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Kai-uwe Hinrichs - One of the best experts on this subject based on the ideXlab platform.
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Thermococcus kodakarensis modulates its Polar Membrane lipids and elemental composition according to growth stage and phosphate availability
Frontiers in microbiology, 2014Co-Authors: Travis B Meador, Emma J Gagen, Marcos Yukio Yoshinaga, Michael E Loscar, Tobias Goldhammer, Jenny Wendt, Michael Thomm, Kai-uwe HinrichsAbstract:We observed significant changes in the elemental and intact Polar lipid (IPL) composition of the archaeon Thermococcus kodakarensis (KOD1) in response to growth stage and phosphorus supply. Reducing the amount of organic supplements and phosphate in growth media resulted in significant decreases in cell size and cellular quotas of carbon (C), nitrogen (N), and phosphorus (P), which coincided with significant increases in cellular IPL quota and IPLs comprising multiple P atoms and hexose moieties. Relatively more cellular P was stored as IPLs in P-limited cells (2-8%) compared to control cells (
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thermococcus kodakarensis modulates its Polar Membrane lipids and elemental composition according to growth stage and phosphate availability
Frontiers in Microbiology, 2014Co-Authors: Travis B Meador, Emma J Gagen, Marcos Yukio Yoshinaga, Michael E Loscar, Tobias Goldhammer, Jenny Wendt, Michael Thomm, Kai-uwe HinrichsAbstract:We observed significant changes in the elemental and intact Polar lipid (IPL) composition of the archaeon Thermococcus kodakarensis (KOD1) in response to growth stage and phosphorus supply. Reducing the amount of organic supplements and phosphate in growth media resulted in significant decreases in cell size and cellular quotas of carbon (C), nitrogen (N), and phosphorus (P), which coincided with significant increases in cellular IPL quota and IPLs comprising multiple P atoms and hexose moieties. Relatively more cellular P was stored as IPLs in P-limited cells (2-8%) compared to control cells (< 0.8%). We also identified a specific IPL biomarker containing a phosphatidyl-N-acetylhexoseamine headgroup that was relatively enriched during rapid cell division. These observations serve as empirical evidence of IPL adaptations in Archaea that will help to interpret the distribution of these biomarkers in natural systems. The reported cell quotas of C, N, and P represent the first such data for a specific archaeon and suggest that thermophiles are C-rich compared to the cell carbon-to-volume relationship reported for planktonic bacteria.
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factors controlling the distribution of anaerobic methanotrophic communities in marine environments evidence from intact Polar Membrane lipids
Geochimica et Cosmochimica Acta, 2011Co-Authors: Pamela E Rossel, Marcus Elvert, Alban Ramette, Antje Boetius, Kai-uwe HinrichsAbstract:Abstract Three distinct types of microbial consortia appear to mediate the anaerobic oxidation of methane with sulfate as electron acceptor in marine sediments and are distributed ubiquitously. These consortia consist of ANerobic MEthanotrophic (ANME) archaea of the ANME-1, ANME-2 and ANME-3 clades and their sulfate-reducing bacterial partners either of the Desulfosarcina–Desulfococcus (ANME-1/DSS and ANME-2/DSS) or Desulfobulbus spp. (ANME-3/DBB) branches. Frequently one consortium type dominates the community, but the selective factors are not well constrained. Here we analyzed patterns in the composition of intact Polar lipids extracted from bacterial and archaeal communities of different marine seep environments. Further, we investigated if different environmental and geographical factors were responsible for the observed patterns, and hence could be important in the selection of seep communities. Intact Polar lipids (IPLs) provide a more robust distinction of the composition of extant communities than their less Polar derivatives. In ANME-1/DSS-dominated communities, glycosidic- and phospho-glyceroldialkylglyceroltetraethers were abundant, while ANME-2/DSS and ANME-3/DBB-dominated communities showed abundant archaeol-based IPLs, either with glycosidic and phospho-headgroups or only phospho-headgroups, respectively. The relative proportion of bacterial IPLs varied widely from 0% to 93% and was generally lower in samples of the ANME-1 type, suggesting lower bacterial biomasses in the respective communities. In addition to these lipid signatures, distinctive features were related to the habitat characteristics of these communities: lower amounts of phosphate-based IPLs were generally observed in communities from calcified microbial mats compared to sediments, which may reflect phosphate limitation. Based on statistical analyses of IPLs and environmental data this study constrained for the first time the occurrence of three environmental factors controlling the distribution of different ANME-associated communities in a wide range of hydrocarbon seep systems. Habitats dominated by ANME-1/DSS communities were characterized by high temperature and low oxygen content in bottom waters (or even anoxia), while ANME-2/DSS and ANME-3/DBB-dominated sediments were located in settings with lower temperatures and higher oxygen content in bottom waters. Furthermore, ANME-2/DSS communities were particularly prominent in environments in which a relatively high supply of sulfate was sustained.
