Ketamine Reduces the Surface Density of the Astroglial Kir4.1 Channel and Inhibits Voltage-Activated Currents in a Manner Similar to the Action of Ba2+ on K+ Currents.

A single sub-anesthetic dose of ketamine evokes rapid and long-lasting beneficial effects in patients with a major depressive disorder. However, the mechanisms underlying this effect are unknown. It has been proposed that astrocyte dysregulation of extracellular K+ concentration ([K+]o) alters neuronal excitability, thus contributing to depression. We examined how ketamine affects inwardly rectifying K+ channel Kir4.1, the principal regulator of K+ buffering and neuronal excitability in the brain. Cultured rat cortical astrocytes were transfected with plasmid-encoding fluorescently tagged Kir4.1 (Kir4.1-EGFP) to monitor the mobility of Kir4.1-EGFP vesicles at rest and after ketamine treatment (2.5 or 25 µM). Short-term (30 min) ketamine treatment reduced the mobility of Kir4.1-EGFP vesicles compared with the vehicle-treated controls (p < 0.05). Astrocyte treatment (24 h) with dbcAMP (dibutyryl cyclic adenosine 5'-monophosphate, 1 mM) or [K+]o (15 mM), which increases intracellular cAMP, mimicked the ketamine-evoked reduction of mobility. Live cell immunolabelling and patch-clamp measurements in cultured mouse astrocytes revealed that short-term ketamine treatment reduced the surface density of Kir4.1 and inhibited voltage-activated currents similar to Ba2+ (300 µM), a Kir4.1 blocker. Thus, ketamine attenuates Kir4.1 vesicle mobility, likely via a cAMP-dependent mechanism, reduces Kir4.1 surface density, and inhibits voltage-activated currents similar to Ba2+, known to block Kir4.1 channels.

Calcium-dependent subquantal peptide release from single docked lawn-resident vesicles of pituitary lactotrophs.

Regulated exocytosis consists of the fusion between vesicles and the plasma membranes, leading to the formation of a narrow fusion pore through which secretions exit the vesicle lumen into the extracellular space. An increase in the cytosolic concentration of free Ca2+ ([Ca2+]i) is considered the stimulus of this process. However, whether this mechanism can be preserved in a simplified system of membrane lawns with docked secretory vesicles, devoid of cellular components, is poorly understood. Here, we studied peptide discharge from individual secretory vesicles docked at the plasma membrane, prepared from primary endocrine pituitary cells (the lactotrophs), releasing hormone prolactin. To label secretory vesicles, we transfected lactotrophs to express the fluorescent atrial natriuretic peptide (ANP.emd), previously shown to be expressed in and released from prolactin-containing vesicles. We used stimulating solutions containing different [Ca2+] to evoke vesicle peptide discharge, which appeared similar in membrane lawns and in intact stimulated lactotrophs. All vesicles examined discharged peptides in a subquantal manner, either exhibiting a unitary or sequential time course. In the membrane lawns, the unitary vesicle peptide discharge was predominant and slightly slower than that recorded in intact cells, but with a shorter delay with respect to the stimulation onset. This study revealed directly that Ca2+ triggers peptide discharge from docked single vesicles in the membrane lawns with a half-maximal response of ∼8 µM [Ca2+], consistent with previous whole-cell patch-clamp studies in endocrine cells where the rapid component of exocytosis, interpreted to represent docked vesicles, was fully activated at <10 µM [Ca2+]. Interestingly, the sequential subquantal peptide vesicle discharge indicates that fluctuations between constricted and dilated fusion pore states are preserved in membrane lawns and that fusion pore regulation appears to be an autonomously controlled process.

Probing single molecule mechanical interactions of syntaxin 1A with native synaptobrevin 2 residing on a secretory vesicle.

