Jernej Jorgačevski

Sphingosine Facilitates SNARE Complex Assembly and Activates Synaptic Vesicle Exocytosis

Summary Synaptic vesicles loaded with neurotransmitters fuse with the plasma membrane to release their content into the extracellular space, thereby allowing neuronal communication. The membrane fusion process is mediated by a conserved set of SNARE proteins: vesicular synaptobrevin and plasma membrane syntaxin and SNAP-25. Recent data suggest that the fusion process may be subject to regulation by local lipid metabolism. Here, we have performed a screen of lipid compounds to identify positive regulators of vesicular synaptobrevin. We show that sphingosine, a releasable backbone of sphingolipids, activates synaptobrevin in synaptic vesicles to form the SNARE complex implicated in membrane fusion. Consistent with the role of synaptobrevin in vesicle fusion, sphingosine upregulated exocytosis in isolated nerve terminals, neuromuscular junctions, neuroendocrine cells and hippocampal neurons, but not in neurons obtained from synaptobrevin-2 knockout mice. Further mechanistic insights suggest that sphingosine acts on the synaptobrevin/phospholipid interface, defining a novel function for this important lipid regulator.

Subnanometer Fusion Pores in Spontaneous Exocytosis of Peptidergic Vesicles

Kiss-and-run exocytosis, consisting of reversible fusion between the vesicle membrane and the plasma membrane, is considered to lead to full fusion after stimulation of vesicles containing classical transmitters. However, whether this is also the case in the fusion of peptidergic vesicles is unknown. Previously, we have observed that spontaneous neuropeptide discharge from a single vesicle is slower than stimulated release, because of the kinetic constraints of fusion pore opening. To explore whether slow spontaneous release also reflects a relatively narrow fusion pore, we analyzed the permeation of FM 4-64 dye and HEPES molecules through spontaneously forming fusion pores in lactotroph vesicles expressing synaptopHluorin, a pH-dependent fluorescent fusion marker. Confocal imaging showed that half of the spontaneous exocytotic events exhibited fusion pore openings associated with a change in synaptopHluorin fluorescence but were impermeable to FM 4-64 and HEPES. Together with membrane capacitance measurements, these findings indicate an open fusion pore diameter <0.5 nm, much smaller than the neuropeptides. In stimulated cells, >70% of exocytotic events exhibited a larger, FM 4-64-permeable pore (>1 nm). Interestingly, capacitance measurements showed that the majority of exocytotic events in spontaneous and stimulated conditions were transient. Stimulation increased the frequency of transient events and the fusion pore dwell time but decreased the fraction of events with lowest measurable fusion pore. Kiss-and-run is the predominant mode of exocytosis in resting and in stimulated peptidergic vesicles. Stimulation prolongs the effective opening of the fusion pore and expands its primary subnanometer diameter to enable hormone secretion without full fusion.

Erratum to “Fusion Pore Diameter Regulation by Cations Modulating Local Membrane Anisotropy”.

1 Laboratory of Biophysics, Faculty of Electrical Engineering, University of Ljubljana, 1000 Ljubljana, Slovenia 2 Laboratory of Neuroendocrinology-Molecular Cell Physiology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia 3 Departamento de Biologia e CESAM, Universidade de Aveiro, 3810-193 Aveiro, Portugal 4 Celica Biomedical Center, Tehnoloski Park 24, 1000 Ljubljana, Slovenia 5 Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia

