Injected cells were allowed to recover for 4-6 hours and were then fixed with RR, processed for EM, and sectioned transverse to the substrate (Fig. 4B). Although endocytic invaginations were observed in uninjected cells and in cells injected with buffer or heat-inactivated antibodies (Fig. 4A), cells injected with Y-27632 clinical trial the native dynamin antibodies displayed many more of these structures that were substantially larger in size and more extensive in length. Indeed, as shown in Fig. 4B, the basal PM of cells in which Dyn2 function was inhibited

was lined with numerous RR-positive membrane structures. To further examine the structure of the hypertrophied endocytic invaginations in Dyn2-inhibited cells, MC65 antibody-injected primary rat hepatocytes were fixed, stained with RR, embedded, and thick-sectioned for viewing with the high-voltage EM. Our objective was to use the advantage of thick sections (0.2-0.4 μm) combined with RR to better define the effects of dynamin antibodies on hot spot morphology

and the relationship of these structures with the PM. We found that inhibition of dynamin function induced several distinct changes in the PM (Fig. 4C,D). Consistent with our previous observations, the LDK378 invaginated structures were not found uniformly along the PM but in distinct foci. The RR-positive endocytic PM was frequently tubulated in close proximity to the cell surface. These tubules often Bacterial neuraminidase extended significant distances (5-10 μm) into the cell interior. Although some of these structures appeared to have spiked clathrin coats, many did not. Interestingly, these tubules were often constricted with numerous varicosities, leading to the formation of a reticularized tubule network with many associated buds (Fig. 4C,D). This vesiculation was often so pronounced as to create endocytic structures that appeared as many vesicle buds attached to tubules, similar to grapes on a vine. These images are consistent with the prediction

that dynamin functions at endocytic hot spots to constrict endocytic PM invaginations into discrete vesicles. Antibody-induced inhibition of dynamin function results in the accumulation of a spectrum of tubules and buds at various stages of the vesiculation process. Stereo 3D images of these structures are provided as Supporting Fig. S1 and reveal the complexity of these very large endocytic structures in comparison to conventional clathrin-coated pits. Although the EM approach described here revealed dramatic changes in the number and size of endocytic hot spot invaginations with dynamin inhibition, these observations are qualitative in nature. Accordingly, we transfected Clone 9 cells with either WT Dyn2 or Dyn2 bearing loss-of-function mutations (Dyn2K44A, Dyn2 Y231/597F) that inhibit activity. Then, to test if inhibiting Dyn2 function leads to an accumulation of hot spots, we examined cells by immunofluorescence (IF) with either Dyn2 or clathrin antibody staining.