The pancreatic islet beta-cell autoantigen of relative molecular mass 64,000 (64K), which is a major target of autoantibodies associated with the development of insulin-dependent diabetes mellitus (IDDM) has been identified as glutamic acid decarboxylase, the biosynthesizing enzyme of the inhibitory neurotransmitter GABA (gamma-aminobutyric acid). Pancreatic beta cells and a subpopulation of central nervous system neurons express high levels of this enzyme. Autoantibodies against glutamic acid decarboxylase with a higher titre and increased epitope recognition compared with those usually associated with IDDM are found in stiff-man syndrome, a rare neurological disorder characterized by a high coincidence with IDDM.

Mutations in the human VPS13 genes are responsible for neurodevelopmental and neurodegenerative disorders including chorea acanthocytosis (VPS13A) and Parkinson's disease (VPS13C). The mechanisms of these diseases are unknown. Genetic studies in yeast hinted that Vps13 may have a role in lipid exchange between organelles. In this study, we show that the N-terminal portion of VPS13 is tubular, with a hydrophobic cavity that can solubilize and transport glycerolipids between membranes. We also show that human VPS13A and VPS13C bind to the ER, tethering it to mitochondria (VPS13A), to late endosome/lysosomes (VPS13C), and to lipid droplets (both VPS13A and VPS13C). These findings identify VPS13 as a lipid transporter between the ER and other organelles, implicating defects in membrane lipid homeostasis in neurological disorders resulting from their mutations. Sequence and secondary structure similarity between the N-terminal portions of Vps13 and other proteins such as the autophagy protein ATG2 suggest lipid transport roles for these proteins as well.

Neurotransmitter containing synaptic vesicles (SVs) form tight clusters at synapses. These clusters act as a reservoir from which SVs are drawn for exocytosis during sustained activity. Several components associated with synaptic vesicles likely to help forming such clusters have been reported, including synapsin. Here we found that synapsin can form a distinct liquid phase in an aqueous environment. Other scaffolding proteins could co-assemble into this condensate, but were not necessary for its formation. Importantly, the synapsin phase could capture small lipid vesicles. The synapsin phase rapidly disassembled upon phosphorylation by calcium/calmodulin-dependent protein kinase II (CaMKII), mimicking the dispersion of synapsin 1 that occurs at presynaptic sites upon stimulation. Thus, principles of liquid-liquid phase separation may apply to the clustering of SVs at synapses.

INTRODUCTION Insulin is secreted by pancreatic β cells in response to glucose stimulation. Its release is controlled by the interplay of calcium and phosphoinositide signaling pathways. A rapid release phase, in which insulin containing granules that are already docked and primed at the plasma membrane (PM) undergo exocytosis, is followed by slow release. In this second phase, granules are docked and primed and then released in a series of bursts, each triggered by a spike in cytosolic Ca2+. RATIONALE To better understand the molecular basis underlying insulin secretion, we characterized TMEM24, a protein enriched in neuroendocrine cells previously suggested to be required for a normal secretory response. RESULTS We found that TMEM24 is an endoplasmic reticulum (ER) protein that concentrates at ER-PM contact sites, where it tethers the two bilayers. TMEM24 binding “in trans” to the PM is negatively regulated by phosphorylation in response to elevation of cytosolic Ca2+, so that TMEM24 transiently dissociates from the PM as Ca2+ concentration spikes and then reassociates with this membrane upon dephosphorylation. Additionally, TMEM24 contains a lipid transport module of the synaptotagmin-like, mitochondrial and lipid-binding protein (SMP) family, which we structurally characterized and showed to bind glycerolipids with a preference for phosphatidylinositol (PI). Thus, TMEM24 helps deliver PI, which is synthesized in the ER, to the PM, where it is converted to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] to replenish pools of this lipid hydrolyzed during glucose-stimulated signaling. Supporting a key role of TMEM24 in the coordination of Ca2+ and phosphoinositide signaling, the lipid transport function of TMEM24 is essential for sustaining the intracellular Ca2+ oscillations that trigger bursts of insulin granule release and hence insulin secretion. PI(4,5)P2 is required for Ca2+-dependent exocytosis. It also controls the activity of PM ion channels that regulate cytosolic Ca2+ levels and is the precursor of IP3, which also helps to modulate cytosolic Ca2+ by triggering Ca2+ release from the ER. Thus, in insulin-secreting cells, TMEM24 participates in coordinating Ca2+ and phosphoinositide signaling pathways to cause pulsatile insulin secretion (see the figure). CONCLUSION Our findings implicate ER-PM contact sites and an ER resident lipid-transfer protein in the direct regulation of PM phosphoinositide pools, offering fresh insights into the mechanisms of cellular phosphoinositide dynamics. More specifically, they elaborate the mechanisms underlying insulin secretion, which is impaired in patients with type II diabetes, and may ultimately have therapeutic ramifications. TMEM24 activity cycle at ER-PM contacts Glucose stimulation of insulin-secreting cells triggers Ca2+ influx, phospholipase C–dependent PI(4,5)P2 cleavage, and granule exocytosis. Ca2+-stimulated phosphorylation causes TMEM24 dissociation from the PM and interruption of SMP-mediated PI transfer that allows PI(4,5)P2 resynthesis. Lower...

The extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER) proteins that bind the plasma membrane (PM) via C2 domains and transport lipids between them via SMP domains. E-Syt1 tethers and transports lipids in a Ca-dependent manner, but the role of Ca in this regulation is unclear. Of the five C2 domains of E-Syt1, only C2A and C2C contain Ca-binding sites. Using liposome-based assays, we show that Ca binding to C2C promotes E-Syt1-mediated membrane tethering by releasing an inhibition that prevents C2E from interacting with PI(4,5)P-rich membranes, as previously suggested by studies in semi-permeabilized cells. Importantly, Ca binding to C2A enables lipid transport by releasing a charge-based autoinhibitory interaction between this domain and the SMP domain. Supporting these results, E-Syt1 constructs defective in Ca binding in either C2A or C2C failed to rescue two defects in PM lipid homeostasis observed in E-Syts KO cells, delayed diacylglycerol clearance from the PM and impaired Ca-triggered phosphatidylserine scrambling. Thus, a main effect of Ca on E-Syt1 is to reverse an autoinhibited state and to couple membrane tethering with lipid transport.

Hippocampal neurons in culture develop extensive axonal and dendritic arbors and form numerous synapses. Presynaptic specializations occur at sites of contact between axons and somata or dendrites but they do not appear until day 3 in culture, even though numerous contacts between cells develop within the first 24 hr (Fletcher et al., 1991). To determine whether this delay in the appearance of presynaptic specializations could be related to maturational events in the presynaptic axon or in the postsynaptic target, “heterochronic” cocultures were prepared by adding newly dissociated neurons to cultures containing mature neurons. The competence of axons to form presynaptic vesicle clusters in response to contact with the somata or dendrites of mature or immature neurons was determined by immunofluorescent staining for synapsin I or synaptophysin. After only 1 d of coculture, there was a fivefold increase in the number of synapses along the somata and dendrites of the mature neurons, compared to mature neurons cultured alone. If newly dissociated neurons were labeled with a fluorescent dye before coculture, dye-labeled axons frequently were colocalized with presynaptic specializations on mature cells. In contrast, when the axons of mature neurons contacted immature neurons, synapses were first observed only after coculture for 3 d. These results suggest that the axons of hippocampal neurons have the capacity to form presynaptic specializations soon after they emerge, provided they encounter appropriate targets, but that the cell bodies and dendrites of hippocampal neurons are not capable of inducing the formation of presynaptic specializations until they reach a critical stage of maturation.

Mitochondria, which are excluded from the secretory pathway, depend on lipid transport proteins for their lipid supply from the ER, where most lipids are synthesized. In yeast, the outer mitochondrial membrane GTPase Gem1 is an accessory factor of ERMES, an ER–mitochondria tethering complex that contains lipid transport domains and that functions, partially redundantly with Vps13, in lipid transfer between the two organelles. In metazoa, where VPS13, but not ERMES, is present, the Gem1 orthologue Miro was linked to mitochondrial dynamics but not to lipid transport. Here we show that Miro, including its peroxisome-enriched splice variant, recruits the lipid transport protein VPS13D, which in turn binds the ER in a VAP-dependent way and thus could provide a lipid conduit between the ER and mitochondria. These findings reveal a so far missing link between function(s) of Gem1/Miro in yeast and higher eukaryotes, where Miro is a Parkin substrate, with potential implications for Parkinson’s disease pathogenesis.

SMP-domains are found in proteins that localize to membrane contact sites. Elucidation of the properties of these proteins gives clues as to the molecular bases underlying processes that occur at such sites. Described here are recent discoveries concerning the structure, function, and regulation of the Extended-Synaptotagmin proteins and ERMES complex subunits, SMP-domain proteins at endoplasmic reticulum (ER)- plasma membrane and ER-mitochondrial contacts, respectively. They act as tethers contributing to the architecture of these sites and as lipid transporters that convey glycerolipids between apposed membranes.