Jakob B. Sørensen

Differential Control of the Releasable Vesicle Pools by SNAP-25 Splice Variants and SNAP-23

The SNARE complex, consisting of synaptobrevin, syntaxin, and SNAP-25, is essential for calcium-triggered exocytosis in neurosecretory cells. Little is known, however, about how developmentally regulated isoforms and other cognate SNARE components regulate vesicular fusion. To address this question, we examined neuroexocytosis from chromaffin cells of Snap25 null mice rescued by the two splice variants SNAP-25a and SNAP-25b and the ubiquitously expressed homolog SNAP-23. In the absence of SNAP-25, vesicle docking persisted, but primed vesicle pools were empty and fast calcium-triggered release abolished. Single vesicular fusion events showed normal characteristics, except for a shorter duration of the fusion pore. Overexpression of SNAP-25a, SNAP-25b, and SNAP-23 resulted in three distinct phenotypes; SNAP-25b induced larger primed vesicle pools than SNAP-25a, whereas SNAP-23 did not support a standing pool of primed vesicles. We conclude that three alternative SNARE components support exocytosis, but they differ in their ability to stabilize vesicles in the primed state.

Sequential N- to C-terminal SNARE complex assembly drives priming and fusion of secretory vesicles

During exocytosis a four‐helical coiled coil is formed between the three SNARE proteins syntaxin, synaptobrevin and SNAP‐25, bridging vesicle and plasma membrane. We have investigated the assembly pathway of this complex by interfering with the stability of the hydrophobic interaction layers holding the complex together. Mutations in the C‐terminal end affected fusion triggering in vivo and led to two‐step unfolding of the SNARE complex in vitro, indicating that the C‐terminal end can assemble/disassemble independently. Free energy perturbation calculations showed that assembly of the C‐terminal end could liberate substantial amounts of energy that may drive fusion. In contrast, similar N‐terminal mutations were without effects on exocytosis, and mutations in the middle of the complex selectively interfered with upstream maturation steps (vesicle priming), but not with fusion triggering. We conclude that the SNARE complex forms in the N‐ to C‐terminal direction, and that a partly assembled intermediate corresponds to the primed vesicle state.

Synaptotagmin-1 docks secretory vesicles to syntaxin-1/SNAP-25 acceptor complexes.

Docking, the initial association of secretory vesicles with the plasma membrane, precedes formation of the SNARE complex, which drives membrane fusion. For many years, the molecular identity of the docked state, and especially the vesicular docking protein, has been unknown, as has the link to SNARE complex assembly. Here, using adrenal chromaffin cells, we identify the vesicular docking partner as synaptotagmin-1, the calcium sensor for exocytosis, and SNAP-25 as an essential plasma membrane docking factor, which, together with the previously known docking factors Munc18-1 and syntaxin, form the minimal docking machinery. Moreover, we show that the requirement for Munc18-1 in docking, but not fusion, can be overcome by stabilizing syntaxin/SNAP-25 acceptor complexes. These findings, together with cross-rescue, double-knockout, and electrophysiological data, lead us to propose that vesicles dock when synaptotagmin-1 binds to syntaxin/SNAP-25 acceptor complexes, whereas Munc18-1 is required for the downstream association of synaptobrevin to form fusogenic SNARE complexes.

