Fusion Of Secretory Vesicles Research Articles

Myosin VI and its binding partner optineurin are involved in secretory vesicle fusion at the plasma membrane.

During constitutive secretion, proteins synthesized at the endoplasmic reticulum (ER) are transported to the Golgi complex for processing and then to the plasma membrane for incorporation or extracellular release. This study uses a unique live-cell constitutive secretion assay to establish roles for the molecular motor myosin VI and its binding partner optineurin in discrete stages of secretion. Small interfering RNA-based knockdown of myosin VI causes an ER-to-Golgi transport delay, suggesting an unexpected function for myosin VI in the early secretory pathway. Depletion of myosin VI or optineurin does not affect the number of vesicles leaving the trans-Golgi network (TGN), indicating that these proteins do not function in TGN vesicle formation. However, myosin VI and optineurin colocalize with secretory vesicles at the plasma membrane. Furthermore, live-cell total internal reflection fluorescence microscopy demonstrates that myosin VI or optineurin depletion reduces the total number of vesicle fusion events at the plasma membrane and increases both the proportion of incomplete fusion events and the number of docked vesicles in this region. These results suggest a novel role for myosin VI and optineurin in regulation of fusion pores formed between secretory vesicles and the plasma membrane during the final stages of secretion.

Differential effect of exocytic SNAREs on the production of recombinant proteins in mammalian cells

Mammalian cells play a dominant role in the industrial production of biopharmaceutical proteins. However, the productivity of producer cells is often hindered by a bottleneck in the saturated secretory pathway, where a sophisticated mechanism of vesicle trafficking is mediated by numerous proteins and their complexes, among which are the cross-kingdom conserved SNAREs [soluble NSF (N-ethylmaleimide-sensitive factor) receptor]. The SNAREs assemble into complexes by means of four interactive α-helices and, thus, trigger the fusion of transport vesicles with the respective target membranes. We report that the transgenic expression of exocytic SNAREs, which control the fusion of secretory vesicles to the plasma membrane, differentially impacts the secretory capacity of HEK-293, HeLa, and CHO-K1 cells. While other exocytic SNAREs have no effect or a negative effect, SNAP-23 [synaptosome-associated protein of 23 kDa] and VAMP8 [vesicle-associated membrane protein 8] specifically increase the production of recombinant proteins when they are ectopically and stably expressed in mammalian cells. The targeted and effective intervention in the secretory capacity of SNARE proteins is a novel engineering strategy, which could lead to the development of new therapies by increasing the production of biopharmaceutical proteins or by boosting the secretion of cell implants in cell therapy initiatives.

Novel Interactions of CAPS (Ca2+-dependent Activator Protein for Secretion) with the Three Neuronal SNARE Proteins Required for Vesicle Fusion

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca(2+)-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis.

Exocytosis, dependent on Ca2+ release from Ca2+ stores, is regulated by Ca2+ microdomains

The relationship between the cellular Ca2+ signal and secretory vesicle fusion (exocytosis) is a key determinant of the regulation of the kinetics and magnitude of the secretory response. Here, we have investigated secretion in cells where the exocytic response is controlled by Ca2+ release from intracellular Ca2+ stores. Using live-cell two-photon microscopy that simultaneously records Ca2+signals and exocytic responses, we provide evidence that secretion is controlled by changes in Ca2+ concentration [Ca2+] in relatively large-volume microdomains. Our evidence includes: (1) long latencies (>2 seconds) between the rise in [Ca2+] and exocytosis, (2) observation of exocytosis all along the lumen and not clustered around Ca2+ release hot-spots, (3) high affinity (Kd=1.75 microM) Ca2+dependence of exocytosis, (4) significant reduction in exocytosis in the presence of cytosolic EGTA, (5) spatial exclusion of secretory granules from the cell membrane by the endoplasmic reticulum, and (6) inability of local Ca2+ responses to trigger exocytosis. These results strongly indicate that the control of exocytosis, triggered by Ca2+ release from stores, is through the regulation of cytosolic[Ca2+] within a microdomain.

