Vesicle mobility studied in cultured astrocytes

Potokar, Maja; Kreft, Marko; Pangršič, Tina; Zorec, Robert

doi:10.1016/j.bbrc.2005.02.030

Cited by 80 publications

(164 citation statements)

References 34 publications

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“…Whether vesicle mobility is observed as directional or non-directional likely depends on their pattern of attachment to the cytoskeleton (35,45,46). Like in our previous study (35), we observed no difference in the apparent shape or size of vesicles in both groups. The mechanism of spontaneous mobility of quinacrine-loaded vesicles is not known.…”

Section: Discussionsupporting

confidence: 44%

“…The mechanism of spontaneous mobility of quinacrine-loaded vesicles is not known. Because most of them were non-directional, we propose that their mobility mainly involved free diffusion (30,35). Stimulation with ionomycin significantly reduced maximal displacement and total track length of all vesicles.…”

Section: Discussionmentioning

confidence: 99%

“…Next, the mobility of quinacrine-loaded vesicles was examined by using confocal microscopy, enabling us to study the mobility of all vesicles in the cytoplasm, including those not detected by TIRF. In our previous study we have shown that ANP-containing vesicles in cortical astrocytes display two types of mobility (35), whereby 35% of vesicles exhibited directional mobility. Interestingly, the quinacrine-loaded vesicles of the present study predominantly showed non-directional mobility with Ͻ1 m maximal displacement.…”

Section: Discussionmentioning

confidence: 99%

“…We calculated the following parameters of vesicle mobility: current time (time from the beginning of single vesicle tracking), step length (displacement of a vesicle in each time interval), track length (the total length of the analyzed vesicle pathway), maximal displacement (a measure of the net translocation of a vesicle) and the directionality index (maximal displacement/total track length) of vesicles as described previously (35,36). The analysis of vesicle mobility was performed for epochs of 30 s.…”

Section: Methodsmentioning

confidence: 99%

“…Vesicle Tracking and Co-localization Analysis-Vesicle mobility was analyzed with ParticleTR software (Celica, Ljubljana, Slovenia) as previously described (35). We calculated the following parameters of vesicle mobility: current time (time from the beginning of single vesicle tracking), step length (displacement of a vesicle in each time interval), track length (the total length of the analyzed vesicle pathway), maximal displacement (a measure of the net translocation of a vesicle) and the directionality index (maximal displacement/total track length) of vesicles as described previously (35,36).…”

Section: Methodsmentioning

confidence: 99%

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Exocytotic Release of ATP from Cultured Astrocytes

Pangršič

Potokar

Stenovec

et al. 2007

Journal of Biological Chemistry

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216

208

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Astrocytes appear to communicate with each other as well as with neurons via ATP. However, the mechanisms of ATP release are controversial. To explore whether stimuli that increase [Ca 2؉ ] i also trigger vesicular ATP release from astrocytes, we labeled ATP-containing vesicles with the fluorescent dye quinacrine, which exhibited a significant co-localization with atrial natriuretic peptide. The confocal microscopy study revealed that quinacrine-loaded vesicles displayed mainly non-directional spontaneous mobility with relatively short track lengths and small maximal displacements, whereas 4% of vesicles exhibited directional mobility. After ionomycin stimulation only non-directional vesicle mobility could be observed, indicating that an increase in [Ca 2؉ ] i attenuated vesicle mobility. Total internal reflection fluorescence (TIRF) imaging in combination with epifluorescence showed that a high percentage of fluorescently labeled vesicles underwent fusion with the plasma membrane after stimulation with glutamate or ionomycin and that this event was Ca 2؉ -dependent. This was confirmed by patchclamp studies on HEK-293T cells transfected with P2X 3 receptor, used as sniffers for ATP release from astrocytes. Glutamate stimulation of astrocytes was followed by an increase in the incidence of small transient inward currents in sniffers, reminiscent of postsynaptic quantal events observed at synapses. Their incidence was highly dependent on extracellular Ca 2؉ . Collectively, these findings indicate that glutamate-stimulated ATP release from astrocytes was most likely exocytotic and that after stimulation the fraction of quinacrine-loaded vesicles, spontaneously exhibiting directional mobility, disappeared.Many recent studies demonstrate that astrocytes play a significant modulatory role in synaptic physiology (1-4). Astrocytes respond to neurotransmitters, integrate different inputs, and signal back to neurons or forward information to neighboring or more distant astrocytes (2). In response to stimulation they release several chemical substances (5-7), termed gliotransmitters, which can interfere with the neuronal communicating pathways (8 -10).One major extracellular messenger important for coordinating the function of astrocytes, as well as for the cross-talk between them and other cell types, is ATP (11). Whereas several lines of evidence support the idea of ATP release from astrocytes (12-14), the release mechanisms are not completely understood. Some studies have described a connexin hemichannel-mediated release (12, 15, 16) in both resting and activated conditions. Other possible mechanisms, like volume-regulated anion channels (17, 18) and ATP-binding cassette transporters (multidrug resistance P-glycoprotein (19), or cystic fibrosis transmembrane conductance regulator (20) have also been reported. On the contrary, only few studies have focused on the possibility of exocytotic, vesicular ATP release mechanism operating in astrocytes (13, 14), even though it has been shown that astrocytes express the element...

