Imaging single membrane fusion events mediated by SNARE proteins

Fix, Marina; Melia, Thomas J.; Jaiswal, Jyoti K.; Rappoport, Joshua Z.; You, Daoqi; Söllner, Thomas H.; Rothman, James E.; Simon, Sanford M.

doi:10.1073/pnas.0401779101

Cited by 155 publications

(179 citation statements)

References 34 publications

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“…Second, when the vesicle fuses, the membrane-bound fluorophores should spread laterally in the plane of the plasma membrane and the fluorescent area should increase linearly with time at a rate that is equal to the diffusion coefficient of the membrane probe. Previously, we have successfully applied this approach to demonstrate vesicle fusion by using fluorescent lipids and membrane proteins such as p75, neural cell adhesion molecule, Glut4, transferrin receptor, CD63, and vesicular stomatitis virus-glycoprotein (2,4,5,(28)(29)(30). Here, by using lysosomal membrane protein CD63 and AO labeling, we have demonstrated the various fates of the vesicle membrane marker as the vesicle undergoes fusion, leakage, or lysis.…”

Section: Discussionmentioning

confidence: 99%

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Resolving vesicle fusion from lysis to monitor calcium-triggered lysosomal exocytosis in astrocytes

Jaiswal

Fix

Takano

et al. 2007

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

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Optical imaging of individual vesicle exocytosis is providing new insights into the mechanism and regulation of secretion by cells. To study calcium-triggered secretion from astrocytes, we used acridine orange (AO) to label vesicles. Although AO is often used for imaging exocytosis, we found that imaging vesicles labeled with AO can result in their photolysis. Here, we define experimental and analytical approaches that permit us to distinguish unambiguously between fusion, leakage, and lysis of individual vesicles. We have used this approach to demonstrate that lysosomes undergo calcium-triggered exocytosis in astrocytes.acridine orange ͉ imaging ͉ secretion ͉ evanescent wave microscopy ͉ ATP E xocytosis, the process by which a vesicle inside of a cell fuses to the cell surface, serves a plethora of important functions in all eukaryotic cells. These include insertion of membrane proteins such as ion channels and receptors, addition of new membranes during neuronal development, and secretion of neurotransmitter at the synapse.Over the past few decades, much has been learned about the physiology of exocytosis in neurons. Through the use of biochemistry, electrophysiology, and genetics, a considerable body of knowledge has been gathered about the molecular participants in exocytosis. However, much remains to be learned about the dynamics of these molecules. Our knowledge has been limited in part by our ability to image single molecules, or even the fusion of single vesicles. Fortunately, the use of total internal reflection fluorescence microscopy (TIR-FM) allows imaging fusion of single vesicles (1-6).Ca 2ϩ -triggered exocytosis, considered a hallmark of excitable cells, has also been reported in nonexcitable cells such as fibroblasts (5,(7)(8)(9). In all nonexcitable cells, Ca 2ϩ -triggered exocytosis is believed to serve a role in membrane repair (10). Additionally, some cells, such as hematopoietic cells, use Ca 2ϩ -triggered exocytosis for specialized functions such as release of cytolytic molecules (11). Astrocytes are nonexcitable cells of the nervous system that were previously thought to have only a protective role for neurons. However, astrocytes have now been shown to be more intricately involved in regulating neuronal physiology (12)(13)(14). This is believed in part to be due to the ability of astrocytes to exocytose a variety of molecules (15).Exocytosis of synaptic vesicles (16), dense core granules (3, 17), and even astrocytic vesicles that undergo Ca 2ϩ -triggered exocytosis (18) have been imaged by using the fluorescence of acridine orange (AO), a weak base that accumulates in acidified vesicles (19). Accumulation at high concentrations causes quenching of AO fluorescence. On release from the vesicle, AO fluorescence is unquenched and thus increases rapidly. Such rapid increase in AO fluorescence followed by its lateral diffusion is called an ''AO flash'' and has been used as a hallmark of exocytosis (3,(20)(21)(22).To examine potential Ca 2ϩ -regulated exocytic vesicles in astrocytes, we incubate...

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Section: Discussionmentioning

confidence: 99%

“…Our knowledge has been limited in part by our ability to image single molecules, or even the fusion of single vesicles. Fortunately, the use of total internal reflection fluorescence microscopy (TIR-FM) allows imaging fusion of single vesicles (1)(2)(3)(4)(5)(6).…”

mentioning

confidence: 99%

Resolving vesicle fusion from lysis to monitor calcium-triggered lysosomal exocytosis in astrocytes

Jaiswal

Fix

Takano

et al. 2007

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

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“…The parallel arrangement of the helices with all amino termini at one end of the helix bundle should bring the membrane anchors of the SNARE proteins and thus the vesicular and target membranes into close proximity. Energy made available from the assembly of the trans-SNARE complexes is used to drive the fusion of lipid bilayers [8][9][10][11]. After membrane fusion, the adapter protein α-SNAP (soluble NSF attachment protein) and the ATPase NSF (Nethylmaleimide-sensitive factor) dissociate the cis-SNARE complexes at the expense of ATP [12,13] to free the SNAREs for the next round of membrane fusion.…”

Section: Introductionmentioning

confidence: 99%

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

Hardee

Minnear

2007

Experimental Cell Research

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Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of α-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the Nterminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the Nterminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins.

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“…In vitro fusion reactions using reconstituted neuronal SNARE proteins are typically performed by using liposomes composed of PC plus an additional 15-25% PS (19,20,22,23). The addition of PA resulted in a concentrationdependent increase in VAMP2 incorporation, whereas PA had no significant effect on the efficiency of Stx4 incorporation ( Fig.…”

Section: Addition Of Pa To the Stx4͞snap23 Acceptor Membranes Enhancesmentioning

confidence: 99%

Asymmetric phospholipid distribution drives in vitro reconstituted SNARE-dependent membrane fusion

Vicogne

Vollenweider

Smith

et al. 2006

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

103

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Insulin-stimulated glucose uptake requires the fusion of GLUT4 transporter-containing vesicles with the plasma membrane, a process that depends on the SNARE (soluble N-ethylmaleimide-sensitive fusion factor attachment receptor) proteins VAMP2 (vesicleassociated membrane protein 2) and syntaxin 4 (Stx4)͞SNAP23 (soluble N-ethylmaleimide-sensitive fusion factor attachment protein 23). Efficient SNARE-dependent fusion has been shown in many settings in vivo to require the generation of both phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidic acid (PA). Addition of PA to Stx4͞SNAP23 vesicles markedly enhanced the fusion rate, whereas its addition to VAMP2 vesicles was inhibitory. In contrast, addition of PIP2 to Stx4͞SNAP23 vesicles inhibited the fusion reaction, and its addition to VAMP2 vesicles was stimulatory. The optimal distribution of phospholipids was found to trigger the progression from the hemifused state to full fusion. These findings reveal an unanticipated dependence of SNARE complex-mediated fusion on asymmetrically distributed acidic phospholipids and provide mechanistic insights into the roles of phospholipase D and PIP kinases in the late stages of regulated exocytosis.SNAP23 ͉ syntaxin 4 ͉ VAMP2

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Imaging single membrane fusion events mediated by SNARE proteins

Cited by 155 publications

References 34 publications

Resolving vesicle fusion from lysis to monitor calcium-triggered lysosomal exocytosis in astrocytes

Resolving vesicle fusion from lysis to monitor calcium-triggered lysosomal exocytosis in astrocytes

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

Asymmetric phospholipid distribution drives in vitro reconstituted SNARE-dependent membrane fusion

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