Munc18-dependent and -independent clustering of syntaxin in the plasma membrane of cultured endocrine cells.

Syntaxin helps in catalyzing membrane fusion during exocytosis. It also forms clusters in the plasma membrane, where both its transmembrane and SNARE domains are thought to homo-oligomerize. To study syntaxin clustering in live PC12 cells, we labeled granules with neuropeptide-Y-mCherry and syntaxin clusters with syntaxin-1a green fluorescent protein (GFP). Abundant clusters appeared under total internal reflection (TIRF) illumination, and some of them associated with granules ("on-granule clusters"). Syntaxin-1a-GFP or its mutants were expressed at low levels and competed with an excess of endogenous syntaxin for inclusion into clusters. On-granule inclusion was diminished by mutations known to inhibit binding to Munc18-1 in vitro. Knock-down of Munc18-1 revealed Munc18-dependent and -independent on-granule clustering. Clustering was inhibited by mutations expected to break salt bridges between syntaxin's Hb and SNARE domains and was rescued by additional mutations expected to restore them. Most likely, syntaxin is in a closed conformation when it clusters on granules, and its SNARE and Hb domains approach to within atomic distances. Pairwise replacements of Munc18-contacting residues with alanines had only modest effects, except that the pair R114A/I115A essentially abolished on-granule clustering. In summary, an on-granule cluster arises from the specific interaction between a granule and a dense cluster of syntaxin-Munc18-1 complexes. Off-granule clusters, by contrast, were resistant to even the strongest mutations we tried and required neither Munc18-1 nor the presence of a SNARE domain. They may well form through the nonstoichiometric interactions with membrane lipids that others have observed in cell-free systems.

Rapid structural change in synaptosomal-associated protein 25 (SNAP25) precedes the fusion of single vesicles with the plasma membrane in live chromaffin cells.

The SNARE complex consists of the three proteins synaptobrevin-2, syntaxin, and synaptosomal-associated protein 25 (SNAP25) and is thought to execute a large conformational change as it drives membrane fusion and exocytosis. The relation between changes in the SNARE complex and fusion pore opening is, however, still unknown. We report here a direct measurement relating a change in the SNARE complex to vesicle fusion on the millisecond time scale. In individual chromaffin cells, we tracked conformational changes in SNAP25 by total internal reflection fluorescence resonance energy transfer (FRET) microscopy while exocytotic catecholamine release from single vesicles was simultaneously recorded using a microfabricated electrochemical detector array. A local rapid and transient FRET change occurred precisely where individual vesicles released catecholamine. To overcome the low time resolution of the imaging frames needed to collect sufficient signal intensity, a method named event correlation microscopy was developed, which revealed that the FRET change was abrupt and preceded the opening of an exocytotic fusion pore by ∼90 ms. The FRET change correlated temporally with the opening of the fusion pore and not with its dilation.

Single secretory granules of live cells recruit syntaxin-1 and synaptosomal associated protein 25 (SNAP-25) in large copy numbers.

Before secretory vesicles undergo exocytosis, they must recruit the proteins syntaxin-1 and synaptosomal associated protein 25 (SNAP-25) in the plasma membrane. GFP-labeled versions of both proteins cluster at sites where secretory granules have docked. Single-particle tracking shows that minority populations of both molecules are strongly hindered in their mobility, consistent with their confinement in nanodomains. We measured the fluorescence of granule-associated clusters, the fluorescence of single molecules, and the numbers of unlabeled syntaxin-1 and SNAP-25 molecules per cell. There was a more than 10-fold excess of SNAP-25 over syntaxin-1. Fifty to seventy copies each of syntaxin-1 and SNAP-25 molecules were associated with a single docked granule, many more than have been reported to be required for fusion.

Syntaxin clusters assemble reversibly at sites of secretory granules in live cells.

Syntaxin resides in the plasma membrane, where it helps to catalyze membrane fusion during exocytosis. The protein also forms clusters in cell-free and granule-free plasma-membrane sheets. We imaged the interaction between syntaxin and single secretory granules by two-color total internal reflection microscopy in PC12 cells. Syntaxin-GFP assembled in clusters at sites where single granules had docked at the plasma membrane. Clusters were intermittently present at granule sites, as syntaxin molecules assembled and disassembled in a coordinated fashion. Recruitment to granules required the N-terminal domain of syntaxin, but not the entry of syntaxin into SNARE complexes. Clusters facilitated exocytosis and disassembled once exocytosis was complete. Syntaxin cluster formation defines an intermediate step in exocytosis.

