Arp2/3 complex distribution in the cytoskeleton of active human platelets.

Human platelets were activated by centrifugation onto poly-l-lysine–coated glass coverslips, permeabilized with 0.5% Triton X-100 in PBS, and fixed and treated with 0.1% SDS. The actin cytoskeleton is labeled with a mixture of affinity-purified rabbit polyclonal anti-Arp3 and anti–p34-Arc antibodies and TRITC-labeled goat antirabbit IgG (A). Anti-Arp2/3 complex staining concentrated at the cell periphery and at foci in the center of the cytoskeletons. The peptides (50 μg/mL) used to raise the antibodies were used mixed with the anti-Arp2/3 complex antibodies as controls and blocked all detectable antibody binding (B). Human platelets were activated by centrifugation onto poly-l-lysine–coated glass coverslips, permeabilized with 0.75% Triton X-100 in PHEM buffer, fixed, and treated with 0.1% SDS (C). Cytoskeletons were labeled with a mixture of rabbit polyclonal anti-Arp3 and anti–p34-Arc antibodies followed by 10 nm colloidal gold coated with goat antirabbit IgG (black spheres). Cytoskeletons were fixed, washed into water, rapidly frozen, freeze-dried, and metal-coated. Immunogold recognizing Arp2/3 complex densely labels spread lamellar regions (lm) of the active cytoskeletons. Some internal structures are also labeled (arrows). The tips of the filopodia are poorly labeled (f). Note that the electron and light microscopy patterns are identical and that there are no filopodia labeled in panel A. Bars represent 5 μm (A) and 200 nm (C).