Interactive mechanical forces between pairs of individual SNARE proteins synaptobrevin 2 (Sb2) and syntaxin 1A (Sx1A) may be sufficient to mediate vesicle docking. This notion, based on force spectroscopy single molecule measurements probing recombinant Sx1A an Sb2 in silico, questioned a predominant view of docking via the ternary SNARE complex formation, which includes an assembly of the intermediate cis binary complex between Sx1A and SNAP25 on the plasma membrane to engage Sb2 on the vesicle. However, whether a trans binary Sx1A-Sb2 complex alone could mediate vesicle docking in a cellular environment remains unclear. To address this issue, we used atomic force microscopy (AFM) in the force spectroscopy mode combined with fluorescence imaging. Using AFM tips functionalized with the full Sx1A cytosolic domain, we probed native Sb2 studding the membrane of secretory vesicles docked at the plasma membrane patches, referred to as "inside-out lawns", identified based on fluorescence stains and prepared from primary culture of lactotrophs. We recorded single molecule Sx1A-Sb2 mechanical interactions and obtained measurements of force (∼183 pN) and extension (∼21.6 nm) necessary to take apart Sx1A-Sb2 binding interactions formed at tip-vesicle contact. Measured interactive force between a single pair of Sx1A-Sb2 molecules is sufficient to hold a single secretory vesicle docked at the plasma membrane within distances up to that of the measured extension. This finding further advances a notion that native vesicle docking can be mediated by a single trans binary Sx1A-Sb2 complex in the absence of SNAP25.

Vesicle cholesterol controls exocytotic fusion pore.

In some lysosomal storage diseases (LSD) cholesterol accumulates in vesicles. Whether increased vesicle cholesterol affects vesicle fusion with the plasmalemma, where the fusion pore, a channel between the vesicle lumen and the extracellular space, is formed, is unknown. Super-resolution microscopy revealed that after stimulation of exocytosis, pituitary lactotroph vesicles discharge cholesterol which transfers to the plasmalemma. Cholesterol depletion in lactotrophs and astrocytes, both exhibiting Ca2+-dependent exocytosis regulated by distinct Ca2+sources, evokes vesicle secretion. Although this treatment enhanced cytosolic levels of Ca2+ in lactotrophs but decreased it in astrocytes, this indicates that cholesterol may well directly define the fusion pore. In an attempt to explain this mechanism, a new model of cholesterol-dependent fusion pore regulation is proposed. High-resolution membrane capacitance measurements, used to monitor fusion pore conductance, a parameter related to fusion pore diameter, confirm that at resting conditions reducing cholesterol increases, while enrichment with cholesterol decreases the conductance of the fusion pore. In resting fibroblasts, lacking the Npc1 protein, a cellular model of LSD in which cholesterol accumulates in vesicles, the fusion pore conductance is smaller than in controls, showing that vesicle cholesterol controls fusion pore and is relevant for pathophysiology of LSD.

Ketamine Action on Astrocytes Provides New Insights into Rapid Antidepressant Mechanisms.

Ketamine, a non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonist, exerts rapid, potent and long-lasting antidepressant effect already after a single administration of a low dose into depressed individuals. Apart from targeting neuronal NMDARs essential for synaptic transmission, ketamine also interacts with astrocytes, the principal homoeostatic cells of the central nervous system. The cellular mechanisms underlying astrocyte-based rapid antidepressant effect are incompletely understood. Here we overview recent data that describe ketamine-dependent changes in astrocyte cytosolic cAMP activity ([cAMP]i) and ketamine-induced modifications of stimulus-evoked Ca2+ signalling. The latter regulates exocytotic release of gliosignalling molecules and stabilizes the vesicle fusion pore in a narrow configuration that obstructs cargo discharge or vesicle membrane recycling. Ketamine also instigates rapid redistribution of cholesterol in the astrocyte plasmalemma that may alter flux of cholesterol to neurones, where it is required for changes in synaptic plasticity. Finally, ketamine attenuates mobility of vesicles carrying the inward rectifying potassium channel (Kir4.1) and reduces the surface density of Kir4.1 channels that control extracellular K+ concentration, which tunes the pattern of action potential firing in neurones of lateral habenula as demonstrated in a rat model of depression. Thus, diverse, but not mutually exclusive, mechanisms act synergistically to evoke changes in synaptic plasticity leading to sustained strengthening of excitatory synapses necessary for rapid antidepressant effect of ketamine.

Astrocyte arborization enhances Ca2+ but not cAMP signaling plasticity.

