Fusion Pore Opening Research Articles

Two modes of lytic granule fusion during degranulation by natural killer cells

Lytic granules in cytotoxic lymphocytes, which include T cells and natural killer (NK) cells, are secretory lysosomes that release their content upon fusion with the plasma membrane (PM), a process known as degranulation. Although vesicle exocytosis has been extensively studied in endocrine and neuronal cells, much less is known about the fusion of lytic granules in cytotoxic lymphocytes. Here, we used total internal reflection fluorescence microscopy to examine lytic granules labeled with fluorescently tagged Fas ligand (FasL) in the NK cell line NKL stimulated with phorbol ester and ionomycin and in primary NK cells activated by physiological receptor–ligand interactions. Two fusion modes were observed: complete fusion, characterized by loss of granule content and rapid diffusion of FasL at the PM; and incomplete fusion, characterized by transient fusion pore opening and retention of FasL at the fusion site. The pH-sensitive green fluorescence protein (pHluorin) fused to the lumenal domain of FasL was used to visualize fusion pore opening with a time resolution of 30 ms. Upon incomplete fusion, pHluorin emission lasted several seconds in the absence of noticeable diffusion. Thus, we conclude that lytic granules in NK cells undergo both complete and incomplete fusion with the PM, and propose that incomplete fusion may promote efficient recycling of lytic granule membrane after the release of cytotoxic effector molecules.

The Final Conformation of the Complete Ectodomain of the HA2 Subunit of Influenza Hemagglutinin Can by Itself Drive Low pH-dependent Fusion

One of the best characterized fusion proteins, the influenza virus hemagglutinin (HA), mediates fusion between the viral envelope and the endosomal membrane during viral entry into the cell. In the initial conformation of HA, its fusogenic subunit, the transmembrane protein HA2, is locked in a metastable conformation by the receptor-binding HA1 subunit of HA. Acidification in the endosome triggers HA2 refolding toward the final lowest energy conformation. Is the fusion process driven by this final conformation or, as often suggested, by the energy released by protein restructuring? Here we explored structural properties as well as the fusogenic activity of the full sized trimeric HA2(1-185) (here called HA2*) that presents the final conformation of the HA2 ectodomain. We found HA2* to mediate fusion between lipid bilayers and between biological membranes in a low pH-dependent manner. Two mutations known to inhibit HA-mediated fusion strongly inhibited the fusogenic activity of HA2*. At surface densities similar to those of HA in the influenza virus particle, HA2* formed small fusion pores but did not expand them. Our results confirm that the HA1 subunit responsible for receptor binding as well as the transmembrane and cytosolic domains of HA2 is not required for fusion pore opening and substantiate the hypothesis that the final form of HA2 is more important for fusion than the conformational change that generates this form.

Modes of Exocytotic and Endocytotic Events in Tobacco BY-2 Protoplasts

To analyze the kinetics and size of single exo- and endocytotic events in BY-2 protoplasts, we employed cell-attached membrane capacitance measurements. These measurements revealed different modes of fusion and fission of single vesicles. In about half of the observed exocytotic events, fusion occurred transiently, which facilitates rapid recycling of vesicles. In addition, transient sequential or multi-vesicular exocytosis observed in some recordings can contribute to an increase in efficiency of secretory product release. Microscopic analysis of the timescale of cellulose and pectin deposition in protoplasts demonstrates that rebuilding of the cell wall starts soon after isolation of protoplasts and that transient fusion events can fully account for secretion of the required soluble material. The capacitance measurements also allowed us to investigate formation of the fusion pore. We speculate that regulation of secretion may involve control of the length and/or size of fusion pore opening. Together, the different kinetic modes of exo- and endocytosis revealed by capacitance measurements underline the complexity of this process in plants and provide a basis for future research into the underlying mechanisms. The fact that similar fusion/fission kinetics are present in plant and animal cells suggests that many of these mechanisms are highly conserved among eukaryotes.