SNARE Conformational Changes that Prepare Vesicles for Exocytosis

When cells release hormones and neurotransmitters through exocytosis, cytosolic Ca(2+) triggers the fusion of secretory vesicles with the plasma membrane. It is well known that this fusion requires assembly of a SNARE protein complex. However, the timing of SNARE assembly relative to vesicle fusion--essential for understanding exocytosis--has not been demonstrated. To investigate this timing, we constructed a probe that detects the assembly of two plasma membrane SNAREs, SNAP25 and syntaxin-1A, through fluorescence resonance energy transfer (FRET). With two-photon imaging, we simultaneously measured FRET signals and insulin exocytosis in beta cells from the pancreatic islet of Langerhans. In some regions of the cell, we found that the SNARE complex was preassembled, which enabled rapid exocytosis. In other regions, SNARE assembly followed Ca(2+) influx, and exocytosis was slower. Thus, SNARE proteins exist in multiple stable preparatory configurations, from which Ca(2+) may trigger exocytosis through distinct mechanisms and with distinct kinetics.

Legionella pneumophila Promotes Functional Interactions between Plasma Membrane Syntaxins and Sec22b

Biogenesis of a specialized organelle that supports intracellular replication of Legionella pneumophila involves the fusion of secretory vesicles exiting the endoplasmic reticulum (ER) with phagosomes containing this bacterial pathogen. Here, we investigated host plasma membrane SNARE proteins to determine whether they play a role in trafficking of vacuoles containing L. pneumophila. Depletion of plasma membrane syntaxins by RNA interference resulted in delayed acquisition of the resident ER protein calnexin and enhanced retention of Rab1 on phagosomes containing virulent L. pneumophila, suggesting that these SNARE proteins are involved in vacuole biogenesis. Plasma membrane-localized SNARE proteins syntaxin 2, syntaxin 3, syntaxin 4 and SNAP23 localized to vacuoles containing L. pneumophila. The ER-localized SNARE protein Sec22b was found to interact with plasma membrane SNAREs on vacuoles containing virulent L. pneumophila, but not on vacuoles containing avirulent mutants of L. pneumophila. The addition of alpha-SNAP and N-ethylmaleimide-sensitive factor (NSF) to the plasma membrane SNARE complexes formed by virulent L. pneumophila resulted in the dissociation of Sec22b, indicating functional pairing between these SNAREs. Thus, L. pneumophila stimulates the non-canonical pairing of plasma membrane t-SNAREs with the v-SNARE Sec22b to promote fusion of the phagosome with ER-derived vesicles. The mechanism by which L. pneumophila promotes pairing of plasma membrane syntaxins and Sec22b could provide unique insight into how the secretory vesicles could provide an additional membrane reserve subverted during phagosome maturation.

Synthesis of fusogenic lipids through activation of phospholipase D1 by GTPases and the kinase RSK2 is required for calcium-regulated exocytosis in neuroendocrine cells

Exocytosis of hormones occurs through the fusion of large dense-core secretory vesicles with the plasma membrane. This highly regulated process involves key proteins such as SNAREs (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors) and also specific lipids at the site of membrane fusion. Among the different lipids required for exocytosis, our recent observations have highlighted the crucial role of PA (phosphatidic acid) in the late stages of membrane fusion in various exocytotic events. An RNAi (RNA interference) strategy coupled with the detection of PA in living cells has pointed to plasma membrane-associated PLD1 (phospholipase D(1)) as the main producer of PA in response to secretagogue stimulation. We have identified several GTPases which regulate the activation level of PLD(1) in neuroendocrine cells. Finally, RSK2 (ribosomal S6 kinase 2) appears to phosphorylate and regulate the activity of PLD(1) in a calcium-dependent manner. Altogether our results have unravelled a complex set of regulatory pathways controlling the synthesis of fusogenic lipids at the secretory granule fusion site by PLD(1).

Protein Farnesylation–Dependent Raf/Extracellular Signal–Related Kinase Signaling Links to Cytoskeletal Remodeling to Facilitate Glucose-Induced Insulin Secretion in Pancreatic β-Cells

