Ca2+-sensor proteins control the secretion of many neuroendocrine substances. Calcium-secretion coupling may involve several mechanisms. First, Ca2+-dependent association of their tandem C2 domains with phosphatidylserine may induce membrane curvature and thereby enhance fusion. Second, their association with SNARE complexes may inhibit membrane fusion in the absence of a Ca2+ trigger. Here we present a method using two optically trapped beads coated with SNARE-free synthetic membranes to elucidate the direct role of the C2AB domain of the soluble Ca2+-sensor Doc2b. Contacting membranes are often coupled by a Doc2b-coated membrane stalk that resists forces up to 600 pN upon bead separation. Stalk formation depends strictly on Ca2+ and phosphatidylserine. Real-time fluorescence imaging shows phospholipid but not content mixing, indicating membrane hemifusion. Thus, Doc2b acts directly on membranes and stabilizes the hemifusion intermediate in this cell-free system. In living cells, this mechanism may co-occur with progressive SNARE complex assembly, together defining Ca2+-secretion coupling.

Synaptotagmins are vesicular proteins implicated in many membrane trafficking events. They are highly conserved in evolution and the mammalian family contains 16 isoforms. We now show that the tandem C2 domains of several calcium-sensitive synaptotagmin isoforms tested, including Drosophila synaptotagmin, rapidly cross-link phospholipid membranes. In contrast to the tandem structure, individual C2 domains failed to trigger membrane cross-linking in several novel assays. Large-scale liposomal aggregation driven by tandem C2 domains in response to calcium was confirmed by the following techniques: turbidity assay, dynamic light-scattering and both confocal and negative stain electron microscopy. Firm cross-linking of membranes was evident from laser trap experiments. High-resolution cryo-electron microscopy revealed that membrane cross-linking by tandem C2 domains results in a constant distance of ∼9 nm between the apposed membranes. Our findings show the conserved nature of this important property of synaptotagmin, demonstrate the significance of the tandem C2 domain structure and provide a plausible explanation for the accelerating effect of synaptotagmins on membrane fusion.

Growing evidence suggests that astrocytes are the active partners of neurons in many brain functions. Astrocytic mitochondria are highly motile organelles which regulate the temporal and spatial patterns of Ca( 2+ ) dynamics, in addition to being a major source of ATP and reactive oxygen species. Previous studies have shown that mitochondria translocate to endoplasmic reticulum during Ca( 2+ ) release from internal stores, but whether a similar spatial interaction between mitochondria and plasma membrane occurs is not known. Using total internal reflection fluorescence (TIRF) microscopy we show that a fraction of mitochondria became trapped near the plasma membrane of cultured hippocampal astrocytes during exposure to the transmitters glutamate or ATP, resulting in net translocation of the mitochondria to the plasma membrane. This translocation was dependent on the intracellular Ca( 2+ ) rise because it was blocked by pre-incubation with BAPTA AM and mimicked by application of the Ca( 2+ ) ionophore ionomycin. Transmembrane Ca( 2+ ) influx induced by raising external Ca( 2+ ) also caused mitochondrial trapping, which occurred more rapidly than that produced by glutamate or ATP. In astrocytes treated with the microtubule-disrupting agent nocodazole, intracellular Ca( 2+ ) rises failed to induce trapping of mitochondria near plasma membrane, suggesting a role for microtubules in this phenomenon. Our data reveal the Ca( 2+ )-dependent trapping of mitochondria near the plasma membrane as a novel form of mitochondrial regulation, which is likely to control the perimembrane Ca( 2+ ) dynamics and regulate signaling by mitochondria-derived reactive oxygen species.

conductor (SC) nanorods (NRs) with Type-II heterojunctions that exhibit a large Quantum Confined Stark Effect (QCSE) at room temperature (1). For using these NRs as voltage sensors, however, one needs to impart them with membraneprotein like properties so that they can be stably inserted into the membrane. We report here spontaneous insertion of SC NRs into liposomes and cell membranes by functionalizing them with specially designed peptides. We provide evidences for insertion from cryo transmission electron microscopy (TEM) and polarized light microscopy. We also report on first attempts to sense membrane potential with these particles with single-particle sensitivity. With further improvements, SC NRs could potentially be used to study signals from whole neural networks in a large field-of-view. Moreover, successful implementation of SC NRs would allow for the analysis of voltage signals at the nano-(single synapse-) scale.