Fast Vesicle Fusion in Living Cells Requires at Least Three SNARE Complexes

Threes the Charm The molecular machinery mediating membrane fusion during secretion from a cell requires a complex of so-called SNARE protein that forms a coiled bundle of four parallel α-helices. Mohrmann et al. (p. 502, published online 16 September) developed an elegant approach to find out how many SNARE complexes are required to promote secretion of individual secretory vesicles in living chromaffin cells by titrating the ratio of wild-type and mutant SNARE proteins expressed. For fast synchronous release, a minimum of three SNARE complexes per vesicle were required. Fewer SNARE complexes resulted in slower release. Membrane fusion proteins cooperate to promote rapid secretory vesicle exocytosis from neuroendocrine cells. Exocytosis requires formation of SNARE [soluble N-ethylmaleimide–sensitive factor attachment protein (SNAP) receptor] complexes between vesicle and target membranes. Recent assessments in reduced model systems have produced divergent estimates of the number of SNARE complexes needed for fusion. Here, we used a titration approach to answer this question in intact, cultured chromaffin cells. Simultaneous expression of wild-type SNAP-25 and a mutant unable to support exocytosis progressively altered fusion kinetics and fusion-pore opening, indicating that both proteins assemble into heteromeric fusion complexes. Expressing different wild-type:mutant ratios revealed a third-power relation for fast (synchronous) fusion and a near-linear relation for overall release. Thus, fast fusion typically observed in synapses and neurosecretory cells requires at least three functional SNARE complexes, whereas slower release might occur with fewer complexes. Heterogeneity in SNARE-complex number may explain heterogeneity in vesicular release probability.

Regulation of releasable vesicle pool sizes by protein kinase A-dependent phosphorylation of SNAP-25

Protein kinase A (PKA) is a key regulator of neurosecretion, but the molecular targets remain elusive. We combined pharmacological manipulations of kinase and phosphatase activities with mutational studies on the exocytotic machinery driving fusion of catecholamine-containing vesicles from chromaffin cells. We found that constitutive PKA activity was necessary to maintain a large number of vesicles in the release-ready, so-called primed, state, whereas calcineurin (protein phosphatase 2B) activity antagonized this effect. Overexpression of the SNARE protein SNAP-25a mutated in a PKA phosphorylation site (Thr-138) eliminated the effect of PKA inhibitors on the vesicle priming process. Another, unidentified, PKA target regulated the relative size of two different primed vesicle pools that are distinguished by their release kinetics. Overexpression of the SNAP-25b isoform increased the size of both primed vesicle pools by a factor of two, and mutations in the conserved Thr-138 site had similar effects as in the a isoform.

Plasmalemmal Phosphatidylinositol-4,5-Bisphosphate Level Regulates the Releasable Vesicle Pool Size in Chromaffin Cells

During exocytosis, certain phospholipids may act as regulators of secretion. Here, we used several independent approaches to perturb the phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] level in bovine chromaffin cells to investigate how changes of plasmalemmal PI(4,5)P2 affect secretion. Membrane levels of PI(4,5)P2 were estimated by analyzing images of lawns of plasma membranes labeled with fluorescent probes specific for PI(4,5)P2. The specific PI(4,5)P2 signal was enriched in submicrometer-sized clusters. In parallel patch-clamp experiments on intact cells, we measured the secretion of catecholamines. Overexpression of phosphatidylinositol-4-phosphate-5-kinase Iγ, or infusion of PI(4,5)P2 through the patch pipette, increased the PI(4,5)P2 level in the plasma membrane and potentiated secretion. Expression of a membrane-targeted inositol 5-phosphatase domain of synaptojanin 1 eliminated PI(4,5)P2 from the membrane and abolished secretion. An inhibitor of phosphatidylinositol-3 kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, led to a transient increase in the PI(4,5)P2 level that was associated with a potentiation of secretion. After prolonged incubation, the level of PI(4,5)P2 decreased and secretion was inhibited. Kinetic analysis showed that changes in PI(4,5)P2 levels led to correlated changes in the size of two releasable vesicle pools, whereas their fusion kinetics remained unaffected. We conclude that during both short- and long-term manipulations of PI(4,5)P2 level secretion scales with plasma membrane PI(4,5)P2 content and that PI(4,5)P2 has an early effect on secretion by regulating the number of vesicles ready for release.

