antibodies against different focal adhesion constituents. ATP uniquely stimulates a rapid breakdown of focal adhesions, and at high ATP concentrations $(>$5mM), many cells are released from the dish. The remaining cells appear contracted with talin, α-actinin and vinculin localized diffusely throughout the cell. Integrin containing tracks of variable intensity outline the regions where cells had resided before they detached from the substratum. At lower ATP concentrations (0.5-5mM) the cells remain spread; however the focal adhesion components, including integrin, show an array of phenotypes ranging from diffusely localized throughout the cell to a localization in small, thin focal adhesions. Okadaic acid, a serine, threonine phosphatase inhibitor, enhances the contracted phenotype, even at low concentrations (0.5mM) of ATP. The localization of focal adhesion components is different in okadaic acid treated cells. In highly contracted cells, integrin is present in tracks where the cells resided prior to the contraction; however focal adhesions are no longer apparent. Talin, vinculin and α-actinin localize in trabecular networks toward the periphery of the cell. Interestingly, phosphotyrosine staining as well as nascent, intracellular integrin precedes the recruitment of focal adhesion constituents into the trabecular network. The ATP-stimulated focal adhesion breakdown appears to operate through two mechanisms. First ATP stimulates the tyrosine phosphorylation of several cytoskeletally associated proteins. These tyrosine phosphorylations correlate well with focal adhesion breakdown. Furthermore, addition of a recombinant, constitutively active tyrosine phosphatase inhibits both the tyrosine phosphorylations and the breakdown of the focal adhesions. None of the major tyrosine phosphoproteins are FAK, integrin, tensin, paxillin or other phosphoproteins implicated in focal adhesion assembly. The second mechanism is cell contraction. High ATP concentrations, or lower ATP concentrations in the presence of okadaic acid induce cell contraction. Inhibiting the contraction by addition of a heptapeptide IRICRKG, which blocks the actin-myosin interaction, also inhibits focal adhesion breakdown. Neither the peptide nor the phosphatase inhibits focal adhesion breakdown under all conditions suggesting that both tension and tyrosine phosphorylations mediate the release of adhesions.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2011

Author dc:creator

• Crowley, Eileen

Contributors dc:contributor

• Horwitz, Alan F.

• Biology, Cell

Statement dc:rights

• Copyright 1996 Crowley, Eileen

Language dc:language

eng

Identifier

9780591197839
AAI9712242
(UMI)AAI9712242

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/21987