The Experts below are selected from a list of 189 Experts worldwide ranked by ideXlab platform
David Zenisek - One of the best experts on this subject based on the ideXlab platform.
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a Membrane Marker leaves synaptic vesicles in milliseconds after exocytosis in retinal bipolar cells
Neuron, 2002Co-Authors: David Zenisek, Jurgen A Steyer, Morris Feldman, Wolfhard AlmersAbstract:Perhaps synaptic vesicles can recycle so rapidly because they avoid complete exocytosis, and release transmitter through a fusion pore that opens transiently. This view emerges from imaging whole terminals where the fluorescent lipid FM1-43 seems unable to leave vesicles during transmitter release. Here we imaged single, FM1-43-stained synaptic vesicles by evanescent field fluorescence microscopy, and tracked the escape of dye from single vesicles by watching the increase in fluorescence after exocytosis. Dye left rapidly and completely during most or all exocytic events. We conclude that vesicles at this terminal allow lipid exchange soon after exocytosis, and lose their dye even if they connected with the plasma Membrane only briefly. At the level of single vesicles, therefore, observations with FM1-43 provide no evidence that exocytosis of synaptic vesicles is incomplete.
Wolfhard Almers - One of the best experts on this subject based on the ideXlab platform.
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a Membrane Marker leaves synaptic vesicles in milliseconds after exocytosis in retinal bipolar cells
Neuron, 2002Co-Authors: David Zenisek, Jurgen A Steyer, Morris Feldman, Wolfhard AlmersAbstract:Perhaps synaptic vesicles can recycle so rapidly because they avoid complete exocytosis, and release transmitter through a fusion pore that opens transiently. This view emerges from imaging whole terminals where the fluorescent lipid FM1-43 seems unable to leave vesicles during transmitter release. Here we imaged single, FM1-43-stained synaptic vesicles by evanescent field fluorescence microscopy, and tracked the escape of dye from single vesicles by watching the increase in fluorescence after exocytosis. Dye left rapidly and completely during most or all exocytic events. We conclude that vesicles at this terminal allow lipid exchange soon after exocytosis, and lose their dye even if they connected with the plasma Membrane only briefly. At the level of single vesicles, therefore, observations with FM1-43 provide no evidence that exocytosis of synaptic vesicles is incomplete.
Reinhard Sailer - One of the best experts on this subject based on the ideXlab platform.
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Fluorescence lifetime imaging (FLIM) of Membrane Markers in living cells
Confocal Multiphoton and Nonlinear Microscopic Imaging, 2003Co-Authors: Herbert Schneckenburger, Wolfgang S. L. Strauss, Michael Wagner, Martina Kretzschmar, Reinhard SailerAbstract:An experimental setup for fluorescence lifetime imaging (FLIM) has been combined with total internal reflection fluorescence microscopy (TIRFM) in order to detect various Membrane Markers within living cells. The method is established using T47D human breast cancer cells transfected by a plasmid encoding for a Membrane associated yellow fluorescent protein (EYFPmem). For further measurements the mitochondrial Marker rhodamine 123 (R123) as well as the Membrane Marker laurdan are used. With increasing concentration R123 is accumulated outside the mitochondria, in particular within the plasma Membrane, whereas mitochondrial fluorescence is quenched. Fluorescence lifetime of laurdan can be used to probe Membrane dynamics, in particular the phase of Membrane lipids. These lipids are in a rigid gel phase at temperatures around 24°C, whereas the gel phases and a liquid crystalline phase coexist at T ≥ 30°C. This phase pattern also depends on the age and the growth phase of the cells and may play a role in the uptake of pharmaceutical agents.
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Time-gated total internal reflection fluorescence spectroscopy (TG-TIRFS): application to the Membrane Marker laurdan.
Journal of Microscopy, 2003Co-Authors: Herbert Schneckenburger, Karl Stock, Joerg Eickholz, Wolfgang S. L. Strauss, Reinhard SailerAbstract:A novel setup for total internal reflection fluorescence microscopy with spectral and temporal (nanosecond) resolution was used to measure the emission spectra of the Membrane Marker laurdan either selectively within the plasma Membrane or in whole living cells, depending on the incident angle of the excitation light. With increasing temperature, the intensity of the fluorescence band around 490 nm increased in comparison with the band around 440 nm, which has previously been assigned to a phase transition of Membrane lipids from gel to liquid crystalline phase. For a better separation of the overlapping spectral bands, time-gated detection with a delay of 10-15 ns with respect to the exciting laser pulse was used. As a parameter of Membrane dynamics the so-called generalized polarization GP = (I440 - I490)/(I440 + I490) was evaluated at temperatures between 24 and 41 degrees C and variable angles of the incident light permitting to excite laurdan molecules either within the plasma Membrane or in the whole cell. A decrease of the GP values by approximately 0.2 units between 28 and 41 degrees C indicated an increase in Membrane fluidity or a decrease in Membrane stiffness with increasing temperature. In addition, higher GP values were observed for the plasma Membrane as compared with intracellular Membranes, probably due to a higher amount of cholesterol. Because properties of the plasma Membrane have a large influence on the uptake or release of certain pharmaceutical agents or metabolites, the direct assessment of the dynamics of the plasma Membrane by total internal reflection fluorescence spectroscopy appears to be important for pharmacology.