Strong, Positive Cooperativity of SNARES For Fusion Pore Opening Studied At the Single-Molecule Level

Single vesicle fluorescence assay detects the fluorescence signals from surface-immobilized nano-scale vesicles (typically diameter of 50 nm). There are three principal labeling positions: vesicle membranes, luminal contents and membrane proteins, each of which allows for study of different aspects of membrane-related biological processes. During the past five years, we have reported two realizations of these possibilities: Measuring the kinetics of single vesicle-vesicle fusion by labeling vesicle membranes [1-3] and detecting fusion pore opening in such single vesicle fusion by encapsulating fluorescently-labeled DNA hairpins inside vesicles [4]. In this work, by using fluorescently labeled SNARE proteins, we report on the kinetics of SNARE complex formation observed at the single-molecule level. For this purpose, we have developed an advanced single-molecule FRET technique, in which we track not one, but up to 10 proteins at the same time while keeping the precision at the single-molecule level. The measured kinetics of SNARE complex formation shows strong, positive cooperativity. We finally discuss whether we can make two single-molecule measurements in one experimental setting, for example, detecting the moment of fusion pore opening while tracking formation of multiple SNARE complexes. Such experiment would reveal quantitative correlation between multimeric structure of SNARE proteins and its functional effect on fusion pore opening.[1] Tae-Young Yoon, Burak Okumus, Fan Zhang, Yeon-Kyun Shin and Taekjip Ha. PNAS 103, 19731 (2006).[2] Tae-Young Yoon, Xiaobing Lu, Jiajie Diao, Taekjip Ha and Yeon-Kyun Shin. Nat. Struct. Mol. Biol. 15, 707 (2008).[3] Han-Ki Lee, Yoosoo Yang, Changbong Hyeon, Tae-Sun Lee, Hong-Won Lee, Dae-Hyuk Kweon, Yeon-Kyun Shin and Tae-Young Yoon. Science 328, 760 (2010).[4] Jiajie Diao, Zengliu Su, Yuji Ishitsuka, Bin Lu, Kyung Suk Lee, Ying Lai, Yeon-Kyun Shin and Taekjip Ha. Nature Communications 1, 54 (2010).

Individual Vesicle-Vesicle and Vesicle-Planar Bilayer Fusion Events Mediated by DNA

We have previously reported that DNA-lipid conjugates, when inserted into lipid vesicles, can mediate interactions between vesicles such as docking and fusion∗. These DNA-mediated interactions are a model for the biological machinery (SNARE proteins) employed in synaptic vesicle fusion. We can also use DNA-lipids to build model membrane platforms, such as tethered vesicles and tethered free-standing membranes, which are employed in these studies to observe individual fusion events using fluorescence microscopy. Two systems will be described: vesicle to vesicle fusion between mobile, tethered vesicles, and vesicle to planar bilayer fusion of small vesicles to a DNA-tethered free-standing bilayer. Fusion of individual vesicles is observed in real time using lipid and content mixing assays, as well as FRET upon hybridization of labeled DNA-lipids. These studies provide insight into the relative timescales and extents of docking, lipid mixing, and content exchange. In addition, using different sequences of fluorescently labeled DNA to mediate fusion gives insight into the importance of the rates and number density of hybrid formation at the fusion pore relative to the rate of fusion pore opening. This fusion machinery can also be used to deliver membrane proteins from small vesicles into GUVs or DNA-tethered planar bilayers, allowing membrane proteins to be studied in a free-standing bilayer. ∗Chan et al, PNAS 2007 and 2009; Chan et al, Biointerphases 2008.

The Conformational Change of SNAP25 during the Exocytosis

It is widely assumed that a conformational change in the SNARE complex induces fusion pore opening and transmitter release. The SNAP-25 FRET construct SCORE (SNARE complex reporter) has CFP as FRET donor inserted at the N-terminal end of SN1 and the fluorescent protein Venus (YFP) as FRET acceptor inserted at the N-terminus of SN2. It was previously shown that SCORE that reports a FRET change in response to stimulation that is calcium dependent but is not abolished by tetanus toxin treatment, which abolishes fusion (An and Almers 2004 Science 306:1042). To determine if the conformational change reported by SCORE is directly associated with fusion we monitored SCORE fluorescence in chromaffin cells in TIRF mode while exocytotic events at the cell bottom were spatially and temporally monitored by a microfabricated electrochemical detector (ECD) array with four Pt electrodes patterned on a glass coverslip (Hafez et al. PNAS 2005 102:13879). After the SCORE-expressing cells were placed on the top of the ECD array, the amperometric currents and fluorescence images were simultaneously recorded. Based on the charge ratios of oxidation currents recorded by the four electrodes, the localization of single exocytotic events was determined. The time course of CFP and YFP fluorescence in the 4 camera pixels at the release site was extracted for 10 s time intervals before and after the amperometric spike. Averaging >300 events revealed a transient increase of YFP and a transient decrease of CFP fluorescence by ∼2.5% at the time of amperometric events, The increased FRET level returned to the pre-exocytotic level within ∼5s. This result directly indicates that the SNAP25 conformational change is directly associated with fusion. Supported by NIH grant R01GM085808.