The plasticity of astrocytes is fundamental for their principal function, maintaining homeostasis of the central nervous system throughout life, and is associated with diverse exposomal challenges. Here, we used cultured astrocytes to investigate at subcellular level basic cell processes under controlled environmental conditions. We compared astroglial functional and signaling plasticity in standard serum-containing growth medium, a condition mimicking pathologic conditions, and in medium without serum, favoring the acquisition of arborized morphology. Using opto-/electrophysiologic techniques, we examined cell viability, expression of astroglial markers, vesicle dynamics, and cytosolic Ca2+ and cAMP signaling. The results revealed altered vesicle dynamics in arborized astrocytes that was associated with increased resting [Ca2+ ]i and increased subcellular heterogeneity in [Ca2+ ]i , whereas [cAMP]i subcellular dynamics remained stable in both cultures, indicating that cAMP signaling is less prone to plastic remodeling than Ca2+ signaling, possibly also in in vivo contexts.

Ketamine Alters Functional Plasticity of Astroglia: An Implication for Antidepressant Effect.

Ketamine, a non-competitive N-methyl-d-aspartate receptor (NMDAR) antagonist, exerts a rapid, potent and long-lasting antidepressant effect, although the cellular and molecular mechanisms of this action are yet to be clarified. In addition to targeting neuronal NMDARs fundamental for synaptic transmission, ketamine also affects the function of astrocytes, the key homeostatic cells of the central nervous system that contribute to pathophysiology of major depressive disorder. Here, I review studies revealing that (sub)anesthetic doses of ketamine elevate intracellular cAMP concentration ([cAMP]i) in astrocytes, attenuate stimulus-evoked astrocyte calcium signaling, which regulates exocytotic secretion of gliosignaling molecules, and stabilize the vesicle fusion pore in a narrow configuration, possibly hindering cargo discharge or vesicle recycling. Next, I discuss how ketamine affects astrocyte capacity to control extracellular K+ by reducing vesicular delivery of the inward rectifying potassium channel (Kir4.1) to the plasmalemma that reduces the surface density of Kir4.1. Modified astroglial K+ buffering impacts upon neuronal firing pattern as demonstrated in lateral habenula in a rat model of depression. Finally, I highlight the discovery that ketamine rapidly redistributes cholesterol in the astrocyte plasmalemma, which may alter the flux of cholesterol to neurons. This structural modification may further modulate a host of processes that synergistically contribute to ketamine's rapid antidepressant action.

The Association Between Antidepressant Effect of SSRIs and Astrocytes: Conceptual Overview and Meta-analysis of the Literature.

Major depressive disorders (MDD) a worldwide psychiatric disease, is yet to be adequately controlled by therapies; while the mechanisms of action of antidepressants are yet to be fully characterised. In the last two decades, an increasing number of studies have demonstrated the role of astrocytes in the pathophysiology and therapy of MDD. Selective serotonin reuptake inhibitors (SSRIs) are the most widely used antidepressants. It is generally acknowledged that SSRIs increase serotonin levels in the central nervous system by inhibiting serotonin transporters, although the SSRIs action is not ideal. The SSRIs antidepressant effect develops with considerable delay; their efficacy is low and frequent relapses are common. Neither cellular nor molecular pharmacological mechanisms of SSRIs are fully characterised; in particular their action on astrocytes remain underappreciated. In this paper we overview potential therapeutic mechanisms of SSRIs associated with astroglia and report the results of meta-analysis of studies dedicated to MDD, SSRIs and astrocytes. In particular, we argue that fluoxetine, the representative SSRI, improves depressive-like behaviours in animals treated with chronic mild stress and reverses depression-associated decrease in astrocytic glial fibrillary acidic protein (GFAP) expression. In addition, fluoxetine upregulates astrocytic mRNA expression of 5-hydroxytriptamin/serotonin2B receptors (5-HT2BR). In summary, we infer that SSRIs exert their anti-depressant effect by regulating several molecular and signalling pathways in astrocytes.

Inhibiting glycolysis rescues memory impairment in an intellectual disability Gdi1-null mouse.

