Increases in intracellular calcium concentration are required for the release of neurotransmitter from presynaptic terminals in all neurons. However, the mechanism by which calcium exerts its effect is not known. A low-sensitivity calcium-dependent photoprotein (n-aequorin-J) was injected into the presynaptic terminal of the giant squid synapse to selectively detect high calcium concentration microdomains. During transmitter release, light emission occurred at specific points or quantum emission domains that remained in the same place during protracted stimulation. Intracellular calcium concentration microdomains on the order of 200 to 300 micromolar occur against the cytoplasmic surface of the plasmalemma during transmitter secretion, supporting the view that the synaptic vesicular fusion responsible for transmitter release is triggered by the activation of a low-affinity calcium-binding site at the active zone.

Multivesicular body morphology and size are controlled in part by PtdIns(3,5)P 2 , produced in mammalian cells by PIKfyve-directed phosphorylation of PtdIns(3)P. Here we identify human Vac14 (hVac14), an evolutionarily conserved protein, present in all eukaryotes but studied principally in yeast thus far, as a novel positive regulator of PIKfyve enzymatic activity. In mammalian cells and tissues, Vac14 is a low-abundance 82-kDa protein, but its endogenous levels could be up-regulated upon ectopic expression of hVac14. PIKfyve and hVac14 largely cofractionated, populated similar intracellular locales, and physically associated. A smallinterfering RNA-directed gene-silencing approach to selectively eliminate endogenous hVac14 rendered HEK293 cells susceptible to morphological alterations similar to those observed upon expression of PIKfyve mutants deficient in PtdIns(3,5)P 2 production. Largely decreased in vitro PIKfyve kinase activity and unaltered PIKfyve protein levels were detected under these conditions. Conversely, ectopic expression of hVac14 increased the intrinsic PIKfyve lipid kinase activity. Concordantly, intracellular PtdIns(3)P-to-PtdIns(3,5)P 2 conversion was perturbed by hVac14 depletion and was elevated upon ectopic expression of hVac14. These data demonstrate a major role of the PIKfyve-associated hVac14 protein in activating PIKfyve and thereby regulating PtdIns(3,5)P 2 synthesis and endomembrane homeostasis in mammalian cells.

Adipocytes serve as the principal energy reservoir of the body; however, the subcellular organization of the machinery regulating lipid trafficking and metabolism is poorly understood. Mobilization of stored triglyceride is thought be controlled by interactions among intracellular lipases and proteins that coat lipid storage droplets. A major limitation of previous studies of hormone-mediated lipolysis, however, is the use of cultured model adipocytes whose three-dimensional architectures do not resemble those in real adipose tissue. To address this limitation, we investigated the intracellular targeting of perilipin, a major lipid coat protein, and hormone-sensitive lipase in three preparations that exhibit more appropriate morphologies: 3T3-L1 adipocytes grown in three-dimensional matrix, dissociated mature adipocytes from mouse adipose tissue, and adipocytes within intact fat pads. High resolution imaging of native and fluorescently tagged proteins indicate that: 1) perilipin preferentially targets a special class of peripheral lipid storage droplets, but not the major or central lipid storage droplets, 2) the peripheral droplets are the sites of attack by hormone-sensitive lipase, and 3) perilipin and hormone-sensitive lipase are continuously colocalized following lipolytic activation. These results indicate that in white adipose tissue, lipolysis takes place in a specialized subcellular domain that is distinct from the major lipid storage site and is defined by perilipin.The white adipose tissue plays a central role in energy storage and utilization. Work in the last decade has established perilipin (Plin) 2 as a critical regulator of lipid stores in adipocytes (reviewed in Refs. 1-3). It has been proposed that Plin covers lipid droplet surfaces and serves as a physical barrier that protects stored triglyceride from hydrolysis by cellular lipases (4, 5). Upon lipolytic stimulation, Plin is phosphorylated at multiple sites by PKA, thereby altering its association with the droplet surface and allowing hormone-sensitive lipase (HSL) access to lipid droplet surface to initiate lipolysis.Although there is strong evidence that Plin is critically involved in regulating access of HSL to stored triglyceride, several observations indicate that a simple barrier/translocation model is insufficient to explain lipolysis in vivo. For example, bulk translocation of HSL and Plin dissociation are not consistent features of lipolytic activation in primary adipocytes (6, 7). Furthermore, lipolysis can be observed in permeabilized fat cells, strongly indicating that the lipolytic machinery is preassembled to such a degree that the soluble pool of HSL may be irrelevant (8 -10). Finally, the barrier/translocation model does not explicitly address the issue of whether all lipid storage droplet (LSD) surfaces are coated with Plin, although the model implies that surfaces not coated by Plin should be subject to attack by HSL.One important difference between the model cells used to develop the barrier/translocation hypothesis and fat...

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