OBJECTIVEPosttranslational prenylation (e.g., farnesylation) of small G-proteins is felt to be requisite for cytoskeletal remodeling and fusion of secretory vesicles with the plasma membrane. Here, we investigated roles of protein farnesylation in the signaling steps involved in Raf-1/extracellular signal–related kinase (ERK1/2) signaling pathway in glucose-induced Rac1 activation and insulin secretion in the pancreatic β-cell.RESEARCH DESIGN AND METHODSThese studies were carried out in INS 832/13 cells and normal rat islets. Molecular biological (e.g., overexpression or small interfering RNA [siRNA]–mediated knockdown) and pharmacologic approaches were used to determine roles for farnesylation in glucose-mediated activation of ERK1/2, Rac1, and insulin secretion. Activation of ERK1/2 was determined by Western blotting. Rac1 activation (i.e., Rac1.GTP) was quantitated by p21-activated kinase pull-down assay. Insulin release was quantitated by enzyme-linked immunosorbent assay.RESULTSCoprovision of structure-specific inhibitors of farnesyl transferase (FTase; e.g., FTI-277 or FTI-2628) or siRNA-mediated knockdown of FTase β-subunit resulted in a significant inhibition of glucose-stimulated ERK1/2 and Rac1 activation and insulin secretion. Pharmacologic inhibition of Raf-1 kinase using GW-5074 markedly reduced the stimulatory effects of glucose on ERK1/2 phosphorylation, Rac1 activation, and insulin secretion, suggesting that Raf-1 kinase activation may be upstream to ERK1/2 and Rac1 activation leading to glucose-induced insulin release. Lastly, siRNA-mediated silencing of endogenous expression of ERK1/2 markedly attenuated glucose-induced Rac1 activation and insulin secretion.CONCLUSIONSTogether, our findings provide the first evidence of a role for protein farnesylation in glucose-mediated regulation of the Raf/ERK signaling pathway culminating in the activation of Rac1, which has been shown to be necessary for cytoskeletal reorganization and exocytotic secretion of insulin.

Primed Vesicles of the Readily Releasable Pool Appear to be in a Hemifused State

The fusion of secretory vesicles with the plasma membrane occurs in three biochemically distinct steps: initial tethering and docking of the vesicle with the membrane, priming of the fusion machinery, and merging of the lipid bilayers. All three processes revolve around the regulated assembly of low-energy complexes formed by the coil-coiling of three members of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein family. According to the hypothesis of distal to proximal (N- to C-terminal) “zipping” of SNAREs during exocytosis, priming of vesicles was suggested to correspond to only partial N-terminal SNARE complex assembly.

Cell Secretion: Current Structural and Biochemical Insights

Essential physiological functions in eukaryotic cells, such as release of hormones and digestive enzymes, neurotransmission, and intercellular signaling, are all achieved by cell secretion. In regulated (calcium-dependent) secretion, membrane-bound secretory vesicles dock and transiently fuse with specialized, permanent, plasma membrane structures, called porosomes or fusion pores. Porosomes are supramolecular, cup-shaped lipoprotein structures at the cell plasma membrane that mediate and control the release of vesicle cargo to the outside of the cell. The sizes of porosomes range from 150nm in diameter in acinar cells of the exocrine pancreas to 12nm in neurons. In recent years, significant progress has been made in our understanding of the porosome and the cellular activities required for cell secretion, such as membrane fusion and swelling of secretory vesicles. The discovery of the porosome complex and the molecular mechanism of cell secretion are summarized in this article.

The vesicle-trafficking protein munc18b increases the secretory capacity of mammalian cells

Heterologous protein production in mammalian cells is often challenged by the bottleneck of the secretory machinery, which prevents producer cells from fully exploiting their physiologic capacity in the production of biopharmaceuticals. Recent advances in the understanding of the molecular mechanisms of vesicle trafficking have enabled the identification of key regulators that control the flow of recombinant proteins along the secretory pathway. Here, we report that transgenic expression of Munc18b, a Sec1/Munc18 (SM) protein regulating the fusion of secretory vesicles to the plasma membrane, enhances the secretory capacity of HeLa, HEK-293 and HT-1080 and so increases overall production of different secreted human glycoproteins as well as the titer of lentiviral particles produced in HEK-293-derived helper cells. Targeted interventions in secretory vesicle trafficking by Munc18b is a novel secretion engineering strategy, which harnesses the full secretory capacity of mammalian cells. Secretion engineering is the latest-generation metabolic engineering strategy, which could improve future therapies by increasing the production of biopharmaceuticals by boosting the secretion performance of cell implants in cell therapy initiatives and by raising the production titers of transgenic viral particles used for gene therapy applications.

Pollen Tube Growth: a Delicate Equilibrium Between Secretory and Endocytic Pathways

