The ESCRT protein complexes are recruited from the cytoplasm and assemble on the endosomal membrane into a protein network that functions in sorting of ubiquitinated transmembrane proteins into the multivesicular body (MVB) pathway. This transport pathway packages cargo proteins into vesicles that bud from the MVB limiting membrane into the lumen of the compartment and delivers these vesicles to the lysosome/vacuole for degradation. The dissociation of ESCRT machinery by the AAA-type ATPase Vps4 is a necessary late step in the formation of MVB vesicles. This ATP-consuming step is regulated by several Vps4-interacting proteins, including the newly identified regulator Ist1. Our data suggest that Ist1 has a dual role in the regulation of Vps4 activity: it localizes to the ESCRT machinery via Did2 where it positively regulates recruitment of Vps4 and it negatively regulates Vps4 by forming an Ist1-Vps4 heterodimer, in which Vps4 cannot bind to the ESCRT machinery. The activity of the MVB pathway might be in part determined by outcome of these two competing activities.

EPI64 is a TBC domain–containing protein that binds the PDZ domains of EBP50, which binds ezrin, a major actin-binding protein of microvilli. High-resolution light microscopy revealed that ezrin and EBP50 localize exclusively to the membrane-surrounded region of microvilli, whereas EPI64 localizes to variable regions in the structures. Overexpressing EPI64 results in its and EBP50's relocalization to the base of microvilli, including to the actin rootlet devoid of ezrin or plasma membrane. Uncoupling EPI64's binding to EBP50, expression of any construct mislocalizing its TBC domain, or knock down of EBP50 results in loss of microvilli. The TBC domain of EPI64 binds directly to Arf6-GTP. Overexpressing the TBC domain increases Arf6-GTP levels, and expressing dominant-active Arf6 results in microvillar loss. These data reveal that microvilli have distinct cytoskeletal subdomains and that EPI64 regulates microvillar structure.

A complex network of interactions mediates the recruitment of Vps4 to ESCRT-III and its subsequent assembly, two key steps in the ESCRT-dependent vesicle formation at the endosome. A model is presented depicting the order of events that lead to active, ESCRT-III–associated Vps4.

Pharmacological approaches and optical recordings have shown that Schwann cells of a myelinating phenotype are activated by 5-HT upon its interaction with the 5-HT(2A) receptor (5-HT(2A)R). In order to further characterize the expression and distribution of this receptor in Schwann cells, we examined rat sciatic nerve and cultured rat Schwann cells using probes specific to 5-HT(2A)R protein mRNA. We also examined the endogenous sources of 5-HT in rat sciatic nerve by employing both histochemical stains and an antibody that specifically recognizes 5-HT. Rat Schwann cells of a myelinating phenotype contained both 5-HT(2A)R protein and mRNA. In the healthy adult rat sciatic nerve, 5-HT(2A)Rs were evenly distributed along the outermost portion of the Schwann cell plasma membrane and within the cytoplasm. The most prominent source of 5-HT was within granules of the endoneurial mast cells, closely juxtaposed to Schwann cells within myelinating sciatic nerves. These results support the hypothesis that the 5-HT receptors expressed by rat Schwann cells in vivo are activated by the release of 5-HT from neighboring mast cells.