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Anastellin, a 76 amino acid polypeptide, derived from the first type-III repeat of the fibronectin (FN) molecule was shown to promote FN matrix assembly, and at higher concentrations to induce disassembly of preformed FN matrix. In our study we analyzed the effects of Anastellin treatment on focal adhesion (FA) organization in cultured Pig Aortic Endothelial Cells.
We specifically examined the effect of this treatment on the organization and local intensity of several FA-associated proteins and on the relationships between them. Cells plated on cover glasses for 16 hrs were treated with 20 µM Anastellin in serum-free medium, for different periods of time, then premeabilized/fixed and immunofluorescently labeled for different FA-associated proteins. The Anastellin treatment induced a dramatic disappearance of FN fibrils within 60 min. Consequently, vinculin labeling in the same cells revealed reduced size of individual FA, but increased number of small adhesions. The morphology of these adhesions differs from the one found in untreated cells, exhibiting elongated shape which largely resembles fibrillar adhesions and arrays of Actin stress fibers. Phosphotyrosine (PY) labeling in matrix adhesions revealed a marked decrease in number and intensity of labeled structures, which were retained only in at the cell periphery. Tensin, which is normally associated with fibrillar adhesions, accumulated, following treatment, in FA-like structures, in association with PY. When Anastellin treatment was performed in the presence of the ser/thr kinase inhibitor H-7, a marked decrease in re-assembly of these tensin-containing adhesions was observed. Live cells movies, showing the distribution of YFP-Paxillin in Anastellin-treated cells, suggest that the assembly of these adhesions is associated with the disassembly of mature FA.
We propose that destruction of FN fibrils by Anastellin, affects the mechanical properties of the underlying matrix and induces the formation of a new form of matrix adhesions, that contain components of both focal- and fibrillar adhesions.
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Anastellin disrupts Fibronectin (FN) filaments and induces the assembly of "Focal Adhesion-like" structures.
Pig Aortic Endothelial Cells (PAEC) were plated on cover-glasses for 16 hr and then treated for 6 hr with 20 µM Anastellin, which induces FN filaments disruption. Then the cells were premeabilized/fixed and double immunolabeled for: Tensin (red) and FN (blue), and Tensin (red) and Phosphotyrosine, PY (blue). The ratios per pixels were calculated and presented by a logarithmic color look-up table, which enable the presentation of ratio value variations spanning two orders of magnitude (from 1 to 10). Anastellin treatment induces the disruption of FN filaments and the formation of increased number of "Focal Adhesion-like" structures, which resemble fibrilar adhesions, which contain increased amounts of PY. |
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