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Distribution of Polar Membrane lipids in permafrost soils and sediments of a small high Arctic catchment
Organic Geochemistry, 2010Co-Authors: Janet Rethemeyer, Kai-uwe Hinrichs, Florence Schubotz, Helen M. Talbot, Martin P. Cooke, Gesine MollenhauerAbstract:The distribution of bacteriohopanepolyols (BHPs) and intact Polar Membrane lipids (IPLs) was studied in different soil profiles and in fluvial and marine sediments along the pathway of transport in a small catchment in the high Arctic at Ny-Alesund, Svalbard (79°N, 12°E). Both groups of cellular Membrane lipids have been used as biomarkers for determining major microbial groups and biogeochemical processes in natural environments. Little is known about their distribution and usefulness as environmental markers in the Arctic. In this study, we show that both Membrane lipid groups are variably distributed in the drainage area of the Broggerbreen Glacier, with highest abundance and diversity in the organic mat overlying the mineral soils, as well as in mossy surface soil. The BHP and IPL composition and abundance both indicate a larger community of phototrophic bacteria in the organic mats and fluvial sediments. The greater diversity and abundance of BHPs relative to IPLs in sub-soils, including probable markers for cyanobacteria, suggest a greater resistance to degradation. In contrast, IPL concentration decreased strongly with soil depth, pointing to their rapid degradation. Bacterial IPLs are thus thought to reflect more reliably the biomass of viable phototrophic bacteria. The high concentrations of proposed soil marker BHPs, adenosylhopane and related structures vs. published results and their relative increase with soil depth suggest better preservation and more effective accumulation of the compounds at low temperature.
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detection of microbial biomass by intact Polar Membrane lipid analysis in the water column and surface sediments of the black sea
Environmental Microbiology, 2009Co-Authors: Florence Schubotz, Kai-uwe Hinrichs, Stuart G Wakeham, Julius S Lipp, Helen F FredricksAbstract:The stratified water column of the Black Sea produces a vertical succession of redox zones, stimulating microbial activity at the interfaces. Our study of intact Polar Membrane lipids (IPLs) in suspended particulate matter and sediments highlights their potential as biomarkers for assessing the taxonomic composition of live microbial biomass. Intact Polar Membrane lipids in oxic waters above the chemocline represent contributions of bacterial and eukaryotic photosynthetic algae, while anoxygenic phototrophic bacteria and sulfate-reducing bacteria comprise a substantial amount of microbial biomass in deeper suboxic and anoxic layers. Intact Polar Membrane lipids such as betaine lipids and glycosidic ceramides suggest unspecified anaerobic bacteria in the anoxic zone. Distributions of Polar head groups and core lipids show planktonic archaea below the oxic zone; methanotrophic archaea are only a minor fraction of archaeal biomass in the anoxic zone, contrasting previous observations based on the aPolar derivatives of archaeal lipids. Sediments contain algal and bacterial IPLs from the water column, but transport to the sediment is selective; bacterial and archaeal IPLs are also produced within the sediments. Intact Polar Membrane lipid distributions in the Black Sea are stratified in accordance with geochemical profiles and provide information on vertical successions of major microbial groups contributing to suspended biomass. This study vastly extends our knowledge of the distribution of complex microbial lipids in the ocean.
Haruo Kasai - One of the best experts on this subject based on the ideXlab platform.
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Sequential compound exocytosis of large dense‐core vesicles in PC12 cells studied with TEPIQ (two‐photon extracellular Polar‐tracer imaging‐based quantification) analysis
The Journal of Physiology, 2005Co-Authors: Takuya Kishimoto, Ting-ting Liu, Hiroyasu Hatakeyama, Tomomi Nemoto, Noriko Takahashi, Haruo KasaiAbstract:We investigated exocytosis of PC12 cells using two-photon excitation imaging and extracellular Polar tracers (TEP imaging) at the basal region of PC12 cells adjacent to the glass cover slip. TEPIQ (two-photon extracellular Polar-tracer imaging-based quantification) analysis revealed that most exocytosis was mediated by large dense-core vesicles (LVs) with a mean diameter of 220 nm, and that exocytosis of LVs occurred slowly with a mean latency of ∼7 s even though exocytosis was induced with large increases in cytosolic Ca2+ concentration by uncaging of a caged-Ca2+ compound. We also found that 97% of exocytic LVs remained poised at the plasma Membrane, 72% maintained their fusion pores in an open conformation for more than 30 s, and 76% triggered sequential compound exocytosis of vesicles that were located deeper in the cytosol. Sequential compound exocytosis by PC12 cells was confirmed by electron microscopic investigation with photoconversion of diaminobenzidine by FM1-43 (a Polar Membrane tracer). Our data suggest that pre-stimulus docking of LVs to the plasma Membrane does not necessarily hasten the fusion reaction, while docking and resulting stability of exocytic LVs facilitates sequential compound exocytosis, and thereby allowing mobilization of deep vesicles.