Dengue Virus Ensures its Fusion in Late Endosomes Using Compartment-Specific Lipids

Many enveloped viruses invade cells via endocytosis and use different environmental factors as triggers for virus-endosome fusion that delivers viral genome into cytosol. Intriguingly, dengue virus (DEN), the most prevalent mosquito-borne virus that infects up to 100 million people each year, fuses only in late endosomes, while activation of DEN protein fusogen glycoprotein E is triggered already at pH characteristic for early endosomes. Are there any cofactors that time DEN fusion to virion entry into late endosomes? Here we show that DEN utilizes bis(monoacylglycero)phosphate (BMP), a lipid specific to late endosomes, as a co-factor for its endosomal acidification-dependent fusion machinery. Effective virus fusion to plasma- and intracellular- membranes, as well as to protein-free liposomes, requires the target membrane to contain anionic lipids such as bis(monoacylglycero)phosphate and phosphatidylserine. Anionic lipids act downstream of low-pH-dependent fusion stages and promote the advance from the earliest hemifusion intermediates to the fusion pore opening. To reach anionic lipid enriched late endosomes, DEN travels through acidified early endosomes but we found that low pH-dependent loss of fusogenic properties of DEN is relatively slow in the presence of anionic lipid-free target membranes. We propose that anionic lipid dependence of DEN fusion machinery protects it against premature irreversible restructuring and inactivation and ensures viral fusion in late endosomes, where the virus encounters anionic lipids for the first time during entry. Currently there are neither vaccines nor effective therapies for DEN and the essential role of the newly identified DEN-BMP interactions in viral genome escape from the endosome suggests a novel target for drug design.

Imaging Single Retrovirus Entry through Alternative Receptor Isoforms and Intermediates of Virus-Endosome Fusion

A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A critical barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA)-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950) internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800). Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor density and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a critical role in establishing low pH-dependent virus entry from within acidic endosomes.

Porosome: the universal secretory portal in cells

In the past 50 years it was believed that during cell secretion, membrane-bound secretory vesicles completely merge at the cell plasma membrane resulting in the diffusion of intra-vesicular contents to the cell exterior and the compensatory retrieval of the excess membrane by endocytosis. This explanation made no sense or logic, since following cell secretion partially empty vesicles accumulate as demonstrated in electron micrographs. Furthermore, with the 'all or none' mechanism of cell secretion by complete merger of secretory vesicle membrane at the cell plasma membrane, the cell is left with little regulation and control of the amount of content release. Moreover, it makes no sense for mammalian cells to possess such 'all or none' mechanism of cell secretion, when even single-cell organisms have developed specialized and sophisticated secretory machinery, such as the secretion apparatus of Toxoplasma gondii, the contractile vacuoles in paramecium, or the various types of secretory structures in bacteria. Therefore, in the 1960's, experimental data concerning neurotransmitter release mechanisms by B. Katz and B. Folkow brilliantly hypothesized that limitation of the quantal packet may be set by the nerve membrane, in which case the size of the packet may actually correspond to just a fraction of the vesicle content. This conundrum in the molecular mechanism of cell secretion was finally resolved in 1997 following discovery of the porosome, the universal secretory machinery in the cell. Porosomes are supramolecular lipoprotein structures at the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to release inravesicular contents to the outside during cell secretion. In the past decade, the composition of the porosome, its structure and dynamics at nanometer (nm) resolution and in real time, and its functional reconstitution into artificial lipid membrane, have all been elucidated. Since porosomes in exocrine and neuroendocrine cells measure 100-180 nm, and only 20-45% increase in porosome diameter is demonstrated following the docking and fusion of secretory vesicles (0.2-1.2 μm in diameter), it is concluded that secretory vesicles "transiently" dock and fuse, rather than completely merge at the base of the porosome complex to release their contents to the outside. In agreement, it has been demonstrated that "secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells"; that "single synaptic vesicles fuse transiently and successively without loss of identity"; and that "zymogen granule (the secretory vesicle in exocrine pancreas) exocytosis is characterized by long fusion pore openings and preservation of vesicle lipid identity". In this review, the discovery of the porosome, resulting in a paradigm shift in our understanding of cell secretion, is briefly presented. Biomedical Reviews 2010; 21: 1-15.