Munc18-1 Tuning of Vesicle Merger and Fusion Pore Properties

The release of hormones and neurotransmitters, mediated by regulated exocytosis, can be modified by regulation of the fusion pore. The fusion pore is considered stable and narrow initially, eventually leading to the complete merger of the vesicle and the plasma membranes. By using the high-resolution patch-clamp capacitance technique, we studied single vesicles and asked whether the Sec1/Munc18 proteins, interacting with the membrane fusion-mediating SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, affect fusion pore properties. Munc18-1 mutants were transfected into lactotrophs to affect the interaction of Munc18-1 with syntaxin1 (Synt1) (R39C), Rab3A (E466K), and Mints (P242S). Compared with wild-type, Munc18-1 E466K increased the frequency of the fusion event. The latter two mutants increased the fusion pore dwell-time. All the mutants stabilized narrow fusion pores and increased the amplitude of fusion events, likely via preferential fusion of larger vesicles, since overexpression of Munc18-1 R39C did not affect the average size of vesicles, as determined by stimulated emission depletion (STED) microscopy. Single-molecule atomic force microscopy experiments revealed that wild-type Munc18-1, but not Munc18-1 R39C, abrogates the interaction between synaptobrevin2 (Syb2) and Synt1 binary trans-complexes. However, neither form of Munc18-1 affected the interaction of Syb2 with the preformed binary cis-Synt1A-SNAP25B complexes. This indicates that Munc18-1 performs a proofing function by inhibiting tethering of Syb2-containing vesicles solely to Synt1 at the plasmalemma and favoring vesicular tethering to the preformed binary cis-complex of Synt1A-SNAP25B. The association of Munc18-1 with the ternary SNARE complex leads to tuning of fusion pores via multiple and converging mechanisms involving Munc18-1 interactions with Synt1A, Rab3A, and Mints.

Fusion pore stability of peptidergic vesicles

Abstract It is believed that in regulated exocytosis the vesicle membrane fuses with the plasma membrane in response to a physiological stimulus. However, in the absence of stimulation, repetitive transient fusion events are also observed, reflecting a stable state. The mechanisms by which the initial fusion pore attains stability are poorly understood. We modelled energetic stability of the fusion pore by taking into account the anisotropic, intrinsic shape of the membrane constituents and their in-plane ordering in the local curvature of the membrane. We used cell-attached membrane capacitance techniques to monitor the appearance and conductance of single fusion pore events in cultured rat lactotrophs. The results revealed a bell-shaped distribution of the fusion pore conductance with a modal value of 25 pS. The experimentally observed increase of the fusion pore stability with decreasing fusion pore radius agrees well with the theoretical predictions. Moreover, the results revealed a correlation between the amplitude of transient capacitance increases and the fusion pore conductance, indicating that larger vesicles may attain a stable fusion pore with larger fusion pore diameters.

Regulation of AQP4 Surface Expression via Vesicle Mobility in Astrocytes

Aquaporin 4 (AQP4) is the predominant water channel in the brain, expressed mainly in astrocytes and involved in water transport in physiologic and pathologic conditions. Besides the classical isoforms M1 (a) and M23 (c), additional ones may be present at the plasma membrane, such as the recently described AQP4b, d, e, and f. Water permeability regulation by AQP4 isoforms may involve several processes, such as channel conformational changes, the extent and arrangement of channels at the plasma membrane, and the dynamics of channel trafficking to/from the plasma membrane. To test whether vesicular trafficking affects the abundance of AQP4 channel at the plasma membrane, we studied the subcellular localization of AQP4 in correlation with vesicle mobility of AQP4e, one of the newly discovered AQP4 isoforms. In cultured rat astrocytes, recombinant AQP4e acquired plasma membrane localization, which resembled that of the antibody labeled endogenous AQP4 localization. Under conditions mimicking reactivation of astrocytes (increase in cytosolic cAMP) and brain edema, an increase in the AQP4 plasma membrane localization was observed. The cytoskeleton remained unaffected with the exception of rearranged actin filaments in the model of reactive astrocytes and vimentin meshwork depolymerization in hypoosmotic conditions. AQP4e vesicle mobility correlated with changes in the plasma membrane localization of AQP4 in all stimulated conditions. Hypoosmotic stimulation triggered a transient reduction in AQP4e vesicle mobility mirrored by the transient changes in AQP4 plasma membrane localization. We suggest that regulation of AQP4 surface expression in pathologic conditions is associated with the mobility of AQP4‐carrying vesicles.