OBJECTIVES: GDI1 gene encodes for αGDI, a protein controlling the cycling of small GTPases, reputed to orchestrate vesicle trafficking. Mutations in human GDI1 are responsible for intellectual disability (ID). In mice with ablated Gdi1, a model of ID, impaired working and associative short-term memory was recorded. This cognitive phenotype worsens if the deletion of αGDI expression is restricted to neurons. However, whether astrocytes, key homeostasis providing neuroglial cells, supporting neurons via aerobic glycolysis, contribute to this cognitive impairment is unclear. METHODS: We carried out proteomic analysis and monitored [18F]-fluoro-2-deoxy-d-glucose uptake into brain slices of Gdi1 knockout and wild type control mice. d-Glucose utilization at single astrocyte level was measured by the Förster Resonance Energy Transfer (FRET)-based measurements of cytosolic cyclic AMP, d-glucose and L-lactate, evoked by agonists selective for noradrenaline and L-lactate receptors. To test the role of astrocyte-resident processes in disease phenotype, we generated an inducible Gdi1 knockout mouse carrying the Gdi1 deletion only in adult astrocytes and conducted behavioural tests. RESULTS: Proteomic analysis revealed significant changes in astrocyte-resident glycolytic enzymes. Imaging [18F]-fluoro-2-deoxy-d-glucose revealed an increased d-glucose uptake in Gdi1 knockout tissue versus wild type control mice, consistent with the facilitated d-glucose uptake determined by FRET measurements. In mice with Gdi1 deletion restricted to astrocytes, a selective and significant impairment in working memory was recorded, which was rescued by inhibiting glycolysis by 2-deoxy-d-glucose injection. CONCLUSIONS: These results reveal a new astrocyte-based mechanism in neurodevelopmental disorders and open a novel therapeutic opportunity of targeting aerobic glycolysis, advocating a change in clinical practice.

Astrocytes in rapid ketamine antidepressant action.

Ketamine, a general anaesthetic and psychotomimetic drug, exerts rapid, potent and long-lasting antidepressant effect, albeit the cellular and molecular mechanisms of this action are yet to be discovered. Besides targeting neuronal NMDARs fundamental for synaptic transmission, ketamine affects the function of astroglia the key homeostatic cells of the central nervous system that contribute to pathophysiology of psychiatric diseases including depression. Here we review studies revealing that (sub)anaesthetic doses of ketamine elevate intracellular cAMP concentration ([cAMP]i) in astrocytes, attenuate stimulus-evoked astrocyte calcium signalling, which regulates exocytotic secretion of gliosignalling molecules, and stabilize the vesicle fusion pore in a narrow configuration possibly hindering cargo discharge or vesicle recycling. Next we discuss how ketamine affects astroglial capacity to control extracellular K+ by reducing cytoplasmic mobility of vesicles delivering the inward rectifying potassium channel (Kir4.1) to the plasmalemma. Modified astroglial K+ buffering impacts upon neuronal excitability as demonstrated in the lateral habenula rat model of depression. Finally, we highlight the recent discovery that ketamine rapidly redistributes cholesterol in the plasmalemma of astrocytes, but not in fibroblasts nor in neuronal cells. This alteration of membrane structure may modulate a host of processes that synergistically contribute to ketamine's rapid and prominent antidepressant action.

Astroglial Mechanisms of Ketamine Action Include Reduced Mobility of Kir4.1-Carrying Vesicles.

The finding that ketamine, an anaesthetic, can elicit a rapid antidepressant effect at low doses that lasts for weeks in patients with depression is arguably a major achievement in psychiatry in the last decades. However, the mechanisms of action are unclear. The glutamatergic hypothesis of ketamine action posits that ketamine is a N-methyl-D-aspartate receptor (NMDAR) antagonist modulating downstream cytoplasmic events in neurons. In addition to targeting NMDARs in synaptic transmission, ketamine may modulate the function of astroglia, key homeostasis-providing cells in the central nervous system, also playing a role in many neurologic diseases including depression, which affects to 20% of the population globally. We first review studies on astroglia revealing that (sub)anaesthetic doses of ketamine attenuate stimulus-evoked calcium signalling, a process of astroglial cytoplasmic excitability, regulating the exocytotic release of gliosignalling molecules. Then we address how ketamine alters the fusion pore activity of secretory vesicles, and how ketamine affects extracellular glutamate and K+ homeostasis, both considered pivotal in depression. Finally, we also provide evidence indicating reduced cytoplasmic mobility of astroglial vesicles carrying the inward rectifying potassium channel (Kir4.1), which may regulate the density of Kir4.1 at the plasma membrane. These results indicate that the astroglial capacity to control extracellular K+ concentration may be altered by ketamine and thus indirectly affect the action potential firing of neurons, as is the case in lateral habenula in a rat disease model of depression. Hence, ketamine-altered functions of astroglia extend beyond neuronal NMDAR antagonism and provide a basis for its antidepressant action through glia.