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Sequential compound exocytosis of large dense-core vesicles in PC12 cells studied with TEPIQ (two-photon extracellular Polar-tracer imaging-based quantification) analysis.
The Journal of physiology, 2005Co-Authors: Takuya Kishimoto, Ting-ting Liu, Hiroyasu Hatakeyama, Tomomi Nemoto, Noriko Takahashi, Haruo KasaiAbstract:We investigated exocytosis of PC12 cells using two-photon excitation imaging and extracellular Polar tracers (TEP imaging) at the basal region of PC12 cells adjacent to the glass cover slip. TEPIQ (two-photon extracellular Polar-tracer imaging-based quantification) analysis revealed that most exocytosis was mediated by large dense-core vesicles (LVs) with a mean diameter of 220 nm, and that exocytosis of LVs occurred slowly with a mean latency of approximately 7 s even though exocytosis was induced with large increases in cytosolic Ca2+ concentration by uncaging of a caged-Ca2+ compound. We also found that 97% of exocytic LVs remained poised at the plasma Membrane, 72% maintained their fusion pores in an open conformation for more than 30 s, and 76% triggered sequential compound exocytosis of vesicles that were located deeper in the cytosol. Sequential compound exocytosis by PC12 cells was confirmed by electron microscopic investigation with photoconversion of diaminobenzidine by FM1-43 (a Polar Membrane tracer). Our data suggest that pre-stimulus docking of LVs to the plasma Membrane does not necessarily hasten the fusion reaction, while docking and resulting stability of exocytic LVs facilitates sequential compound exocytosis, and thereby allowing mobilization of deep vesicles.
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A new quantitative (two-photon extracellular Polar-tracer imaging-based quantification (TEPIQ)) analysis for diameters of exocytic vesicles and its application to mouse pancreatic islets.
The Journal of physiology, 2005Co-Authors: Haruo Kasai, Takuya Kishimoto, Ting-ting Liu, Hiroyasu Hatakeyama, Tomomi Nemoto, Noriko TakahashiAbstract:We have developed an imaging approach to estimate the diameter of exocytic vesicles that are smaller than the resolution of an optical microscope and present within intact tissue. This approach is based on two-photon excitation imaging of Polar tracers in the extracellular medium, is designated TEPIQ (two-photon extracellular Polar-tracer imaging-based quantification), and has three variants. TEPIQ analysis of DeltaV measures vesicle volume with a fluid-phase tracer, sulforhodamine B (SRB). TEPIQ analysis of DeltaS determines vesicle surface area with a Polar Membrane tracer, FM1-43. TEPIQ analysis of DeltaV/DeltaS estimates vesicle diameter from the SRB/FM1-43 fluorescence ratio. TEPIQ analysis is insensitive to microscope settings because the same setup is used for calibration and actual experiments. We tested the validity of TEPIQ with glucose-induced exocytosis from beta-cells within pancreatic islets. The three TEPIQ variants yielded estimates for the mean diameter of exocytic vesicles of between 340 and 390 nm, consistent with the size of insulin granules. TEPIQ analysis relies on the combination of two-photon excitation imaging, the narrow intercellular spaces of intact tissue, and the presence of diffusible Polar tracers in the extracellular medium. It allows quantitative imaging of exocytosis within secretory organs, yielding estimates of vesicle diameter with nanometer resolution.
Noriko Takahashi - One of the best experts on this subject based on the ideXlab platform.
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Sequential compound exocytosis of large dense‐core vesicles in PC12 cells studied with TEPIQ (two‐photon extracellular Polar‐tracer imaging‐based quantification) analysis
The Journal of Physiology, 2005Co-Authors: Takuya Kishimoto, Ting-ting Liu, Hiroyasu Hatakeyama, Tomomi Nemoto, Noriko Takahashi, Haruo KasaiAbstract:We investigated exocytosis of PC12 cells using two-photon excitation imaging and extracellular Polar tracers (TEP imaging) at the basal region of PC12 cells adjacent to the glass cover slip. TEPIQ (two-photon extracellular Polar-tracer imaging-based quantification) analysis revealed that most exocytosis was mediated by large dense-core vesicles (LVs) with a mean diameter of 220 nm, and that exocytosis of LVs occurred slowly with a mean latency of ∼7 s even though exocytosis was induced with large increases in cytosolic Ca2+ concentration by uncaging of a caged-Ca2+ compound. We also found that 97% of exocytic LVs remained poised at the plasma Membrane, 72% maintained their fusion pores in an open conformation for more than 30 s, and 76% triggered sequential compound exocytosis of vesicles that were located deeper in the cytosol. Sequential compound exocytosis by PC12 cells was confirmed by electron microscopic investigation with photoconversion of diaminobenzidine by FM1-43 (a Polar Membrane tracer). Our data suggest that pre-stimulus docking of LVs to the plasma Membrane does not necessarily hasten the fusion reaction, while docking and resulting stability of exocytic LVs facilitates sequential compound exocytosis, and thereby allowing mobilization of deep vesicles.