Although pollen tube growth is a prerequisite for higher plant fertilization and seed production, the processes leading to pollen tube emission and elongation are crucial for understanding the basic mechanisms of tip growth. It was generally accepted that pollen tube elongation occurs by accumulation and fusion of Golgi-derived secretory vesicles (SVs) in the apical region, or clear zone, where they were thought to fuse with a restricted area of the apical plasma membrane (PM), defining the apical growth domain. Fusion of SVs at the tip reverses outside cell wall material and provides new segments of PM. However, electron microscopy studies have clearly shown that the PM incorporated at the tip greatly exceeds elongation and a mechanism of PM retrieval was already postulated in the mid-nineteenth century. Recent studies on endocytosis during pollen tube growth showed that different endocytic pathways occurred in distinct zones of the tube, including the apex, and led to a new hypothesis to explain vesicle accumulation at the tip; namely, that endocytic vesicles contribute substantially to V-shaped vesicle accumulation in addition to SVs and that exocytosis does not involve the entire apical domain. New insights suggested the intriguing hypothesis that modulation between exo- and endocytosis in the apex contributes to maintain PM polarity in terms of lipid/protein composition and showed distinct degradation pathways that could have different functions in the physiology of the cell. Pollen tube growth in vivo is closely regulated by interaction with style molecules. The study of endocytosis and membrane recycling in pollen tubes opens new perspectives to studying pollen tube-style interactions in vivo.

Cab45b, a Munc18b-interacting Partner, Regulates Exocytosis in Pancreatic β-Cells

Cab45b is a cytosolic Ca(2+)-binding protein reported to regulate zymogen secretion in pancreatic acini. We now show that Cab45b is also expressed in pancreatic islet beta-cells and interacts there with the Sec1-Munc18 protein Munc18b. We employed patch clamp cell capacitance measurements to show that antibodies against Cab45b inhibited depolarization-evoked membrane capacitance increments, suggesting an impact on beta-cell granule exocytosis, both the readily releasable granule pool and refilling of this pool. Site-specific mutants in the Cab45b EF-hands were used to dissect the molecular interactions involved in Cab45b function. Mutants in EF-hands 2 and 3 had no detectable effects on interaction of Cab45b with Munc18b and did not affect the depolarization-evoked calcium currents, but remarkably, they facilitated the complex formation of Munc18b with syntaxin-2 and -3. As a result, these two EF-hand mutants inhibited beta-cell membrane capacitance increments. This inhibition is mediated via Munc18b because Munc18b silencing with small interfering RNA abolished the effects of these two mutants. The results suggest a mechanism for Cab45b action that involves regulating the dynamic association of Munc18b with SNAREs to impact beta-cell granule exocytosis.

Analysis of SEC9 Suppression Reveals a Relationship of SNARE Function to Cell Physiology

BackgroundGrowth and division of Saccharomyces cerevisiae is dependent on the action of SNARE proteins that are required for membrane fusion. SNAREs are regulated, through a poorly understood mechanism, to ensure membrane fusion at the correct time and place within a cell. Although fusion of secretory vesicles with the plasma membrane is important for yeast cell growth, the relationship between exocytic SNAREs and cell physiology has not been established.Methodology/Principal FindingsUsing genetic analysis, we identified several influences on the function of exocytic SNAREs. Genetic disruption of the V-ATPase, but not vacuolar proteolysis, can suppress two different temperature-sensitive mutations in SEC9. Suppression is unlikely due to increased SNARE complex formation because increasing SNARE complex formation, through overexpression of SRO7, does not result in suppression. We also observed suppression of sec9 mutations by growth on alkaline media or on a non-fermentable carbon source, conditions associated with a reduced growth rate of wild-type cells and decreased SNARE complex formation.Conclusions/SignificanceThree main conclusions arise from our results. First, there is a genetic interaction between SEC9 and the V-ATPase, although it is unlikely that this interaction has functional significance with respect to membrane fusion or SNAREs. Second, Sro7p acts to promote SNARE complex formation. Finally, Sec9p function and SNARE complex formation are tightly coupled to the physiological state of the cell.

The Coffin‐Lowry syndrome‐associated protein RSK2 is implicated in calcium‐regulated exocytosis through the regulation of PLD1

Exocytosis of neurotransmitters and hormones occurs through the fusion of secretory vesicles with the plasma membrane. This highly regulated process involves key proteins such as SNAREs, and also specific lipids at the site of membrane fusion. Phospholipase D (PLD) has recently emerged as a promoter of membrane fusion in various exocytotic events potentially by providing fusogenic cone‐shaped phosphatidic acid. We show here that PLD1 is regulated by ribosomal S6 kinase 2 (RSK2)‐dependent phosphorylation. RSK2 is activated by a high K+induced rise in cytosolic calcium. Expression of inactive RSK2 mutants or selective knock down of endogenous RSK2 dramatically affects the different kinetic components of the exocytotic response in chromaffin cells. RSK2 physically interacts with and stimulates PLD activity through the phosphorylation of Thr147 in the PLD1 amino‐terminal PX‐domain. Expression of PLD1 phosphomimetic mutants fully restores secretion in cells depleted of RSK2, suggesting that RSK2 is a critical upstream signaling element in the activation of PLD1 to produce the lipids required for exocytosis. We propose that PLD‐related defects in neuronal and endocrine activities could contribute to the effect observed after the loss of function mutations in Rsk2 that leads to Coffin‐Lowry syndrome (CLS), an X‐linked form of growth and mental retardation.