Astrocytic Vesicle Mobility in Health and Disease

Astrocytes are no longer considered subservient to neurons, and are, instead, now understood to play an active role in brain signaling. The intercellular communication of astrocytes with neurons and other non-neuronal cells involves the exchange of molecules by exocytotic and endocytotic processes through the trafficking of intracellular vesicles. Recent studies of single vesicle mobility in astrocytes have prompted new views of how astrocytes contribute to information processing in nervous tissue. Here, we review the trafficking of several types of membrane-bound vesicles that are specifically involved in the processes of (i) intercellular communication by gliotransmitters (glutamate, adenosine 5′-triphosphate, atrial natriuretic peptide), (ii) plasma membrane exchange of transporters and receptors (EAAT2, MHC-II), and (iii) the involvement of vesicle mobility carrying aquaporins (AQP4) in water homeostasis. The properties of vesicle traffic in astrocytes are discussed in respect to networking with neighboring cells in physiologic and pathologic conditions, such as amyotrophic lateral sclerosis, multiple sclerosis, and states in which astrocytes contribute to neuroinflammatory conditions.

Elementary properties of spontaneous fusion of peptidergic vesicles: fusion pore gating

The release of hormones and neurotransmitters by regulated exocytosis requires the delivery of secretory vesicles to the plasma membrane, where they dock and become primed for fusion with the plasma membrane. Upon stimulation a fusion pore is formed through which cargo molecules diffuse out of the vesicle lumen into the extracellular space. After the cargo release the fusion pore either closes (kiss‐and‐run, transient exocytosis), fluctuates between an open and a closed state (for short times, fusion pore flickering, or for rather longer periods, ‘pulsing pore’) or expands irreversibly (full fusion exocytosis). In almost all secretory cells spontaneous secretion of vesicle cargo can be detected in the absence of stimulation. Spontaneous and stimulated exocytosis were thought to exhibit similar properties at elementary level, differing only in the probability of occurrence. However, recent studies indicate that spontaneous exocytosis differs from the stimulated one in many respects, therefore opening questions about the physiological role of spontaneous exocytosis. In this report we address the elementary properties of spontaneous and stimulated peptidergic vesicle discharge which appears to be modulated by fusion pore conductance (diameter) and fusion pore gating.

High-resolution membrane capacitance measurements for the study of exocytosis and endocytosis

In order to understand exocytosis and endocytosis, it is necessary to study these processes directly. An elegant way to do this is by measuring plasma membrane capacitance (Cm), a parameter proportional to cell surface area, the fluctuations of which are due to fusion and fission of secretory and other vesicles. Here we describe protocols that enable high-resolution Cm measurements in macroscopic and microscopic modes. Macroscopic mode, performed in whole-cell configuration, is used for measuring bulk Cm changes in the entire membrane area, and it enables the introduction of exocytosis stimulators or inhibitors into the cytosol through the patch pipette. Microscopic mode, performed in cell-attached configuration, enables measurements of Cm with attofarad resolution and allows characterization of fusion pore properties. Although we usually apply these protocols to primary pituitary cells and astrocytes, they can be adapted and used for other cell types. After initial hardware setup and culture preparation, several Cm measurements can be performed daily.

Single-vesicle architecture of synaptobrevin2 in astrocytes

Exocytic transmitter release is regulated by the SNARE complex, which contains a vesicular protein, synaptobrevin2 (Sb2). However, Sb2 vesicular arrangement is unclear. Here we use super-resolution fluorescence microscopy to study the prevalence and distribution of endogenous and exogenous Sb2 in single vesicles of astrocytes, the most abundant glial cells in the brain. We tag Sb2 protein at C- and N termini with a pair of fluorophores, which allows us to determine the Sb2 length and geometry. To estimate total number of Sb2 proteins per vesicle and the quantity necessary for the formation of fusion pores, we treat cells with ATP to stimulate Ca2+-dependent exocytosis, increase intracellular alkalinity to enhance the fluorescence presentation of yellow-shifted pHluorin (YpH), appended to the vesicle lumen domain of Sb2, and perform photobleaching of YpH fluorophores. Fluorescence intensity analysis reveals that the total number of endogenous Sb2 units or molecules per vesicle is ≤25.