Dissection of SNARE-driven membrane fusion and neuroexocytosis by wedging small hydrophobic molecules into the SNARE zipper

Neuronal SNARE proteins mediate neurotransmitter release at the synapse by facilitating the fusion of vesicles to the presynaptic plasma membrane. Cognate v-SNAREs and t-SNAREs from the vesicle and the plasma membrane, respectively, zip up and bring about the apposition of two membranes attached at the C-terminal ends. Here, we demonstrate that SNARE zippering can be modulated in the midways by wedging with small hydrophobic molecules. Myricetin, which intercalated into the hydrophobic inner core near the middle of the SNARE complex, stopped SNARE zippering in motion and accumulated the trans-complex, where the N-terminal region of v-SNARE VAMP2 is in the coiled coil with the frayed C-terminal region. Delphinidin and cyanidin inhibited N-terminal nucleation of SNARE zippering. Neuronal SNARE complex in PC12 cells showed the same pattern of vulnerability to small hydrophobic molecules. We propose that the half-zipped trans-SNARE complex is a crucial intermediate waiting for a calcium trigger that leads to fusion pore opening.

Push-and-pull regulation of the fusion pore by synaptotagmin-7

In chromaffin cells, Ca(2+) binding to synaptotagmin-1 and -7 triggers exocytosis by promoting fusion pore opening and fusion pore expansion. Synaptotagmins contain two C2 domains that both bind Ca(2+) and contribute to exocytosis; however, it remains unknown whether the C2 domains act similarly or differentially to promote opening and expansion of fusion pores. Here, we use patch amperometry measurements in WT and synaptotagmin-7-mutant chromaffin cells to analyze the role of Ca(2+) binding to the two synaptotagmin-7 C2 domains in exocytosis. We show that, surprisingly, Ca(2+) binding to the C2A domain suffices to trigger fusion pore opening but that the resulting fusion pores are unstable and collapse, causing a dramatic increase in kiss-and-run fusion events. Thus, synaptotagmin-7 controls fusion pore dynamics during exocytosis via a push-and-pull mechanism in which Ca(2+) binding to both C2 domains promotes fusion pore opening, but the C2B domain is selectively essential for continuous expansion of an otherwise unstable fusion pore.

Dengue Virus Ensures Its Fusion in Late Endosomes Using Compartment-Specific Lipids

Many enveloped viruses invade cells via endocytosis and use different environmental factors as triggers for virus-endosome fusion that delivers viral genome into cytosol. Intriguingly, dengue virus (DEN), the most prevalent mosquito-borne virus that infects up to 100 million people each year, fuses only in late endosomes, while activation of DEN protein fusogen glycoprotein E is triggered already at pH characteristic for early endosomes. Are there any cofactors that time DEN fusion to virion entry into late endosomes? Here we show that DEN utilizes bis(monoacylglycero)phosphate, a lipid specific to late endosomes, as a co-factor for its endosomal acidification-dependent fusion machinery. Effective virus fusion to plasma- and intracellular- membranes, as well as to protein-free liposomes, requires the target membrane to contain anionic lipids such as bis(monoacylglycero)phosphate and phosphatidylserine. Anionic lipids act downstream of low-pH-dependent fusion stages and promote the advance from the earliest hemifusion intermediates to the fusion pore opening. To reach anionic lipid-enriched late endosomes, DEN travels through acidified early endosomes, but we found that low pH-dependent loss of fusogenic properties of DEN is relatively slow in the presence of anionic lipid-free target membranes. We propose that anionic lipid-dependence of DEN fusion machinery protects it against premature irreversible restructuring and inactivation and ensures viral fusion in late endosomes, where the virus encounters anionic lipids for the first time during entry. Currently there are neither vaccines nor effective therapies for DEN, and the essential role of the newly identified DEN-bis(monoacylglycero)phosphate interactions in viral genome escape from the endosome suggests a novel target for drug design.

