Fig. 8. MARCKS localization and translocation in 5-expressing cells plated on FN. (A) MARCKS is localized to focal adhesion sites during the initial phases of muscle cell attachment and spreading. 5-expressing cells were plated on FN, and cells were fixed and co-stained for MARCKS and the focal adhesion protein paxillin. The cells shown here were fixed 30 minutes after plating and show colocalization of MARCKS and paxillin, demonstrating the localization of MARCKS at focal adhesion sites (arrow). (B) MARCKS translocates from the membrane to the cytosol during muscle cell spreading. 5-expressing cells were plated on FN for various times prior to fixation and immunocytochemical assessment of MARCKS localization. MARCKS localization to focal adhesion sites (arrows in each panel) is most prominent at early time points, decreasing in intensity as the cells spread. With time, MARCKS becomes more diffusely distributed in the cytosol. (C) MARCKS translocation is mediated by integrin activation. To confirm the immunocytochemical translocation in B and to assess the role of integrin activation in the process, 5-expressing and 5-deficient cells were plated on FN, and MARCKS translocation was assessed by cellular fractionation. In 5-expressing cells, MARCKS is initially localized predominantly in the membrane compartment, consistent with the localization seen in B. With time, MARCKS translocates to the cytosolic fraction such that by 90 minutes, nearly all of the protein is in this compartment. By contrast, there is almost no translocation of MARCKS from the membrane to the cytosol in 5-deficient cells plated on FN.