Nestin affects fusion pore dynamics in mouse astrocytes.

AIM: Astrocytes play a homeostatic role in the central nervous system and influence numerous aspects of neurophysiology via intracellular trafficking of vesicles. Intermediate filaments (IFs), also known as nanofilaments, regulate a number of cellular processes including organelle trafficking and adult hippocampal neurogenesis. We have recently demonstrated that the IF protein nestin, a marker of neural stem cells and immature and reactive astrocytes, is also expressed in some astrocytes in the unchallenged hippocampus and regulates neurogenesis through Notch signalling from astrocytes to neural stem cells, possibly via altered trafficking of vesicles containing the Notch ligand Jagged-1. METHODS: We thus investigated whether nestin affects vesicle dynamics in astrocytes by examining single vesicle interactions with the plasmalemma and vesicle trafficking with high-resolution cell-attached membrane capacitance measurements and confocal microscopy. We used cell cultures of astrocytes from nestin-deficient (Nes-/- ) and wild-type (wt) mice, and fluorescent dextran and Fluo-2 to examine vesicle mobility and intracellular Ca2+ concentration respectively. RESULTS: Nes-/- astrocytes exhibited altered sizes of vesicles undergoing full fission and transient fusion, altered vesicle fusion pore geometry and kinetics, decreased spontaneous vesicle mobility and altered ATP-evoked mobility. Purinergic stimulation evoked Ca2+ signalling that was slightly attenuated in Nes-/- astrocytes, which exhibited more oscillatory Ca2+ responses than wt astrocytes. CONCLUSION: These results demonstrate at the single vesicle level that nestin regulates vesicle interactions with the plasmalemma and vesicle trafficking, indicating its potential role in astrocyte vesicle-based communication.

Exocytosis of large-diameter lysosomes mediates interferon γ-induced relocation of MHC class II molecules toward the surface of astrocytes.

Astrocytes are the key homeostatic cells in the central nervous system; initiation of reactive astrogliosis contributes to neuroinflammation. Pro-inflammatory cytokine interferon γ (IFNγ) induces the expression of the major histocompatibility complex class II (MHCII) molecules, involved in antigen presentation in reactive astrocytes. The pathway for MHCII delivery to the astrocyte plasma membrane, where MHCII present antigens, is unknown. Rat astrocytes in culture and in organotypic slices were exposed to IFNγ to induce reactive astrogliosis. Astrocytes were probed with optophysiologic tools to investigate subcellular localization of immunolabeled MHCII, and with electrophysiology to characterize interactions of single vesicles with the plasmalemma. In culture and in organotypic slices, IFNγ augmented the astrocytic expression of MHCII, which prominently co-localized with lysosomal marker LAMP1-EGFP, modestly co-localized with Rab7, and did not co-localize with endosomal markers Rab4A, EEA1, and TPC1. MHCII lysosomal localization was corroborated by treatment with the lysosomolytic agent glycyl-L-phenylalanine-ß-naphthylamide, which reduced the number of MHCII-positive vesicles. The surface presence of MHCII was revealed by immunolabeling of live non-permeabilized cells. In IFNγ-treated astrocytes, an increased fraction of large-diameter exocytotic vesicles (lysosome-like vesicles) with prolonged fusion pore dwell time and larger pore conductance was recorded, whereas the rate of endocytosis was decreased. Stimulation with ATP, which triggers cytosolic calcium signaling, increased the frequency of exocytotic events, whereas the frequency of full endocytosis was further reduced. In IFNγ-treated astrocytes, MHCII-linked antigen surface presentation is mediated by increased lysosomal exocytosis, whereas surface retention of antigens is prolonged by concomitant inhibition of endocytosis.

Correction to: Slow Release of HIV-1 Protein Nef from Vesicle-like Structures Is Inhibited by Cytosolic Calcium Elevation in Single Human Microglia.

The original version of this article unfortunately contained a mistake in Author name. In Pia Puzar Dominkus, "Puzar" should be classified as Familyname.

Astrocyte Specific Remodeling of Plasmalemmal Cholesterol Composition by Ketamine Indicates a New Mechanism of Antidepressant Action.