Rat and Drosophila Synaptotagmin 4 Have Opposite Effects during SNARE-catalyzed Membrane Fusion

Synaptotagmins (Syt) are a large family of proteins that regulate membrane traffic in neurons and other cell types. One isoform that has received considerable attention is SYT4, with apparently contradictory reports concerning the function of this isoform in fruit flies and mice. SYT4 was reported to function as a negative regulator of neurotrophin secretion in mouse neurons and as a positive regulator of secretion of a yet to be identified growth factor from muscle cells in flies. Here, we have directly compared the biochemical and functional properties of rat and fly SYT4. We report that rat SYT4 inhibited SNARE-catalyzed membrane fusion in both the absence and presence of Ca2+. In marked contrast, fly SYT4 stimulated SNARE-mediated membrane fusion in response to Ca2+. Analysis of chimeric molecules, isolated C2 domains, and point mutants revealed that the C2B domain of the fly protein senses Ca2+ and is sufficient to stimulate fusion. Rat SYT4 was able to stimulate fusion in response to Ca2+ when the conserved Asp-to-Ser Ca2+ ligand substitution in its C2A domain was reversed. In summary, rat SYT4 serves as an inhibitory isoform, whereas fly SYT4 is a bona fide Ca2+ sensor capable of coupling Ca2+ to membrane fusion.

Duration of fusion pore opening and the amount of hormone released are regulated by myosin II during kiss-and-run exocytosis

Since the fusion pore of the secretory vesicle is resealed before complete dilation during 'kiss-and-run' exocytosis, their cargoes are not completely released. Although the transient fusion pore is kept open for several seconds, the precise mechanisms that control fusion pore maintenance, and their physiological significance, are not well understood. Using dual-colour TIRF (total internal reflection fluorescence) microscopy in neuroendocrine PC12 cells, we show that myosin II regulates the fusion pore dynamics during kiss-and-run exocytosis. The release kinetics of mRFP (monomeric red fluorescent protein)-tagged tPA (tissue plasminogen activator) and Venus-tagged BDNF (brain-derived neurotrophic factor), which show slower release kinetics than NPY (neuropeptide Y)-mRFP and insulin-mRFP, were prolonged by the overexpression of a wild-type form of the RLC (myosin II regulatory light chain). In contrast, overexpression of a dominant-negative form of RLC shortened the release kinetics. Using spH (synapto-pHluorin), a green fluorescent protein-based pH sensor inside the vesicles, we confirmed that the modulation of the release kinetics by myosin II is due to changes in the duration of fusion pore opening. In addition, we revealed that the amount of hormone released into the extracellular space upon stimulation was increased by overexpression of wild-type RLC. We propose that the duration of fusion pore opening is regulated by myosin II to control the amount of hormone released from a single vesicle.

V-ATPase Membrane Sector Associates with Synaptobrevin to Modulate Neurotransmitter Release

Acidification of synaptic vesicles by the vacuolar proton ATPase is essential for loading with neurotransmitter. Debated findings have suggested that V-ATPase membrane domain (V0) also contributes to Ca(2+)-dependent transmitter release via a direct role in vesicle membrane fusion, but the underlying mechanisms remain obscure. We now report a direct interaction between V0 c-subunit and the v-SNARE synaptobrevin, constituting a molecular link between the V-ATPase and SNARE-mediated fusion. Interaction domains were mapped to the membrane-proximal domain of VAMP2 and the cytosolic 3.4 loop of c-subunit. Acute perturbation of this interaction with c-subunit 3.4 loop peptides did not affect synaptic vesicle proton pump activity, but induced a substantial decrease in neurotransmitter release probability, inhibiting glutamatergic as well as cholinergic transmission in cortical slices and cultured sympathetic neurons, respectively. Thus, V-ATPase may ensure two independent functions: proton transport by a fully assembled V-ATPase and a role in SNARE-dependent exocytosis by the V0 sector.

The actin cytoskeleton inhibits pore expansion during PIV5 fusion protein-promoted cell-cell fusion.

Paramyxovirus fusion (F) proteins promote both virus-cell fusion, required for viral entry, and cell-cell fusion, resulting in syncytia formation. We used the F-actin stabilizing drug, jasplakinolide, and the G-actin sequestrant, latrunculin A, to examine the role of actin dynamics in cell-cell fusion mediated by the parainfluenza virus 5 (PIV5) F protein. Jasplakinolide treatment caused a dose-dependent increase in cell-cell fusion as measured by both syncytia and reporter gene assays, and latrunculin A treatment also resulted in fusion stimulation. Treatment with jasplakinolide or latrunculin A partially rescued a fusion pore opening defect caused by deletion of the PIV5 F protein cytoplasmic tail, but these drugs had no effect on fusion inhibited at earlier stages by either temperature arrest or by a PIV5 heptad repeat peptide. These data suggest that the cortical actin cytoskeleton is an important regulator of fusion pore enlargement, an energetically costly stage of viral fusion protein-mediated membrane merger.

