Sonja Grilc

Properties of Ca2+-dependent exocytosis in cultured astrocytes

Astrocytes, a subtype of glial cells, have numerous characteristics that were previously considered exclusive for neurons. One of these characteristics is a cytosolic [Ca2+] oscillation that controls the release of the chemical transmitter glutamate and atrial natriuretic peptide. These chemical messengers appear to be released from astrocytes via Ca2+‐dependent exocytosis. In the present study, patch‐clamp membrane capacitance measurements were used to monitor changes in the membrane area of a single astrocyte, while the photolysis of caged calcium compounds by a UV flash was used to elicit steps in [Ca2+]i to determine the exocytotic properties of astrocytes. Experiments show that astrocytes exhibit Ca2+‐dependent increases in membrane capacitance, with an apparent Kd value of ∼20 μM [Ca2+]i. The delay between the flash delivery and the peak rate in membrane capacitance increase is in the range of tens to hundreds of milliseconds. The pretreatment of astrocytes by the tetanus neurotoxin, which specifically cleaves the neuronal/neuroendocrine type of SNARE protein synaptobrevin, abolished flash‐induced membrane capacitance increases, suggesting that Ca2+‐dependent membrane capacitance changes involve tetanus neurotoxin‐sensitive SNARE‐mediated vesicular exocytosis. Immunocytochemical experiments show distinct populations of vesicles containing glutamate and atrial natriuretic peptide in astrocytes. We conclude that the recorded Ca2+‐dependent changes in membrane capacitance represent regulated exocytosis from multiple types of vesicles, about 100 times slower than the exocytotic response in neurons.

Intermediate filaments attenuate stimulation-dependent mobility of endosomes/lysosomes in astrocytes.

Intermediate filament (IF) proteins upregulation is a hallmark of astrocyte activation and reactive gliosis, but its pathophysiological implications remain incompletely understood. A recently reported association between IFs and directional mobility of peptidergic vesicles allows us to hypothesize that IFs affect vesicle dynamics and exocytosis‐mediated astrocyte communication with neighboring cells. Here, we ask whether the trafficking of recycling vesicles (i.e., those fused to and then retrieved from the plasma membrane) and endosomes/lysosomes depends on IFs. Recycling vesicles were labeled by antibodies against vesicle glutamate transporter 1 (VGLUT1) and atrial natriuretic peptide (ANP), respectively, and by lysotracker, which labels endosomes/lysosomes. Quantitative fluorescence microscopy was used to monitor the mobility of labeled vesicles in astrocytes, derived from either wild‐type (WT) mice or mice deficient in glial fibrillary acidic protein and vimentin (GFAP−/−Vim−/−), the latter lacking astrocyte IFs. Stimulation with ionomycin or ATP enhanced the mobility of VGLUT1‐positive vesicles and reduced the mobility of ANP‐positive vesicles in WT astrocytes. In GFAP−/−Vim−/− astrocytes, both vesicle types responded to stimulation, but the relative increase in mobility of VGLUT1‐positive vesicles was more prominent compared with nonstimulated cells, whereas the stimulation‐dependent attenuation of ANP‐positive vesicles mobility was reduced compared with nonstimulated cells. The mobility of endosomes/lysosomes decreased following stimulation in WT astrocytes. However, in GFAP−/−Vim−/− astrocytes, a small increase in the mobility of endosomes/lysosomes was observed. These findings show that astrocyte IFs differentially affect the stimulation‐dependent mobility of vesicles. We propose that upregulation of IFs in pathologic states may alter the function of astrocytes by deregulating vesicle trafficking.

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.