Cytopathology and Exocrine Dysfunction Induced in Ex Vivo Rabbit Lacrimal Gland Acinar Cell Models by Chronic Exposure to Histamine or Serotonin

Lacrimal immunohistopathology has diverse clinical presentations, suggesting that inflammatory mediators exert diverse influences. Chronic exposure to agonistic acetylcholine receptor autoantibodies has been studied previously; the present work addressed mediators that signal through other G protein-coupled receptors. Acinus-like structures and reconstituted acinar epithelial monolayers from rabbit lacrimal glands were exposed to varying concentrations of histamine or 5-hydroxytryptamine (5-HT) for 20 hours. Net and vectorial beta-hexosaminidase secretion, cytosolic Ca(2+) (Ca(i)) elevation, apical recruitment of p150(Glued), actin microfilament meshwork organization, and ultrastructure were assessed. Histamine and 5-HT acutely stimulated beta-hexosaminidase secretion at lower, but not higher, concentrations. Neither of them acutely elevated Ca(i) levels. Both recruited p150(Glued) at concentrations that failed to induce secretion. Chronic exposure to 10 mM histamine inhibited carbachol (CCh)-induced beta-hexosaminidase secretion and prevented the formation of continuous monolayers; 1 mM 5-HT partially inhibited secretion at the apical medium. Neither altered secretion to the basal medium. Chronic exposure to histamine or 5-HT partially decreased CCh induced Ca(i) elevations and p150(Glued) recruitment, even at concentrations that did not inhibit secretion. Both expanded acinar lumina and thickened microfilament meshworks, and both caused homotypic fusion of secretory vesicles and formation of aqueous vacuoles in the apical and basal cytoplasm. Chronic exposure to forskolin, which activates adenylyl cyclase, induced similar cytopathologic changes but impaired secretion modestly and only at the highest concentration tested. Inflammatory mediators that signal through G protein-coupled receptors cause acinar cell cytopathology and dose-dependent reductions of CCh-induced beta-hexosaminidase secretion. Although agonistic acetylcholine receptor autoantibodies may cause pervasive functional quiescence, inflammatory mediators may cause varying degrees of exocrine dysfunction.

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Conservation of Helical Bundle Structure between the Exocyst Subunits

BackgroundThe exocyst is a large hetero-octomeric protein complex required for regulating the targeting and fusion of secretory vesicles to the plasma membrane in eukaryotic cells. Although the sequence identity between the eight different exocyst subunits is less than 10%, structures of domains of four of the subunits revealed a similar helical bundle topology. Characterization of several of these subunits has been hindered by lack of soluble protein for biochemical and structural studies.Methodology/Principal FindingsUsing advanced hidden Markov models combined with secondary structure predictions, we detect significant sequence similarity between each of the exocyst subunits, indicating that they all contain helical bundle structures. We corroborate these remote homology predictions by identifying and purifying a predicted domain of yeast Sec10p, a previously insoluble exocyst subunit. This domain is soluble and folded with approximately 60% α-helicity, in agreement with our predictions, and capable of interacting with several known Sec10p binding partners.Conclusions/SignificanceAlthough all eight of the exocyst subunits had been suggested to be composed of similar helical bundles, this has now been validated by our hidden Markov model structure predictions. In addition, these predictions identified protein domains within the exocyst subunits, resulting in creation and characterization of a soluble, folded domain of Sec10p.

The Functions of Munc18‐1 in Regulated Exocytosis

The activation of regulated exocytosis occurs by a rise in cytosolic Ca(2+) concentration. Synaptotagmins act as the Ca(2+) sensors, whereas the machinery that allows fusion of secretory vesicles with the plasma membrane consists of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, including syntaxin 1, SNAP-25, and VAMP. Within the pathway leading to exocytosis, there is an essential requirement for a member of the conserved Sec1/Munc18 (SM) protein family, which in neurotransmitter and neurohormone release in mammalian cells is Munc18-1. The exact role of Munc18-1 and the steps within exocytosis in which it acts have been intensively investigated. Current evidence suggests that Munc18-1 acts via distinct modes of interactions with syntaxin 1 and the other SNARE proteins and influences all of the steps leading to exocytosis, including vesicle recruitment, tethering, docking, priming, and membrane fusion.