The SM protein Vps33 and the t-SNARE Habc domain promote fusion pore opening

Intracellular membrane fusion proceeds via distinct stages of membrane docking, hemifusion and fusion pore opening and depends on interacting families of Rab, SNARE and SM proteins. Trans-SNARE complexes dock the membranes in close apposition. Efficient fusion requires further SNARE-associated proteins. They might increase the number of trans-SNARE complexes or the fusogenic potential of a single SNARE complex. We investigated the contributions of the SM protein Vps33 to hemifusion and pore opening between yeast vacuoles. Mutations in Vps33 that weaken its interactions with the SNARE complex allowed normal trans-SNARE pairing and lipid mixing but retarded content mixing. Deleting the H(abc) domain of the vacuolar t-SNARE Vam3, which interacts with Vps33, had the same effect. This suggests that SM proteins promote fusion pore opening by enhancing the fusogenic activity of a SNARE complex. They should thus be considered integral parts of the fusion machinery.

Single Molecule Content Mixing Analysis of SNARE-Mediated Membrane Fusion

A single liposome fusion assay has been developed in our lab to unambiguously detect different stages of fusion including docking, hemi and full fusion via fluorescence resonance energy transfer. [1, 2] While this assay provides the extent of lipid mixing, the presence of the fusion pore opening cannot be detected. In order to further push the technique and to reveal the minimal machinery of SNARE-mediated membrane fusion, we have developed a single molecule assay based on content mixing of a large DNA probe.

Differential Activity-Dependent Secretion of Brain-Derived Neurotrophic Factor from Axon and Dendrite

Brain-derived neurotrophic factor (BDNF) is essential for neuronal survival and differentiation during development and for synaptic function and plasticity in the mature brain. BDNF-containing vesicles are widely distributed and bidirectionally transported in neurons, and secreted BDNF can act on both presynaptic and postsynaptic cells. Activity-dependent BDNF secretion from neuronal cultures has been reported, but it remains unknown where the primary site of BDNF secretion is and whether neuronal activity can trigger BDNF secretion from axons and dendrites with equal efficacy. Using BDNF fused with pH-sensitive green fluorescent protein to visualize BDNF secretion, we found that BDNF-containing vesicles exhibited markedly different properties of activity-dependent exocytic fusion at the axon and dendrite of cultured hippocampal neurons. Brief spiking activity triggered a transient fusion pore opening, followed by immediate retrieval of vesicles without dilation of the fusion pore, resulting in very little BDNF secretion at the axon. On the contrary, the same brief spiking activity induced "full-collapse" vesicle fusion and substantial BDNF secretion at the dendrite. However, full vesicular fusion with BDNF secretion could occur at the axon when the neuron was stimulated by prolonged high-frequency activity, a condition neurons may encounter during epileptic discharge. Thus, activity-dependent axonal secretion of BDNF is highly restricted as a result of incomplete fusion of BDNF-containing vesicles, and normal neural activity induces BDNF secretion from dendrites, consistent with the BDNF function as a retrograde factor. Our study also revealed a novel mechanism by which differential exocytosis of BDNF-containing vesicles may regulate BDNF-TrkB signaling between connected neurons.

Phosphatidylserine Regulation of Ca2+-triggered Exocytosis and Fusion Pores in PC12 Cells

The synaptic vesicle protein synaptotagmin I (Syt I) binds phosphatidylserine (PS) in a Ca(2+)-dependent manner. This interaction is thought to play a role in exocytosis, but its precise functions remain unclear. To determine potential roles for Syt I-PS binding, we varied the PS content in PC12 cells and liposomes and studied the effects on the kinetics of exocytosis and Syt I binding in parallel. Raising PS produced a steeply nonlinear, saturating increase in Ca(2+)-triggered fusion, and a graded slowing of the rate of fusion pore dilation. Ca(2+)-Syt I bound liposomes more tightly as PS content was raised, with a steep increase in binding at low PS, and a further gradual increase at higher PS. These two phases in the PS dependence of Ca(2+)-dependent Syt I binding to lipid may correspond to the two distinct and opposing kinetic effects of PS on exocytosis. PS influences exocytosis in two ways, enhancing an early step leading to fusion pore opening, and slowing a later step when fusion pores dilate. The possible relevance of these results to Ca(2+)-triggered Syt I binding is discussed along with other possible roles of PS.