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Time-resolved total internal reflection fluorescence spectroscopy: application to the Membrane Marker laurdan
Laser Microscopy, 2000Co-Authors: Herbert Schneckenburger, Karl Stock, Joerg Eickholz, Wolfgang S. L. Strauss, Marco Lyttek, Reinhard SailerAbstract:A compact device for variable-angle total internal reflection flourescent microscopy was developed. A pulsed Nd:YAG laser operated at 355 nm was adapted using a multimode quartz fiber and collimating optics with a variable angle of incidence between 64 degrees and 72 degrees. Fluorescence spectra of BKEz-7 endothelial cells incubated with the Membrane Marker 6-dodecanoyl-2- dimethylamino-naphthalene were measured under TIR illumination as a function of the angle of incidence, incubation time and temperature. Emission bands around 440 nm and 490 nm were detected corresponding to laurdan locate within the gel phase or liquid crystalline phase of cellular lipids, respectively. The generalized polarization GP = (I440-I490)/(I440+I490) was used as a measure of intracellular temperature with a precision of ±1°C in the physiologically interesting range between 35°C and 38°C. Following pulsed laser excitation, the time delay between excitation and fluorescence detection was varied. A time gate at 10-15 ns after laser excitation revealed to be an optimum for spectral discrimination of the two emission bands. Fluorescence intensity IF of both bands decreased continuously when the angle of incidence Θ was increased. Between Θ = 68° and 72° the angular dependence corresponded to a fluorophore located within a thin layer (plasma Membrane) at 150-200nm distance from the light reflecting surface. Between Θ=65° and 68° additional contributions from intracellular Membranes were observed.© (2000) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.
Regis B. Kelly - One of the best experts on this subject based on the ideXlab platform.
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redistribution of synaptic vesicles and their proteins in temperature sensitive shibire ts1 mutant drosophila
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Justin Van Goor, Mani Ramaswami, Regis B. KellyAbstract:Abstract From an extract of Drosophila melanogaster head homogenates, a Membrane fraction can be isolated that has the same sedimentation properties as vertebrate synaptic vesicles and contains Drosophila synaptotagmin. The fraction disappears from homogenates of temperature-sensitive (ts) mutant shibire(ts1) (shi(ts1)) flies paralyzed by exposure to non-permissive temperatures, and reappears on return to permissive temperatures. Since reversible, temperature-dependent depletion of synaptic vesicles is known to occur in shibire(ts1) flies, we conclude that the fraction we have identified contains synaptic vesicles. We have examined the fate of synaptic vesicle Membrane proteins in shibire flies at nonpermissive temperatures and found that all of these vesicle antigens are transferred to rapidly sedimenting Membranes and codistribute with a plasma Membrane Marker by both glycerol velocity and metrizamide density sedimentation and by confocal microscopy. Three criteria were used to establish that other neuron-specific antigens--neuronal synaptobrevin and cysteine-string proteins--are legitimate components of synaptic vesicles: cosedimentation with Drosophila synaptotagmin, immunoadsorption, and disappearance of these antigens from the vesicle fractions in paralyzed shibire flies.
Bjorn Olsen - One of the best experts on this subject based on the ideXlab platform.
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col2 cre recombinase is co expressed with endogenous type ii collagen in embryonic renal epithelium and drives development of polycystic kidney disease following inactivation of ciliary genes
Matrix Biology, 2008Co-Authors: Elona Kolpakovahart, Claudia Nicolae, Jing Zhou, Bjorn OlsenAbstract:Here we report on the severe defects in renal epithelium induced by the transgenic Col2-Cre line used previously for skeletal tissue-specific gene targeting. We demonstrate that conditional ablation of the Kif3a or Pkd1 genes encoding primary cilium/intraflagellar transport-associated proteins using type II collagen-specific Cre transgenic strain results in a severe form of polycystic kidney disease in mice. We detect Col2-Cre recombinase expression in kidney epithelium, which reflects expression of the endogenous Col1α(II) gene in the embryonic renal tubules. We determine the exon 2-containing splice variant of the Col1α(II) gene as a major transcript expressed in kidney. Furthermore, the confocal immunocytochemical analysis demonstrates deposition of the type II collagen within the mesenchymal–epithelial renal tissue interfaces and its colocalization with the basement Membrane Marker collagen IV during embryonic kidney morphogenesis. © 2008 Elsevier B.V. All rights reserved.