Sensing muscle ischemia: coincident detection of acid and ATP via interplay of two ion channels.

Ischemic pain--examples include the chest pain of a heart attack and the leg pain of a 30 s sprint--occurs when muscle gets too little oxygen for its metabolic need. Lactic acid cannot act alone to trigger ischemic pain because the pH change is so small. Here, we show that another compound released from ischemic muscle, adenosine tri-phosphate (ATP), works together with acid by increasing the pH sensitivity of acid-sensing ion channel number 3 (ASIC3), the molecule used by sensory neurons to detect lactic acidosis. Our data argue that ATP acts by binding to P2X receptors that form a molecular complex with ASICs; the receptor on sensory neurons appears to be P2X5, an electrically quiet ion channel. Coincident detection of acid and ATP should confer sensory selectivity for ischemia over other conditions of acidosis.

Release of the styryl dyes from single synaptic vesicles in hippocampal neurons.

In small presynaptic boutons in brain, synaptic vesicles are thought not to merge with the plasma membrane when they release transmitter, but instead to close their fusion pores and survive intact for future use (kiss-and-run exocytosis). The strongest evidence for this idea is the slow and incomplete release of the fluorescent membrane marker, FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide], from single vesicles. We investigated the release of FM1-43 from sparse cultures of hippocampal neurons grown on coverslips with no glia. This allowed presynaptic boutons to be imaged at favorable signal-to-noise ratio. Sparingly stained boutons were imaged at high time resolution, while high-frequency electrical stimulation caused exocytosis. The release of FM1-43 was quantal and occurred in abrupt steps, each representing a single fusion event. The fluorescence of vesicle clusters traveling along axons had a distribution with the same quantal size, indicating that a vesicle releases all the dye it contains. In most fusion events, the time constant of dye release was <100 ms, and slower release was rarely observed. After exocytosis, no FM1-43 could be detected in the axon to either side of a bouton, indicating that dye was released before it could spread. Our results are consistent with synaptic vesicles fusing fully with the plasma membrane during high-frequency stimulation.

Stimulated exocytosis of endosomes in goldfish retinal bipolar neurons.

After exocytosis, synaptic vesicle components are selectively retrieved by clathrin-mediated endocytosis and then re-used in future rounds of transmitter release. Under some conditions, synaptic terminals in addition perform bulk endocytosis of large membranous sacs. Bulk endocytosis is less selective than clathrin-mediated endocytosis and probably internalizes components normally targeted to the plasma membrane. Nonetheless, this process plays a major role in some tonic ribbon-type synapses, which release neurotransmitter for prolonged periods of time. We show here, that large endosomes formed after strong and prolonged stimulation undergo stimulated exocytosis in retinal bipolar neurons. The result suggests how cells might return erroneously internalized components to the plasma membrane, and also demonstrates that synaptic vesicles are not the only neuronal organelle that stains with styryl dyes and undergoes stimulated exocytosis.

Two ribeye genes in teleosts: the role of Ribeye in ribbon formation and bipolar cell development.

Ribeye is the only known protein specific to synaptic ribbon, but its function is unclear. We show that the teleost fish, Fugu and zebrafish, have two ribeye genes, ribeye a and ribeye b. Whole-mount in situ hybridization revealed that ribeye a is expressed in tissues containing synaptic ribbons, including the pineal gland, inner ear, and retina. Ribeye b is absent in the pineal gland. In the retina, ribeye a is expressed in both photoreceptors and bipolar cells, whereas ribeye b is detected only in photoreceptors. To study the function of Ribeye a in retina, we depleted it by morpholino antisense oligos. Fish deficient in Ribeye a lack an optokinetic response and have shorter synaptic ribbons in photoreceptors and fewer synaptic ribbons in bipolar cells. Their bipolar cells still target Syntaxin-3 proteins to the inner plexiform layer and have abundant vsx1 mRNA. However, they lack large synaptic terminals and show increased apoptosis. Rod bipolar cells are fewer in number and/or deficient in PKCalpha. Recovery of Ribeye a levels rescues the optokinetic response, increases the number of PKCalpha-positive bipolar cells, and stops apoptosis. We conclude that Ribeye a is important for late steps in bipolar cell development.

Tracking SNARE complex formation in live endocrine cells.