EAAT2 density at the astrocyte plasma membrane and Ca2 + -regulated exocytosis

We studied whether regulated exocytosis affects the glutamate transporter density in cultured astrocytes, in which the expression of a fluorescently labeled excitatory amino acid transporter 2 (EAAT2-EGFP) predominantly labeled the plasma membrane. The addition of ionomycin that elevates cytosolic Ca2+ strongly increased the fluorescence of FM 4-64 membrane area dye, confirming the presence of regulated exocytosis in transfected astrocytes. However, concomitant with Ca2+-dependent FM 4-64 fluorescence increase, ionomycin induced a significant steady-state decrease in EAAT2-EGFP fluorescence. This is likely due to a secondary inner filter effect since,(i) in the absence of FM 4-64, ionomycin stimulation was ineffective in changing the EAAT2-EGFP fluorescence, and (ii) fluorescence changes in FM 4-64 and EAAT2-EGFP were inversely correlated. To test whether subcellular EAAT2-EGFP structures are translocated from the cytoplasm to the plasma membrane during ionomycin stimulation, EAAT2-EGFP fluorescence was monitored locally at the plasma membrane and a few microns away in the adjacent cytoplasm. Measurements revealed sites with an increase in EAAT2-EGFP plasma membrane fluorescence correlated with a fluorescence decrease beneath the plasma membrane, and sites with plasma membrane fluorescence decrease correlated with fluorescence increase within the adjacent cytoplasm. The sites of rapid translocation/retrieval of EAAT2-EGFP structures to/from the plasma membrane appeared to be distributed in a punctuate pattern around the cell perimeter. The density of EAAT2-EGFP was regulated in a Ca2+-dependent manner, since in the absence of extracellular Ca2+ local translocation/retrieval events were absent, revealing rapid surface density regulation of EAAT2 in astrocytes by regulated exo/endocytosis.

The Fusion Pore and Vesicle Cargo Discharge Modulation

Exocytosis, the merger of the vesicle membrane with the plasma membrane, is thought to mediate the release of hormones and neurotransmitters from secretory vesicles. The work of Bernard Katz and colleagues decades ago considered that vesicle cargo discharge initially requires the delivery of secretory vesicles to the plasma membrane where vesicles dock and are primed for fusion with the plasma membrane. Then, upon stimulation, the vesicle and the plasma membranes fuse to form a transient fusion pore through which cargo molecules diffuse out of the vesicle lumen into the extracellular space. Katz and colleagues considered this process to occur in an all‐or‐none fashion. However, recent studies show that this may not be so simple. The aim of this overview is to highlight the novel findings that indicate that fusion pores are subject to regulations, which affect the release competence of a single vesicle. Here we discuss the elementary properties of spontaneous and stimulated peptidergic vesicle discharge, which appears to be modulated, at least in pituitary lactotrophs, by fusion pore conductance (pore diameter) and fusion pore gating (kinetics).

Prolactin Secretion Sites Contain Syntaxin-1 and Differ from Ganglioside Monosialic Acid Rafts in Rat Lactotrophs

In neuroendocrine cells, discharge of hormones follows the fusion of exocytotic vesicles with the plasma membrane at confined sites; however, the molecular nature of these distinct sites remains poorly understood. We studied intact pituitary lactotrophs and plasma membrane lawns by confocal microscopy in conjunction with antibodies against rat prolactin (rPRL), soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) proteins (syntaxin-1 and synaptobrevin-2,) and fluorescent cholera toxin subunit B (CT-B), a marker of ganglioside monosialic acid (GM1) lipid rafts, to examine 1) whether rPRL vesicles discharge cargo at GM1 rafts, 2) whether discharging rPRL vesicles interact with SNAREs, and 3) to examine the overlap of GM1 rafts, rPRL, and syntaxin-1 sites in plasma membrane lawns. In intact cells, immunofluorescently labeled rPRL poorly colocalized (<6%) with CT-B. In conditions favoring endocytotic trafficking, vesicle SNARE synaptobrevin-2 modestly colocalized (35%) with CT-B, whereas it highly colocalized (58%) with retrieved rPRL. Although partial mixing between rPRL and CT-B intracellular trafficking pathways is likely, our results indicated that rPRL discharge involves interactions with plasma membrane SNAREs, but not with GM1 rafts. In support of this, the plasma membrane SNARE syntaxin-1 poorly colocalized with CT-B (<5%), whereas it highly colocalized (75%) with rPRL in inside-out plasma membrane lawns. Spontaneous and stimulated rPRL discharge in live lactotrophs is thus associated with plasma membrane sites enriched with SNARE proteins, however, spatially confined to plasma membrane areas other than GM1 rafts.

Correlated ATP-induced changes in membrane area and membrane conductance in single rat adipocytes.

