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      Dynamic visualization of membrane-inserted fraction of pHluorin-tagged channels using repetitive acidification technique

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          Abstract

          Background

          Changes in neuronal excitability, synaptic efficacy and generally in cell signaling often result from insertion of key molecules into plasma membrane (PM). Many of the techniques used for monitoring PM insertion lack either spatial or temporal resolution.

          Results

          We improved the imaging method based on time-lapse total internal reflection fluorescence (TIRF) microscopy and pHluorin tagging by supplementing it with a repetitive extracellular acidification protocol. We illustrate the applicability of this method by showing that brief activation of NMDA receptors ("chemical LTP") in cultured hippocampal neurons induced a persistent PM insertion of glutamate receptors containing the pHluorin-tagged GluR-A(flip) subunits.

          Conclusion

          The repetitive acidification technique provides a more accurate way of monitoring the PM-inserted fraction of fluorescently tagged molecules and offers a good temporal and spatial resolution.

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          Most cited references 21

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          Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.

          In neural systems, information is often carried by ensembles of cells rather than by individual units. Optical indicators provide a powerful means to reveal such distributed activity, particularly when protein-based and encodable in DNA: encodable probes can be introduced into cells, tissues, or transgenic organisms by genetic manipulation, selectively expressed in anatomically or functionally defined groups of cells, and, ideally, recorded in situ, without a requirement for exogenous cofactors. Here we describe sensors for secretion and neurotransmission that fulfil these criteria. We have developed pH-sensitive mutants of green fluorescent protein ('pHluorins') by structure-directed combinatorial mutagenesis, with the aim of exploiting the acidic pH inside secretory vesicles to monitor vesicle exocytosis and recycling. When linked to a vesicle membrane protein, pHluorins were sorted to secretory and synaptic vesicles and reported transmission at individual synaptic boutons, as well as secretion and fusion pore 'flicker' of single secretory granules.
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            The elementary unit of store-operated Ca2+ entry: local activation of CRAC channels by STIM1 at ER–plasma membrane junctions

            The activation of store-operated Ca2+ entry by Ca2+ store depletion has long been hypothesized to occur via local interactions of the endoplasmic reticulum (ER) and plasma membrane, but the structure involved has never been identified. Store depletion causes the ER Ca2+ sensor stromal interacting molecule 1 (STIM1) to form puncta by accumulating in junctional ER located 10–25 nm from the plasma membrane (see Wu et al. on p. 803 of this issue). We have combined total internal reflection fluorescence (TIRF) microscopy and patch-clamp recording to localize STIM1 and sites of Ca2+ influx through open Ca2+ release–activated Ca2+ (CRAC) channels in Jurkat T cells after store depletion. CRAC channels open only in the immediate vicinity of STIM1 puncta, restricting Ca2+ entry to discrete sites comprising a small fraction of the cell surface. Orai1, an essential component of the CRAC channel, colocalizes with STIM1 after store depletion, providing a physical basis for the local activation of Ca2+ influx. These studies reveal for the first time that STIM1 and Orai1 move in a coordinated fashion to form closely apposed clusters in the ER and plasma membranes, thereby creating the elementary unit of store-operated Ca2+ entry.
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              Regulated ATP release from astrocytes through lysosome exocytosis.

              Release of ATP from astrocytes is required for Ca2+ wave propagation among astrocytes and for feedback modulation of synaptic functions. However, the mechanism of ATP release and the source of ATP in astrocytes are still not known. Here we show that incubation of astrocytes with FM dyes leads to selective labelling of lysosomes. Time-lapse confocal imaging of FM dye-labelled fluorescent puncta, together with extracellular quenching and total-internal-reflection fluorescence microscopy (TIRFM), demonstrated directly that extracellular ATP or glutamate induced partial exocytosis of lysosomes, whereas an ischaemic insult with potassium cyanide induced both partial and full exocytosis of these organelles. We found that lysosomes contain abundant ATP, which could be released in a stimulus-dependent manner. Selective lysis of lysosomes abolished both ATP release and Ca2+ wave propagation among astrocytes, implicating physiological and pathological functions of regulated lysosome exocytosis in these cells.
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                Author and article information

                Affiliations
                [1 ]Neuroscience Center, University of Helsinki, FIN-00014, Helsinki, Finland
                [2 ]Department of Biological and Environmental Sciences, Division of Biochemistry, University of Helsinki, FIN-00014, Helsinki, Finland
                [3 ]Allen Institute for Brain Science, Seattle, WA 98103, USA
                Contributors
                Journal
                BMC Neurosci
                BMC Neuroscience
                BioMed Central
                1471-2202
                2009
                30 November 2009
                : 10
                : 141
                2794868
                1471-2202-10-141
                19948025
                10.1186/1471-2202-10-141
                Copyright ©2009 Khiroug et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Categories
                Methodology article

                Neurosciences

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