The Use of pHluorins for Optical Measurements of Presynaptic Activity

Sankaranarayanan, Sethuraman; Angelis, Dino De; Rothman, James E.; Ryan, Timothy A.

doi:10.1016/s0006-3495(00)76468-x

Cited by 541 publications

(628 citation statements)

References 15 publications

Supporting

Mentioning

595

Contrasting

Unclassified

Order By: Relevance

“…On the basis of these measurements, we estimate that 30% of axonal SpH resides on the cell surface. This amount was similar to the surface abundance previously determined in cultured hippocampal neurons (25) and in Torpedo axons (14). The surface fraction calculation assumes that SVs are acidic, and it may underestimate the true surface percentage if SVs are closer to a neutral pH, as has been observed in Drosophila terminals (34).…”

Section: Resultssupporting

confidence: 89%

“…SpH is highly pH-sensitive such that fluorescence is expected to be quenched in the acidic environment of the SV lumen, whereas SpH residing on the plasma membrane will be unquenched, producing a 20-fold increased fluorescence per SpH molecule (25). Individual animals were dissected, and axonal fluorescence from a 20-to 100-m region of the dorsal nerve cord was focused onto a photodiode.…”

Section: Resultsmentioning

confidence: 99%

“…Is the spatial distribution of surface synaptobrevin restricted in some manner? To address these questions, we used synaptopHluorin (SpH), a pH-sensitive variant of GFP fused to the luminal domain of synaptobrevin (25)(26)(27)(28)(29), to analyze genes that regulate the surface pool of v-SNAREs in the nematode Caenorhabditis elegans. …”

mentioning

confidence: 99%

See 2 more Smart Citations

Factors regulating the abundance and localization of synaptobrevin in the plasma membrane

Dittman

Kaplan

2006

Proc. Natl. Acad. Sci. U.S.A.

141

138

View full text Add to dashboard Cite

After synaptic vesicle fusion, vesicle proteins must be segregated from plasma membrane proteins and recycled to maintain a functional vesicle pool. We monitored the distribution of synaptobrevin, a vesicle protein required for exocytosis, in Caenorhabditis elegans motor neurons by using a pH-sensitive synaptobrevin GFP fusion protein, synaptopHluorin. We estimated that 30% of synaptobrevin was present in the plasma membrane. By using a panel of endocytosis and exocytosis mutants, we found that the majority of surface synaptobrevin derives from fusion of synaptic vesicles and that, in steady state, synaptobrevin equilibrates throughout the axon. The surface synaptobrevin was enriched near active zones, and its spatial extent was regulated by the clathrin adaptin AP180. These results suggest that there is a plasma membrane reservoir of synaptobrevin that is supplied by the synaptic vesicle cycle and available for retrieval throughout the axon. The size of the reservoir is set by the relative rates of exo-and endocytosis.AP180 ͉ endocytosis ͉ pHluorin ͉ synaptic vesicle N eurotransmitter released at synapses originates from a recycling pool of synaptic vesicles (SVs) (1-3). Several processes are required for neurotransmitter secretion, including biogenesis of SVs, docking with the plasma membrane, ATPdependent priming of SVs to make them fusion-competent, calcium-evoked fusion, and endocytic recycling (4-6). The fusion step is believed to be mediated by the SNARE complex, a four-helix coiled-coil structure consisting of a vesicle SNARE (v-SNARE), synaptobrevin͞VAMP, and two target membrane SNARE (t-SNARE) proteins, syntaxin 1 and SNAP-25, on the plasma membrane (7-10).Accurate sorting of SNAREs to vesicle and plasma membranes is critical for the coordination of SV fusion (11, 12). The t-SNAREs syntaxin and SNAP-25 are abundant in the plasma membrane but are excluded from recycling SVs (13, 14), although syntaxin has been found in some intracellular compartments (13,(15)(16)(17). Several studies have documented a significant fraction of endogenous synaptobrevin (14) or synaptobrevin GFP (18,19) in the plasma membrane. Surface synaptobrevin could be derived from fusion of SV precursors undergoing anterograde transport via the constitutive secretory pathway (20-23). Alternatively, surface synaptobrevin could reflect diffusion within the plasma membrane after vesicle fusion (19) or ''stranded'' vesicles that fail to undergo endocytosis (24). Finally, some authors have argued that surface synaptobrevin results from missorting, particularly in cases in which synaptobrevin is overexpressed (22).Several questions remain concerning the surface pool of synaptobrevin. Does this pool arise from v-SNAREs that escape retrieval after exocytosis? Which endocytic pathways regulate this pool of synaptobrevin? Is the spatial distribution of surface synaptobrevin restricted in some manner? To address these questions, we used synaptopHluorin (SpH), a pH-sensitive variant of GFP fused to the luminal domain of synaptobrevin (25)(26)(27)...

show abstract

Section: Resultssupporting

confidence: 89%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Factors regulating the abundance and localization of synaptobrevin in the plasma membrane

Dittman

Kaplan

2006

Proc. Natl. Acad. Sci. U.S.A.

