Time–resolved capacitance measurements: monitoring exocytosis in single cells

Lindau, Manfred

doi:10.1017/s0033583500003279

Cited by 87 publications

(95 citation statements)

References 72 publications

(105 reference statements)

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“…Nerve endings were obtained from the hypophysis of male Sprague-Dawley rats (260-500 g) (12 [8][9][10][11][12][13][14][15] ,um. In the experiments described here most nerve endings had a diameter of [8][9][10] pm and an initial capacitance of 1.5-2.5 pF.…”

Section: Methodsmentioning

confidence: 99%

“…Following patch disruption the sine wave was switched on. The phase was determined automatically by phase tracking (13) and the capacitive and ohmic changes were calculated on-line in the computer (14,15 When [Ca2+]i is elevated by Ca2+ entry through voltagedependent Ca2+ channels during step depolarization, an instantaneous capacitance increase, which occurs during the first 80 Ms, is followed by a second phase with a slow increase extending over several seconds (8 Fig. 1, presumably reflecting endocytosis of large vacuoles.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Exo-endocytosis and closing of the fission pore during endocytosis in single pituitary nerve terminals internally perfused with high calcium concentrations.

Rosenboom

Lindau

1994

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

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An increase in free Ca2+ triggers exocytosis in pituitary nerve terminals leading to an increase in membrane area and membrane capacitance. When Ca2+ is increased by step depolarization, an instantaneous capacitance increase during the first 80 ms is followed by a slow increase extending over several seconds. We measured capacitance changs associated with exocytosis and endocytosis in single pituitary nerve terminals internally perfused with hig Ca2+. At 50 SM Ca2+ the capacitance increased by up to 2%/s, similar to the slow phase observed during depolarization. Our results Indicate that at the site of fusion very high Ca+ is required. Following exocytosis, large downward capacitance steps were measured, reflecting endocytosis of large vacuoles. These events were not abrupt but reflected a gradual decrease of fission pore conductance from 8 nS to <40 pS during 500 ms, revealing the dynamics of individual fission pore closures. Above 300 pS, narrowing of the endocytotic fission pore was :10 times slower than the previously reported expansion of the exocytotic fusion pore. The transition between 300 pS and 0 pS took =-200 ms, whereas it has been reported that the exocytotic fusion pore measuredin mast cells opens from 0 to 280 pS in <100 as. The time course of closing of the fission pore may be explained by an exponential decrease in pore diameter occurring at a constant rate.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Exo-endocytosis and closing of the fission pore during endocytosis in single pituitary nerve terminals internally perfused with high calcium concentrations.

Rosenboom

Lindau

1994

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

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“…The specific phase angle for lock-in measurements (Joshi and Fernandez, 1988;Gillis, 1995) was found by "capacitance dithering" (Neher and Marty, 1982). Fusion pore conductances were calculated off-line as G P ϭ (Y 0 2 ϩ Y 90 2 )/Y 0 (Lindau, 1991;Ratinov et al, 1998). The evolution of the average pore conductance was characterized by aligning individual pore conductance records at the moment of pore opening and calculating a mean pore conductance every 5 or 20 ms. For some pores, conductances increased beyond measurable values, usually ϳ20 nS, sooner than the time interval selected to build the average pore profile (e.g., 20 s).…”

Section: Electrical Measurementsmentioning

confidence: 99%

Amino Acid Sequence Requirements of the Transmembrane and Cytoplasmic Domains of Influenza Virus Hemagglutinin for Viable Membrane Fusion

Melikyan

Lin

Roth

et al. 1999

MBoC

120

135

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The amino acid sequence requirements of the transmembrane (TM) domain and cytoplasmic tail (CT) of the hemagglutinin (HA) of influenza virus in membrane fusion have been investigated. Fusion properties of wild-type HA were compared with those of chimeras consisting of the ectodomain of HA and the TM domain and/or CT of polyimmunoglobulin receptor, a nonviral integral membrane protein. The presence of a CT was not required for fusion. But when a TM domain and CT were present, fusion activity was greater when they were derived from the same protein than derived from different proteins. In fact, the chimera with a TM domain of HA and truncated CT of polyimmunoglobulin receptor did not support full fusion, indicating that the two regions are not functionally independent. Despite the fact that there is wide latitude in the sequence of the TM domain that supports fusion, a point mutation of a semiconserved residue within the TM domain of HA inhibited fusion. The ability of a foreign TM domain to support fusion contradicts the hypothesis that a pore is composed solely of fusion proteins and supports the theory that the TM domain creates fusion pores after a stage of hemifusion has been achieved. INTRODUCTIONMembrane fusion is common to many cellular processes, such as exocytosis and intracellular trafficking. But only in the case of viruses have the proteins responsible for fusion been unambiguously identified. A number of common features have been found among viral fusion proteins. All are oligomerized. The monomer of each oligomer always contains a critical stretch of nonpolar amino acids, known as the fusion peptide (Hernandez et al., 1996;Durell et al., 1997). As crystallographically determined, fusion proteins from several different virus families have the same backbone structure of extended coiled-coil ␣-helices (Bullough et al., 1994;Chan et al., 1997). Because viral fusion proteins of unrelated viruses have common structural patterns, it is logical to assume that their mechanisms of fusion are similar as well. Furthermore, a complex of SNARES, proteins thought to be responsible for the constitutive fusion of intracellular trafficking and the regulated fusion that occurs in exocytosis (Sü dhof, 1995), has been crystallographically determined (Poirier et al., 1998;Sutton et al., 1998). It too displays a long coiled-coil region. Therefore it may well be that the mechanisms of viral fusion pertain more generally to cellular fusion. Influenza virus has long been the prototypic virus for study of fusion mechanisms. It ‡ Corresponding author. E-mail address: fcohen@rush.edu. Abbreviations used: CF, carboxyfluorescein; CPZ, chlorpromazine; CT, cytoplasmic tail; DMEM, Dulbecco's modified Eagle's medium; GPI, glycosylphosphatidylinositol; HA, hemagglutinin; PBS-A-S, PBS supplemented with 0.1% sodium azide and 5% calf serum; p.i., postinfection; pIgR, polyimmunoglobulin receptor; R18, octadecylrhodamine B; RBC, red blood cell; RD, tetramethylrhodamine-tagged dextran; TM, transmembrane; VSV, vesicular stomatitis virus; WT,...