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Section: Discussionsupporting

confidence: 44%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Exocytotic Release of ATP from Cultured Astrocytes

Pangršič

Potokar

Stenovec

et al. 2007

Journal of Biological Chemistry

Self Cite

216

208

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Vesicular transmitter release from astrocytes

Montana

Malarkey

Verderio

et al. 2006

Glia

293

234

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Astrocytes can release a variety of transmitters, including glutamate and ATP, in response to stimuli that induce increases in intracellular Ca(2+) levels. This release occurs via a regulated, exocytotic pathway. As evidence of this, astrocytes express protein components of the vesicular secretory apparatus, including synaptobrevin 2, syntaxin, and SNAP-23. Additionally, astrocytes possess vesicular organelles, the essential morphological elements required for regulated Ca(2+)-dependent transmitter release. The location of specific exocytotic sites on these cells, however, remains to be unequivocally determined.

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Sub‐micromolar increase in [Ca²⁺]_i triggers delayed exocytosis of ATP in cultured astrocytes

Pryazhnikov

Khiroug

2007

Glia

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Astrocytes release a variety of transmitter molecules, which mediate communication between glial cells in the brain and modulate synaptic transmission. ATP is a major glia-derived transmitter, but the mechanisms and kinetics of ATP release from astrocytes remain largely unknown. Here, we combined epifluorescence and total internal reflection fluorescence microscopy to monitor individual quinacrine-loaded ATP-containing vesicles undergoing exocytosis in cultured astrocytes. In resting cells, vesicles exhibited three-dimensional motility, spontaneous docking and release at low rate. Extracellular ATP application induced a Ca(2+)-dependent increase in the rate of exocytosis, which persisted for several minutes. Using UV flash photolysis of caged Ca(2+), the threshold [Ca(2+)](i) for ATP exocytosis was found to be approximately 350 nM. Subthreshold [Ca(2+)](i) transients predominantly induced vesicle docking at plasma membrane without subsequent release. ATP exocytosis triggered either by purinergic stimulation or by Ca(2+) uncaging occurred after a substantial delay ranging from tens to hundreds of seconds, with only approximately 4% of release occurring during the first 30 s. The time course of the cargo release from vesicles had two peaks centered on

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Vesicle mobility studied in cultured astrocytes

Cited by 80 publications

References 34 publications

Exocytotic Release of ATP from Cultured Astrocytes

Exocytotic Release of ATP from Cultured Astrocytes

Vesicular transmitter release from astrocytes

Sub‐micromolar increase in [Ca²⁺]_i triggers delayed exocytosis of ATP in cultured astrocytes

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Vesicle mobility studied in cultured astrocytes

Cited by 80 publications

References 34 publications

Exocytotic Release of ATP from Cultured Astrocytes

Exocytotic Release of ATP from Cultured Astrocytes

Vesicular transmitter release from astrocytes

Sub‐micromolar increase in [Ca2+]i triggers delayed exocytosis of ATP in cultured astrocytes

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Sub‐micromolar increase in [Ca²⁺]_i triggers delayed exocytosis of ATP in cultured astrocytes