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Fig. 6. FAK is required for the formation of fibrillar adhesions. (A) Tensin immunostaining shows lack of fibrillar adhesions (arrowheads) on the dorsal surface of cells that do not have either FAK or PYK2/FAK chimeras localized in focal adhesions. (B). Anti-active ß₁ integrin 9EG7 Ab-chase experiments demonstrate defects in formation of fibrillar adhesions and organized FN in FAK^-/- cells. Live cells were exposed to the anti-active ß₁ integrin 9EG7 mAb for 45 minutes. After the Ab was washed away, one set of cells was fixed immediately (t=0) and a second set 2 hours later (t=2). Procedures for subsequent labeling of FN, active ß₁ integrin and paxillin are given in Materials and Methods. In overlaid images paxillin, a marker of focal adhesions, is colored blue, FN is red, and chased 9EG7 mAb is green. Within 2 hours ß₁ integrin and FN were translocated backward to the dorsal surface in cells that have FAK in focal contacts (FAK^+/+ and DA2). In FAK^-/- cells only a few ß₁ integrin/FN patches were detectable at the margins of spread cells. Integrin/antibody complexes are endocytosed at a much higher rate in cells that do not have either FAK or FAK/PYK2 chimera in focal adhesions. These complexes are not visualized because secondary anti-rat antibody is incubated with non-permeabilized cells.

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