Several minutes later, the same cell assembled additional hot spots at different locations
along the cell base that also generated many vesicles and then disappeared (images not shown). Many of these dynamin-associated vesicles were seen translocating in a linear path, as if along selleckchem cytoskeletal filaments. This observation suggests that dynamin may remain on the motile vesicle, rather than immediately dissociating as predicted.13 We counted the number of vesicles generated during a typical hot spot lifetime (10-15 minutes) and observed an average steady state release of 4-6 vesicles/min (4 cells counted). Most remarkable is that each hot spot undergoes a 2 to 3-minute burst that generates 15-20 vesicles per minute during which the structure physically “dissolves.” This is exceptionally rapid
when compared with the release of multiple vesicles from conventional clathrin pits (1-2 vesicles/min).20 The rapid vesicle formation from Dyn2/clathrin/AP2 hot spots suggests that these are endocytic structures that are either continuous with the PM or reside as an internal endocytic sorting compartment. Because the HRP marker used to label hot spots by EM (Fig. 1C) was internalized by cells over a 45-minute time period, it is possible that the endocytic hot spots, while in intimate proximity with the PM, represent internal endocytic sorting compartments. To test if these endocytic structures are distinct from or continuous with the PM, we utilized Ruthenium red (RR), an electron-dense dye, to label the cell surface and
any invaginations DMXAA continuous with the external environment. Because the dye is included in the primary fixative, thereby preventing its internalization, membrane compartments stained with the dye represent extensions of the PM. As expected, cells stained with RR, processed for EM, and sectioned transverse to the substrate showed very dark apical and basal PMs, whereas internal membrane Urease systems were only lightly stained (Fig. 4A,B). In control cells (Fig. 4A), small patches of dark, tubular invaginations were observed extending from the basal, but not the apical, PM. These structures were nearly identical in dimensions to those observed in the HRP-labeled cells that were sectioned en face (Fig. 1) and appear as a “side view” of the endocytic hot spots shown in the previous figures. Most important, these endocytic structures were darkly stained with the RR dye, indicating that they are continuous invaginations of the PM. Because GFP-tagged Dyn2 associates with the endocytic hot spots (Figs. 1-3), we predicted that disruption of Dyn2 function would alter the number and complexity of the RR-positive PM invaginations. To test this hypothesis, cultured cells were microinjected with one of two purified polyclonal antibodies that we have used in previous studies to inhibit dynamin function.