Abstract: In the past few years it has been shown that, like many other non‐neuroendocrine cells, adipocytes possess a mechanism for triggered exocytosis. Endocytosis and exocytosis affect the plasma membrane surface area, which can be directly monitored with electrophysiological patch‐clamp techniques by measuring membrane capacitance, a parameter linearly related to the plasma membrane area. In this study we used the whole‐cell mode of the patch‐clamp technique to measure changes in membrane capacitance to monitor the effect of extracellular adenosine triphosphate (ATP) on the dynamics of membrane area changes in single adipocytes. Experimental evidence shows that extracellular application of ATP (100 μM) increases membrane capacitance for 30 ± 2%. In controls a significantly smaller increase of 3 ± 2% was measured, which is due to a slow exocytic‐endocytic membrane cycling rate of 0/3%/min. We found that ATP induces a transient increase in membrane current, temporally associated with the peak rate in membrane capacitance increase. These results show directly the presence of ATP‐induced increase in membrane area correlated to the increase in membrane current in single adipocytes.

Glutamate stimulation increases hormone release in rat melanotrophs

In melanotrophs, neuroendocrine cells from the intermediate lobe of the rat pituitary gland, glutamate causes a rise in intracellular [Ca2+] suggesting the presence of ionotropic NMDA and non-NMDA AMPA/K receptors. However, the Ca(2+)-dependent release of the major peptide hormone, alpha-melanocyte stimulating hormone (alpha-MSH), in response to glutamate stimulation has not been studied yet in this cell model. Significant spontaneous secretion of the peptide, which results in hormone deposits on the perimeter of the cells, has been confirmed by using confocal microscopy. Co-staining with a membrane area marker FM 1-43, which co-localized with the immunocytochemically marked hormone deposits, showed that fusion-competent sites on the plasma membrane coincided with secretion-competent sites. Stimulation of the cells with glutamate and high K+ saline induced a significant increase in the plasma membrane area covered with alpha-MSH deposits compared to control cells incubated with glutamate and CNQX, a glutamate channel blocker. The optical approach to monitor the secretory activity of a single neuroendocrine cell revealed that glutamate stimulates the release of alpha-MSH at distinct exocytotic membrane domains only.

FM1–43 measurements of local exocytotic events in rat melanotrophs

We have explored the existence of fusion‐ and secretion‐competent sites on the plasma membrane of peptide secreting rat pituitary melanotrophs at rest, and following stimulation with glutamate. We monitored changes in fluorescence of FM1–43, a styryl dye which labels plasma membrane. The results show spontaneous local increases in FM1–43 reporting changes in membrane surface area due to cumulative exocytosis. Addition of glutamate, further increased the occurrence of these events. Statistical analysis of local FM1–43 fluorescence changes suggests that this is due to the recruitment of inactive exocytotic domains and due to the stimulation of already active exocytotic domains.

Chapter 12 Exocytosis: The Pulsing Fusion Pore

Abstract The elaborate intracellular membrane system of eukaryotic cells participates in vesicle trafficking and represents an important basis exploited in cell-to-cell signaling. Communication between cells involves the release of neurotransmitters, hormones and other chemical messengers that are stored in secretory vesicles and granules. A key event in the release of these primary messengers is exocytosis, consisting of fusion between the vesicle and the plasma membrane. This leads to the formation of a fusion pore through which a diffusional continuum between the vesicle lumen and the extracellular space is established. In the past, in vitro studies of biological membrane fusion considered this an almost impossible process, because large pressures had to be delivered to counteract the electrostatic repulsion owing to negatively charged membrane surfaces. It is only a decade or so that the omnipresent fusion between biological membranes started to be understood in greater detail. Since the SNARE hypothesis was proposed about a decade ago, several proteins have been identified to play a role in exocytosis, and attempts to define minimal molecular machinery for regulated exocytosis have been considered. However, several studies provided evidence for multiple modes of exocytosis, and that exocytosis may not necessarily lead to the release of vesicle cargo. The aim of this chapter is to review the results obtained on pituitary cells, specialized to release a number of important hormones and to highlight that there are multiple mechanisms of exocytosis present in the same cell. Moreover, the goal is to address elementary properties of exocytosis, consisting of the interaction between a single vesicle and the plasma membrane. These studies indicate that the long-thought concept of membrane fusion as an irreversible process will have to be changed. Here we discuss an unusually regular reversible opening of the fusion pore termed “the pulsing pore”.