141

138

View full text Add to dashboard Cite

show abstract

“…Not only are genetic and reverse genetic screens possible, techniques such as double-stranded RNA-mediated gene interference allow us to phenocopy a null allele in the space of days. With the introduction of transgenic pH-sensitive forms of GFP (38,39), it should eventually be possible to study pH regulation by the nhx exchangers in an intact nematode. The identification of the cellular and intracellular expression patterns for each of the NHX proteins is an important first step toward studying their physiological roles.…”

Section: Discussionmentioning

confidence: 99%

The NHX Family of Na+-H+ Exchangers in Caenorhabditis elegans

Nehrke

Melvin

2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

Na ؉ -H ؉ exchangers prevent cellular acidification by catalyzing the electroneutral exchange of extracellular sodium for an intracellular proton. To date, seven Na ؉ -H ؉ exchangers have been identified in mammals, and although several members of this family have been extensively studied and characterized, it is clear that there are major gaps in our understanding with respect to the remaining family members. To initiate the study of Na ؉ -H ؉ exchangers in a genomically defined and genetically tractable model system, we have cloned the complete cDNAs and analyzed splice site variation for nine putative homologs from the nematode Caenorhabditis elegans, which we have called NHX-1 through -9. The expression patterns and cellular distributions of the NHX proteins were determined using transcriptional and translational promoter-transgene fusion constructs to green fluorescent protein. Four of the putative exchangers were expressed at the cell surface, whereas five of the exchangers were associated with the membranes of intracellular organelles. Individual isoforms were expressed exclusively in the intestine, seam cells, hypodermal cells of the main body syncytium, and the excretory cell, all of which are polarized epithelial cells, suggesting a role for these proteins in epithelial membrane transport processes in the nematode. Other isoforms were found to express either ubiquitously or in a pan-neural pattern, suggesting a more conserved role in cell pH regulation or neuronal function. Finally, we show that recombinant NHX-4, the ubiquitous nematode Na ؉ -H ؉ exchanger, mediates Na ؉ -dependent pH recovery after intracellular acidification. NHX-4 has a K a for Na ؉ of ϳ32 mM, is not Cl ؊ -dependent, and is relatively insensitive to the amiloride analog EIPA.

show abstract

“…Recent investigations have used vesicle-associated f luorescent dyes and GFP-tagged LDCV-associated proteins or insulin itself, and imaging with conventional, total internal reflection fluorescence (TIRF), or confocal microscopy (7)(8)(9)(10). Fusion events are inferred by the loss of fluorescence at the cell surface (fluorescent vesicle proteins, ''recycled'' membrane dyes, or acidophilic dyes) or by the increased fluorescence associated with neutralization of granule pH (11). These studies have generally concluded that LDCVs are recruited to the plasma membrane and may make multiple partial fusions before fully fusing and releasing the granule contents (f lickering and͞or kiss-and-run fusions) (6,12,14).…”

mentioning

confidence: 99%

Direct imaging shows that insulin granule exocytosis occurs by complete vesicle fusion

Bindokas

Kuznetsov

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Confocal imaging of GFP-tagged secretory granules combined with the use of impermeant extracellular dyes permits direct observation of insulin packaged in secretory granules, trafficking of these granules to the plasma membrane, exocytotic fusion of granules with the plasma membrane, and eventually the retrieval of membranes by endocytosis. Most such studies have been done in tumor cell lines, using either confocal methods or total internal reflectance microscopy. Here we compared these methods by using GFP-syncollin or PC3-GFP plus rhodamine dextrans to study insulin granule dynamics in insulinoma cells, normal mouse islets, and primary pancreatic beta cells. We found that most apparently docked granules did not fuse with the plasma membrane after stimulation. Granules that did fuse typically fused completely, but a few dextran-filled granules lingered at the membrane. Direct recycling of granules occurred only rarely. Similar results were obtained with both confocal and total internal reflection microscopy, although each technique had advantages for particular aspects of the granule life cycle. We conclude that insulin exocytosis involves a prolonged interaction of secretory granules with the plasma membrane, and that the majority of exocytotic events occur by full, not partial, fusion.

show abstract

The Use of pHluorins for Optical Measurements of Presynaptic Activity

Cited by 541 publications

References 15 publications

Factors regulating the abundance and localization of synaptobrevin in the plasma membrane

Factors regulating the abundance and localization of synaptobrevin in the plasma membrane

The NHX Family of Na+-H+ Exchangers in Caenorhabditis elegans

Direct imaging shows that insulin granule exocytosis occurs by complete vesicle fusion

Contact Info

Product

Resources

About