Formation, stabilisation and fusion of the readily releasable pool of secretory vesicles

Calcium-triggered exocytosis of neurotransmitter or hormone-filled vesicles has developed as the main mechanism for cell-to-cell communication in animals. Consequently, in the course of evolution this form of exocytosis has been optimized for speed. Since many of the maturation processes of vesicles are intrinsically slow, the solution has been to develop a pool of vesicles that are fully matured and can be fused very rapidly upon stimulation. Vesicles in this readily releasable pool are characterized by very low release rate constants at the resting cytosolic [Ca2+] ([Ca2+]i) and very high release rate constants at stimulated [Ca2+]i. Here I review the kinetic and molecular requirements for the existence of such a pool of vesicles, focusing on chromaffin cells of the adrenal medulla. I discuss how the use of assay methods with different time resolution may lead to fundamentally different conclusions about the role of proteins in exocytosis. Finally, I review recent evidence that the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, formed between proteins residing in the vesicle and the plasma membrane, is involved in formation and stabilization of the readily releasable vesicle pool, whereas synaptotagmin, a Ca2+- and phospholipid-binding vesicular protein, is involved in setting the Ca2+ dependence of the fusion process itself. Future studies are likely to focus on the interaction between these two classes of proteins.

Dissecting docking and tethering of secretory vesicles at the target membrane

Secretory vesicles dock at their target in preparation for fusion. Using single‐vesicle total internal reflection fluorescence microscopy in chromaffin cells, we show that most approaching vesicles dock only transiently, but that some are captured by at least two different tethering modes, weak and strong. Both vesicle delivery and tethering depend on Munc18‐1, a known docking factor. By decreasing the amount of cortical actin by Latrunculin A application, morphological docking can be restored artificially in docking‐deficient munc18‐1 null cells, but neither strong tethering nor fusion, demonstrating that morphological docking is not sufficient for secretion. Deletion of the t‐SNARE and Munc18‐1 binding partner syntaxin, but not the v‐SNARE synaptobrevin/VAMP, also reduces strong tethering and fusion. We conclude that docking vesicles either undock immediately or are captured by minimal tethering machinery and converted in a munc18‐1/syntaxin‐dependent, strongly tethered, fusion‐competent state.

The SNARE protein SNAP-25 is linked to fast calcium triggering of exocytosis

Synchronous neurotransmission depends on the tight coupling between Ca2+ influx and fusion of neurotransmitter-filled vesicles with the plasma membrane. The vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein synaptobrevin 2 and the plasma membrane SNAREs syntaxin 1 and synaptosomal protein of 25 kDa (SNAP-25) are essential for calcium-triggered exocytosis. However, the link between calcium triggering and SNARE function remains elusive. Here we describe mutations in two sites on the surface of the SNARE complex formed by acidic and hydrophilic residues of SNAP-25 and synaptobrevin 2, which were found to coordinate divalent cations in the neuronal SNARE complex crystal structure. By reducing the net charge of the site in SNAP-25 we identify a mutation that interferes with calcium triggering of exocytosis when overexpressed in chromaffin cells. Exocytosis was elicited by photorelease of calcium from a calcium cage and evaluated by using patch-clamp capacitance measurements at millisecond time resolution. We present a method for monitoring the dependence of exocytotic rate upon calcium concentration at the release site and demonstrate that the mutation decreased the steepness of this relationship, indicating that the number of sequential calcium-binding steps preceding exocytosis is reduced by one. We conclude that the SNARE complex is linked directly to calcium triggering of exocytosis, most likely in a complex with auxiliary proteins.

Vesicle docking in regulated exocytosis

In electron micrographs, many secretory and synaptic vesicles are found ‘docked’ at the target membrane, but it is unclear why and how. It is generally assumed that docking is a necessary first step in the secretory pathway before vesicles can acquire fusion competence (through ‘priming’), but recent studies challenge this. New biophysical methods have become available to detect how vesicles are tethered at the target membrane, and genetic manipulations have implicated many genes in tethering, docking and priming. However, these studies have not yet led to consistent working models for these steps. In this study, we review recent attempts to characterize these early steps and the cellular factors to orchestrate them. We discuss whether assays for docking, tethering and priming report on the same phenomena and whether all vesicles necessarily follow the same linear docking–priming–fusion pathway. We conclude that most evidence to date is consistent with such a linear pathway assuming several refinements that imply that some vesicles can be nonfunctionally docked (‘dead‐end’ docking) or, conversely, that the linear pathway can be greatly accelerated (crash fusion).