Ketamine is an antidepressant with rapid therapeutic onset and long-lasting effect, although the underlying mechanism(s) remain unknown. Using FRET-based nanosensors we found that ketamine increases [cAMP]i in astrocytes. Membrane capacitance recordings, however, reveal fundamentally distinct mechanisms of effects of ketamine and [cAMP]i on vesicular secretion: a rise in [cAMP]i facilitated, whereas ketamine inhibited exocytosis. By directly monitoring cholesterol-rich membrane domains with a fluorescently tagged cholesterol-specific membrane binding domain (D4) of toxin perfringolysin O, we demonstrated that ketamine induced cholesterol redistribution in the plasmalemma in astrocytes, but neither in fibroblasts nor in PC 12 cells. This novel mechanism posits that ketamine affects density and distribution of cholesterol in the astrocytic plasmalemma, consequently modulating a host of processes that may contribute to ketamine's rapid antidepressant action.

Fingolimod Suppresses the Proinflammatory Status of Interferon-γ-Activated Cultured Rat Astrocytes.

Astroglia, the primary homeostatic cells of the central nervous system, play an important role in neuroinflammation. They act as facultative immunocompetent antigen-presenting cells (APCs), expressing major histocompatibility complex (MHC) class II antigens upon activation with interferon (IFN)-γ and possibly other proinflammatory cytokines that are upregulated in disease states, including multiple sclerosis (MS). We characterized the anti-inflammatory effects of fingolimod (FTY720), an established drug for MS, and its phosphorylated metabolite (FTY720-P) in IFN-γ-activated cultured rat astrocytes. The expression of MHC class II compartments, ß2 adrenergic receptor (ADR-ß2), and nuclear factor kappa-light-chain enhancer of activated B cells subunit p65 (NF-κB p65) was quantified in immunofluorescence images acquired by laser scanning confocal microscopy. In addition, MHC class II-enriched endocytotic vesicles were labeled by fluorescent dextran and their mobility analyzed in astrocytes subjected to different treatments. FTY720 and FTY720-P treatment significantly reduced the number of IFN-γ-induced MHC class II compartments and substantially increased ADR-ß2 expression, which is otherwise small or absent in astrocytes in MS. These effects could be partially attributed to the observed decrease in NF-κB p65 expression, because the NF-κB signaling cascade is activated in inflammatory processes. We also found attenuated trafficking and secretion from dextran-labeled endo-/lysosomes that may hinder efficient delivery of MHC class II molecules to the plasma membrane. Our data suggest that FTY720 and FTY720-P at submicromolar concentrations mediate anti-inflammatory effects on astrocytes by suppressing their action as APCs, which may further downregulate the inflammatory process in the brain, constituting the therapeutic effect of fingolimod in MS.

Nestin Regulates Neurogenesis in Mice Through Notch Signaling From Astrocytes to Neural Stem Cells.

The intermediate filament (nanofilament) protein nestin is a marker of neural stem cells, but its role in neurogenesis, including adult neurogenesis, remains unclear. Here, we investigated the role of nestin in neurogenesis in adult nestin-deficient (Nes-/-) mice. We found that the proliferation of Nes-/- neural stem cells was not altered, but neurogenesis in the hippocampal dentate gyrus of Nes-/- mice was increased. Surprisingly, the proneurogenic effect of nestin deficiency was mediated by its function in the astrocyte niche. Through its role in Notch signaling from astrocytes to neural stem cells, nestin negatively regulates neuronal differentiation and survival; however, its expression in neural stem cells is not required for normal neurogenesis. In behavioral studies, nestin deficiency in mice did not affect associative learning but was associated with impaired long-term memory.

Slow Release of HIV-1 Protein Nef from Vesicle-like Structures Is Inhibited by Cytosolic Calcium Elevation in Single Human Microglia.