Syntaxin, synaptosome-associated protein of 25 kD (SNAP25), and vesicle-associated membrane protein/synaptobrevin are collectively called SNAP receptor (SNARE) proteins, and they catalyze neuronal exocytosis by forming a "core complex." The steps in core complex formation are unknown. Here, we monitored SNARE complex formation in vivo with the use of a fluorescent version of SNAP25. In PC12 cells, we found evidence for a syntaxin-SNAP25 complex that formed with high affinity, required only the amino-terminal SNARE motif of SNAP25, tolerated a mutation that blocks formation of other syntaxin-SNAP25 complexes, and assembled reversibly when Ca2+ entered cells during depolarization. The complex may represent a precursor to the core complex formed during a Ca2+-dependent priming step of exocytosis.

Recapture after exocytosis causes differential retention of protein in granules of bovine chromaffin cells.

After exocytosis, chromaffin granules release essentially all their catecholamines in small fractions of a second, but it is unknown how fast they release stored peptides and proteins. Here we compare the exocytic release of fluorescently labelled neuropeptide Y (NPY) and tissue plasminogen activator from single granules. Exocytosis was tracked by measuring the membrane capacitance, and single granules in live cells were imaged by evanescent field microscopy. Neuropeptide Y left most granules in small fractions of a second, while tissue plasminogen activator remained in open granules for minutes. Taking advantage of the dependence on pH of the fluorescence of green fluorescent protein, we used rhythmic external acidification to determine whether and when granules re-sealed. One-third of them re-sealed within 100 s and retained significant levels of tissue plasminogen activator. Re-sealing accounts for only a fraction of the endocytosis monitored in capacitance measurements. When external [Ca2+] was raised, even neuropeptide Y remained in open granules until they re-sealed. It is concluded that a significant fraction of chromaffin granules re-seal after exocytosis, and retain those proteins that leave granules slowly. We suggest that granules vary the stoichiometry of release by varying both granule re-sealing and the association of proteins with the granule matrix.

Bilayers merge even when exocytosis is transient.

During exocytosis, the lumen of secretory vesicles connects with the extracellular space. In some vesicles, this connection closes again, causing the vesicle to be recaptured mostly intact. The degree to which the bilayers of such vesicles mix with the plasma membrane is unknown. Work supporting the kiss-and-run model of transient exocytosis implies that synaptic vesicles allow neither lipid nor protein to escape into the plasma membrane, suggesting that the two bilayers never merge. Here, we test whether neuroendocrine granules behave similarly. Using two-color evanescent field microscopy, we imaged the lipid probe FM4-64 and fluorescent proteins in single dense core granules. During exocytosis, granules lost FM4-64 into the plasma membrane in small fractions of a second. Although FM4-64 was lost, granules retained the membrane protein, GFP-phogrin. By using GFP-phogrin as a probe for resealing, it was found that even granules that reseal lose FM4-64. We conclude that the lipid bilayers of the granule and the plasma membrane become continuous even when exocytosis is transient.

Neural Wiskott Aldrich Syndrome Protein (N-WASP) and the Arp2/3 complex are recruited to sites of clathrin-mediated endocytosis in cultured fibroblasts.

Several findings suggest that actin-mediated motility can play a role in clathrin-mediated endocytosis but it remains unclear whether and when key proteins required for this process are recruited to endocytic sites. Here we investigate this question in live Swiss 3T3 cells using two-colour evanescent field (EF) microscopy. We find that Arp3, a component of the Arp2/3 complex, appears transiently while single clathrin-coated pits internalize. There is also additional recruitment of Neural-Wiskott Aldrich Syndrome Protein (N-WASP), a known activator of the Arp2/3 complex. Both proteins appear at about the same time as actin. We suggest that N-WASP and the Arp2/3 complex trigger actin polymerization during a late step in clathrin-mediated endocytosis, and propel clathrin-coated pits or vesicles from the plasma membrane into the cytoplasm.

Imaging calcium entry sites and ribbon structures in two presynaptic cells.

We investigated the location of calcium entry sites and synaptic ribbons in the type-Mb goldfish bipolar neuron and the bullfrog saccular hair cell. Cells were loaded with a fast calcium indicator (Fluo-3 or Fluo-5F) and an excess of a high-affinity but slow Ca buffer (EGTA). The cell surface was imaged by evanescent field microscopy. Small fluorescent "hot spots" representing calcium entry sites appeared abruptly when a voltage step opened Ca channels and disappeared or dimmed abruptly when Ca channels closed. In bipolar cells, the fluorescence of hot spots tracked the calcium influx. Hair cells showed similar Ca hot spots. Synaptic ribbons or dense bodies were labeled by immunofluorescence with an antibody that recognizes the ribbon protein ribeye. The antibody labeled punctate structures beneath the plasma membrane. In both bipolar neurons and hair cells, the number of Ca entry sites was similar or identical to that of ribbons or dense bodies, consistent with the idea that calcium-channel clusters reside near ribbons, and that both mark active zones. In bipolar cells, the number of Ca entry sites and ribeye-positive fluorescent spots is also strikingly similar to that of exocytic active zones but significantly less than the number of total exocytic sites including solitary fusion events outside active zones. We suggest that in bipolar terminals, active zones, Ca entry sites, and synaptic ribbons all colocalize, but also that a significant number of vesicles can fuse outside active zones and, hence, independently of synaptic ribbons.

Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells.

Classical cell biology teaches that exocytosis causes the membrane of exocytic vesicles to disperse into the cell surface and that a cell must later retrieve by molecular sorting whatever membrane components it wishes to keep inside. We have tested whether this view applies to secretory granules in intact PC-12 cells. Three granule proteins were labeled with fluorescent proteins in different colors, and two-color evanescent-field microscopy was used to view single granules during and after exocytosis. Whereas neuro-peptide Y was lost from granules in seconds, tissue plasminogen activator (tPA) and the membrane protein phogrin remained at the granule site for over 1 min, thus providing markers for postexocytic granules. When tPA was imaged simultaneously with cyan fluorescent protein (CFP) as a cytosolic marker, the volume occupied by the granule appeared as a dark spot where it excluded CFP. The spot remained even after tPA reported exocytosis, indicating that granules failed to flatten into the cell surface. Phogrin was labeled with GFP at its luminal end and used to sense the pH in granules. When exocytosis caused the acidic granule interior to neutralize, GFP-phogrin at first brightened and later dimmed again as the interior separated from the extracellular space and reacidified. Reacidification and dimming could be reversed by application of NH(4)Cl. We conclude that most granules reseal in <10 s after releasing cargo, and that these empty or partially empty granules are recaptured otherwise intact.

A membrane marker leaves synaptic vesicles in milliseconds after exocytosis in retinal bipolar cells.

Perhaps synaptic vesicles can recycle so rapidly because they avoid complete exocytosis, and release transmitter through a fusion pore that opens transiently. This view emerges from imaging whole terminals where the fluorescent lipid FM1-43 seems unable to leave vesicles during transmitter release. Here we imaged single, FM1-43-stained synaptic vesicles by evanescent field fluorescence microscopy, and tracked the escape of dye from single vesicles by watching the increase in fluorescence after exocytosis. Dye left rapidly and completely during most or all exocytic events. We conclude that vesicles at this terminal allow lipid exchange soon after exocytosis, and lose their dye even if they connected with the plasma membrane only briefly. At the level of single vesicles, therefore, observations with FM1-43 provide no evidence that exocytosis of synaptic vesicles is incomplete.

Imaging actin and dynamin recruitment during invagination of single clathrin-coated pits.

As a final step in endocytosis, clathrin-coated pits must separate from the plasma membrane and move into the cytosol as a coated vesicle. Because these events involve minute movements that conventional light microscopy cannot resolve, they have not been observed directly and their dynamics remain unexplored. Here, we used evanescent field (EF) microscopy to observe single clathrin-coated pits or vesicles as they draw inwards from the plasma membrane and finally lose their coats. This inward movement occurred immediately after a brief burst of dynamin recruitment and was accompanied by transient actin assembly. Therefore, dynamin may provide the trigger and actin may provide the force for movement into the cytosol.

Morphology and dynamics of the endocytic pathway in Dictyostelium discoideum.

Dictyostelium discoideum is a genetically and biochemically tractable social amoeba belonging to the crown group of eukaryotes. It performs some of the tasks characteristic of a leukocyte such as chemotactic motility, macropinocytosis, and phagocytosis that are not performed by other model organisms or are difficult to study. D. discoideum is becoming a popular system to study molecular mechanisms of endocytosis, but the morphological characterization of the organelles along this pathway and the comparison with equivalent and/or different organelles in animal cells and yeasts were lagging. Herein, we used a combination of evanescent wave microscopy and electron microscopy of rapidly frozen samples to visualize primary endocytic vesicles, vesicular-tubular structures of the early and late endo-lysosomal system, such as multivesicular bodies, and the specialized secretory lysosomes. In addition, we present biochemical and morphological evidence for the existence of a micropinocytic pathway, which contributes to the uptake of membrane along side macropinocytosis, which is the major fluid phase uptake process. This complex endosomal compartment underwent continuous cycles of tubulation/vesiculation as well as homo- and heterotypic fusions, in a way reminiscent of mechanisms and structures documented in leukocytes. Finally, egestion of fluid phase from the secretory lysosomes was directly observed.