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“…The V-command was a 50-mV sine wave (root mean square, 1 kHz). Capacitance and conductance values were estimated from the real and imaginary components of the complex admittance, which was obtained by a Lock-In amplifier (SR-830, Stanford Research) (20). We set the phase manually at the beginning of each recording.…”

Section: Molecularmentioning

confidence: 99%

Analysis of SCAMP1 Function in Secretory Vesicle Exocytosis by Means of Gene Targeting in Mice

Fernández‐Chacón¹,

Toledo²,

Hammer³

et al. 1999

Journal of Biological Chemistry

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Secretory carrier membrane proteins (SCAMPs) comprise a family of ubiquitous membrane proteins of transport vesicles with no known function. Their universal presence in all cells suggests a fundamental role in membrane traffic. SCAMPs are particularly highly expressed in organelles that undergo regulated exocytosis, such as synaptic vesicles and mast cell granules. Of the three currently known SCAMPs, SCAMP1 is the most abundant. To investigate the possible functions of SCAMP1, we generated mice that lack SCAMP1. SCAMP1-deficient mice are viable and fertile. They exhibit no changes in the overall architecture or the protein composition of the brain or alterations in peripheral organs. Capacitance measurements in mast cells demonstrated that exocytosis could be triggered reliably by GTP␥S in SCAMP1-deficient cells. The initial overall capacitance of mast cells was similar between wild type and mutant mice, but the final cell capacitance after completion of exocytosis, was significantly smaller in SCAMP1-deficient cells than in wild type cells. Furthermore, there was an increased proportion of reversible fusion events, which may have caused the decrease in the overall capacitance change observed after exocytosis. Our data show that SCAMP1 is not essential for exocytosis, as such, and does not determine the stability or size of secretory vesicles, but is required for the full execution of stable exocytosis in mast cells. This phenotype could be the result of a function of SCAMP1 in the formation of stable fusion pores during exocytosis or of a role of SCAMP1 in the regulation of endocytosis after formation of fusion pores. SCAMPs1 represent a family of membrane proteins present in transport vesicles (1-7). The three SCAMPs that have been identified in mammals (2) are proteins of 35-40 kDa that are composed of an N-terminal cytoplasmic sequence followed by four transmembrane regions and a short cytoplasmic tail. SCAMPs are highly conserved evolutionarily and very homologous to each other. Although they were originally discovered as components of secretory granules in exocrine glands (8), SCAMPs were later found to be present in all cells tested on post-Golgi transport vesicles (3-7). SCAMPs are particularly abundant in vesicles that are subject to regulated exocytosis, such as mast cell granules, exocrine granules, and synaptic vesicles. The three SCAMP proteins are differentially expressed in tissues and are not universally co-expressed in all cells (2). Only one of the three SCAMPs, SCAMP1, appears to be present at high levels on synaptic vesicles that lack SCAMPs 2 and 3 (Refs. 1 and 2 and footnote 2).The function of SCAMPs are unknown. Their ubiquitous presence in transport vesicles in all cells indicates a fundamental role in membrane traffic. The enrichment of SCAMPs in secretory vesicles that are subject to regulated exocytosis followed by rapid endocytosis suggests a function in exo-or endocytosis. Interestingly, SCAMP1 and SCAMP3 are tyrosinephosphorylated in vivo by the epidermal growth factor receptor tyr...

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Time–resolved capacitance measurements: monitoring exocytosis in single cells

Cited by 87 publications

References 72 publications

Exo-endocytosis and closing of the fission pore during endocytosis in single pituitary nerve terminals internally perfused with high calcium concentrations.

Exo-endocytosis and closing of the fission pore during endocytosis in single pituitary nerve terminals internally perfused with high calcium concentrations.

Amino Acid Sequence Requirements of the Transmembrane and Cytoplasmic Domains of Influenza Virus Hemagglutinin for Viable Membrane Fusion

Analysis of SCAMP1 Function in Secretory Vesicle Exocytosis by Means of Gene Targeting in Mice

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