Ca-dependent mobility of vesicles capturing anti-VGLUT1 antibodies

Celica Biomedical Center, Proletarska 4, 1000 Ljubljana, Slovenia.
Experimental Cell Research (Impact Factor: 3.37). 12/2007; 313(18):3809-18. DOI: 10.1016/j.yexcr.2007.08.020
Source: PubMed

ABSTRACT Several aspects of secretory vesicle cycle have been studied in the past, but vesicle trafficking in relation to the fusion site is less well understood. In particular, the mobility of recaptured vesicles that traffic back toward the central cytoplasm is still poorly defined. We exposed astrocytes to antibodies against the vesicular glutamate transporter 1 (VGLUT1), a marker of glutamatergic vesicles, to fluorescently label vesicles undergoing Ca(2+)-dependent exocytosis and examined their number, fluorescence intensity, and mobility by confocal microscopy. In nonstimulated cells, immunolabeling revealed discrete fluorescent puncta, indicating that VGLUT1 vesicles, which are approximately 50 nm in diameter, cycle slowly between the plasma membrane and the cytoplasm. When the cytosolic Ca(2+) level was raised with ionomycin, the number and fluorescence intensity of the puncta increased, likely because the VGLUT1 epitopes were more accessible to the extracellularly applied antibodies following Ca(2+)-triggered exocytosis. In nonstimulated cells, the mobility of labeled vesicles was limited. In stimulated cells, many vesicles exhibited directional mobility that was abolished by cytoskeleton-disrupting agents, indicating dependence on intact cytoskeleton. Our findings show that postfusion vesicle mobility is regulated and may likely play a role in synaptic vesicle cycle, and also more generally in the genesis and removal of endocytic vesicles.

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    • "Increases in [Ca 2þ ] i Decrease the Mobility of AQP4e Vesicles An increase in [Ca 2þ ] i differentially affects vesicle mobility in rat astrocytes and appears to be vesicle type specific (Potokar et al., 2007, 2008, 2010; Stenovec et al., 2007). The speed of AQP4e vesicles was unaffected by increases in [Ca 2þ ] i , as observed in vesicles on their way to the plasma membrane (Potokar et al., 2005, 2007). "
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    ABSTRACT: 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.
    Glia 06/2013; 61(6). DOI:10.1002/glia.22485 · 6.03 Impact Factor
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    • "To test that the vesicle mobility recorded in cultured astrocytes also occurs in vivo, vesicle dynamics was studied in brain slices in which cell-to-cell contacts are preserved and tissue architecture is closer to the one present in the brain (Potokar et al., 2009). The mobility of specifically labelled recycling glutamatergic and peptidergic vesicles (immunopositive for VGLUT1 or ANP respectively) was similar to that in cultured astrocytes (Stenovec et al., 2007; Potokar et al., 2008). "
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    ABSTRACT: Astroglial cells, due to their passive electrical properties, were long considered subservient to neurons and to merely provide the framework and metabolic support of the brain. Although astrocytes do play such structural and housekeeping roles in the brain, these glial cells also contribute to the brain's computational power and behavioural output. These more active functions are endowed by the Ca(2+)-based excitability displayed by astrocytes. An increase in cytosolic Ca(2+) levels in astrocytes can lead to the release of signalling molecules, a process termed gliotransmission, via the process of regulated exocytosis. Dynamic components of astrocytic exocytosis include the vesicular-plasma membrane secretory machinery, as well as the vesicular traffic, which is governed not only by general cytoskeletal elements but also by astrocyte-specific IFs (intermediate filaments). Gliotransmitters released into the ECS (extracellular space) can exert their actions on neighbouring neurons, to modulate synaptic transmission and plasticity, and to affect behaviour by modulating the sleep homoeostat. Besides these novel physiological roles, astrocytic Ca(2+) dynamics, Ca(2+)-dependent gliotransmission and astrocyte-neuron signalling have been also implicated in brain disorders, such as epilepsy. The aim of this review is to highlight the newer findings concerning Ca(2+) signalling in astrocytes and exocytotic gliotransmission. For this we report on Ca(2+) sources and sinks that are necessary and sufficient for regulating the exocytotic release of gliotransmitters and discuss secretory machinery, secretory vesicles and vesicle mobility regulation. Finally, we consider the exocytotic gliotransmission in the modulation of synaptic transmission and plasticity, as well as the astrocytic contribution to sleep behaviour and epilepsy.
    ASN Neuro 02/2012; 4(2). DOI:10.1042/AN20110061 · 4.44 Impact Factor
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    • "TIRFM were estimated to be ∼40 nm based on comparison to 40 nm diameter fluorescent beads ( Cali et al . 2008 ; Marchaland et al . 2008 ) . Glutamatergic vesicles labelled by capturing an extracellular antibody against VGLUT1 in a Ca 2+ - dependent manner while recycling with the plasma membrane are electron - lucent and have diameters of ∼50 nm ( Stenovec et al . 2007 ) . Vesicles found within gliosomes , subcellular components of astrocytic processes isolated from brain by fractionation ( Nakamura et al . 1993 ) , express synaptobrevin 2 and VGLUT1 and measure ∼30 nm in diameter ( Stigliani et al . 2006 ) ."
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    ABSTRACT: Astrocytes can release various gliotransmitters in response to stimuli that cause increases in intracellular Ca(2+) levels; this secretion occurs via a regulated exocytosis pathway. Indeed, astrocytes express protein components of the vesicular secretory apparatus. However, the detailed temporal characteristics of vesicular fusions in astrocytes are not well understood. In order to start addressing this issue, we used total internal reflection fluorescence microscopy (TIRFM) to visualize vesicular fusion events in astrocytes expressing the fluorescent synaptobrevin 2 derivative, synapto-pHluorin. Although our cultured astrocytes from visual cortex express synaptosome-associated protein of 23 kDa (SNAP23), but not of 25 kDa (SNAP25), these glial cells exhibited a slow burst of exocytosis under mechanical stimulation; the expression of SNAP25B did not affect bursting behaviour. The relative amount of two distinct types of events observed, transient and full fusions, depended on the applied stimulus. Expression of exogenous synaptotagmin 1 (Syt1) in astrocytes endogenously expressing Syt4, led to a greater proportion of transient fusions when astrocytes were stimulated with bradykinin, a stimulus otherwise resulting in more full fusions. Additionally, we studied the stability of the transient fusion pore by measuring its dwell time, relation to vesicular size, flickering and decay slope; all of these characteristics were secretagogue dependent. The expression of SNAP25B or Syt1 had complex effects on transient fusion pore stability in a stimulus-specific manner. SNAP25B obliterated the appearance of flickers and reduced the dwell time when astrocytes were mechanically stimulated, while astrocytes expressing SNAP25B and stimulated with bradykinin had a reduction in decay slope. Syt1 reduced the dwell time when astrocytes were stimulated either mechanically or with bradykinin. Our detailed study of temporal characteristics of astrocytic exocytosis will not only aid the general understanding of this process, but also the interpretation of the events at the tripartite synapse, both in health and disease.
    The Journal of Physiology 07/2011; 589(Pt 17):4271-300. DOI:10.1113/jphysiol.2011.210435 · 4.54 Impact Factor
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