Once infected by HIV-1, microglia abundantly produce accessory protein Nef that enhances virus production and infectivity, but little is known about its intracellular compartmentalization, trafficking mode(s), and release from microglia. Here, we transfected immortalized human microglia with a plasmid encoding Nef tagged with green fluorescent protein (Nef.GFP) to biochemically and microscopically identify Nef.GFP-associated cellular compartments and examine their mobility and Nef release from cultured cells. Immunoblotting revealed that Nef.GFP confined to subcellular fractions with a buoyant density similar to organelles positive for lysosomal-associated membrane protein 1 (LAMP1) but structurally segregated from dextran-laden and LysoTracker-laden endo-/lysosomes in live cells. As revealed by confocal microscopy, Nef.GFP-positive vesicle-like structures were smaller than dextran-laden vesicles and displayed slow and non-directional mobility, in contrast to the faster and directional mobility of dextran-laden vesicles. Ionomycin-evoked elevation in intracellular free Ca2+ concentration ([Ca2+]i) negligibly affected mobility of Nef.GFP structures but strongly and irrecoverably attenuated mobility of dextran-laden vesicles. A slow time-dependent decrease in the number of Nef.GFP-positive structures was observed in non-stimulated controls (5 ± 1 structures/min), but not in ionomycin-stimulated cells (0 ± 2 structures/min; P < 0.05), indicating that elevated [Ca2+]i inhibits the release of Nef.GFP structures. The latter significantly co-localized with membrane sites immunopositive for the tetraspanins CD9 (36 ± 4%) and CD81 (22 ± 1%). This is the first report to demonstrate that microglial CD9- and CD81-positive plasma membrane-derived compartments are associated with biogenesis and Nef release.

PKH26 labeling of extracellular vesicles: Characterization and cellular internalization of contaminating PKH26 nanoparticles.

PKH lipophilic dyes are highly fluorescent and stain membranes by intercalating their aliphatic portion into the exposed lipid bilayer. They have established use in labeling and tracking of cells in vivo and in vitro. Despite wide use of PKH-labeled extracellular vesicles (EVs) in cell targeting and functional studies, nonEV-associated fluorescent structures have never been examined systematically, nor was their internalization by cells. Here, we have characterized PKH26-positive particles in lymphoblastoid B exosome samples and exosome-free controls stained by ultracentrifugation, filtration, and sucrose-cushion-based and sucrose-gradient-based procedures, using confocal imaging and asymmetric-flow field-flow fractionation coupled to multi-angle light-scattering detector analysis. We show for the first time that numerous PKH26 nanoparticles (nine out of ten PKH26-positive particles) are formed during ultracentrifugation-based exosome staining, which are almost indistinguishable from PKH26-labeled exosomes in terms of size, surface area, and fluorescence intensity. When PKH26-labeled exosomes were purified through sucrose, PKH26 nanoparticles were differentiated from PKH26-labeled exosomes based on their reduced size. However, PKH26 nanoparticles were only physically removed from PKH26-labeled exosomes when separated on a sucrose gradient, and at the expense of low PKH26-labeled exosome recovery. Overall, low PKH26-positive particle recovery is characteristic of filtration-based exosome staining. Importantly, PKH26 nanoparticles are internalized by primary astrocytes into similar subcellular compartments as PKH26-labeled exosomes. Altogether, PKH26 nanoparticles can result in false-positive signals for stained EVs that can compromise the interpretation of EV internalization. Thus, for use in EV uptake and functional studies, sucrose-gradient-based isolation should be the method of choice to obtain PKH26-labeled exosomes devoid of PKH26 nanoparticles.

Ångstrom-size exocytotic fusion pore: Implications for pituitary hormone secretion.

In the past, vesicle content release was thought to occur immediately and completely after triggering of exocytosis. However, vesicles may merge with the plasma membrane to form an Ångstrom diameter fusion pore that prevents the exit of secretions from the vesicle lumen. The advantage of such a narrow pore is to minimize the delay between the trigger and the release. Instead of stimulating a sequence of processes, leading to vesicle merger with the plasma membrane and a formation of a fusion pore, the stimulus only widens the pre-established fusion pore. The fusion pore may be stable and may exhibit repetitive opening of the vesicle lumen to the cell exterior accompanied by a content discharge. Such release of vesicle content is partial (subquantal), and depends on fusion pore open time, diameter and the diffusibility of the cargo. Such transient mode of fusion pore opening was not confirmed until the development of the membrane capacitance patch-clamp technique, which enables high-resolution measurement of changes in membrane surface area. It allows millisecond dwell-time measurements of fusion pores with subnanometer diameters. Currently, the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) proteins are considered to be key entities in end-stage exocytosis, and the SNARE complex assembly/disassembly may regulate the fusion pore. Moreover, lipids or other membrane constituents with anisotropic (non-axisymmetric) geometry may also favour the establishment of stable narrow fusion pores, if positioned in the neck of the fusion pore.