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Sequential compound exocytosis of large dense-core vesicles in PC12 cells studied with TEPIQ (two-photon extracellular Polar-tracer imaging-based quantification) analysis.
The Journal of physiology, 2005Co-Authors: Takuya Kishimoto, Ting-ting Liu, Hiroyasu Hatakeyama, Tomomi Nemoto, Noriko Takahashi, Haruo KasaiAbstract:We investigated exocytosis of PC12 cells using two-photon excitation imaging and extracellular Polar tracers (TEP imaging) at the basal region of PC12 cells adjacent to the glass cover slip. TEPIQ (two-photon extracellular Polar-tracer imaging-based quantification) analysis revealed that most exocytosis was mediated by large dense-core vesicles (LVs) with a mean diameter of 220 nm, and that exocytosis of LVs occurred slowly with a mean latency of approximately 7 s even though exocytosis was induced with large increases in cytosolic Ca2+ concentration by uncaging of a caged-Ca2+ compound. We also found that 97% of exocytic LVs remained poised at the plasma Membrane, 72% maintained their fusion pores in an open conformation for more than 30 s, and 76% triggered sequential compound exocytosis of vesicles that were located deeper in the cytosol. Sequential compound exocytosis by PC12 cells was confirmed by electron microscopic investigation with photoconversion of diaminobenzidine by FM1-43 (a Polar Membrane tracer). Our data suggest that pre-stimulus docking of LVs to the plasma Membrane does not necessarily hasten the fusion reaction, while docking and resulting stability of exocytic LVs facilitates sequential compound exocytosis, and thereby allowing mobilization of deep vesicles.
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A new quantitative (two-photon extracellular Polar-tracer imaging-based quantification (TEPIQ)) analysis for diameters of exocytic vesicles and its application to mouse pancreatic islets.
The Journal of physiology, 2005Co-Authors: Haruo Kasai, Takuya Kishimoto, Ting-ting Liu, Hiroyasu Hatakeyama, Tomomi Nemoto, Noriko TakahashiAbstract:We have developed an imaging approach to estimate the diameter of exocytic vesicles that are smaller than the resolution of an optical microscope and present within intact tissue. This approach is based on two-photon excitation imaging of Polar tracers in the extracellular medium, is designated TEPIQ (two-photon extracellular Polar-tracer imaging-based quantification), and has three variants. TEPIQ analysis of DeltaV measures vesicle volume with a fluid-phase tracer, sulforhodamine B (SRB). TEPIQ analysis of DeltaS determines vesicle surface area with a Polar Membrane tracer, FM1-43. TEPIQ analysis of DeltaV/DeltaS estimates vesicle diameter from the SRB/FM1-43 fluorescence ratio. TEPIQ analysis is insensitive to microscope settings because the same setup is used for calibration and actual experiments. We tested the validity of TEPIQ with glucose-induced exocytosis from beta-cells within pancreatic islets. The three TEPIQ variants yielded estimates for the mean diameter of exocytic vesicles of between 340 and 390 nm, consistent with the size of insulin granules. TEPIQ analysis relies on the combination of two-photon excitation imaging, the narrow intercellular spaces of intact tissue, and the presence of diffusible Polar tracers in the extracellular medium. It allows quantitative imaging of exocytosis within secretory organs, yielding estimates of vesicle diameter with nanometer resolution.
Hiroshi Naraoka - One of the best experts on this subject based on the ideXlab platform.
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simultaneous total analysis of core and Polar Membrane lipids in archaea by high performance liquid chromatography high resolution mass spectrometry coupled with heated electrospray ionization
Rapid Communications in Mass Spectrometry, 2019Co-Authors: Shunsuke Horai, Noriaki Yamauchi, Hiroshi NaraokaAbstract:RATIONALE Archaea have characteristic Membrane lipids including diether and/or tetraether isoprenoidal core lipids with various Polar head groups. Since the Polar group is removed soon after the end of archaeal activity, the occurrences of core and Polar lipids are regarded as dead and active signals, respectively. The core and Polar lipids have generally been analyzed separately using atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI), respectively, coupled with mass spectrometry. METHODS In this study, simultaneous analyses of core and Polar archaeal lipids have been examined using heated electrospray ionization (HESI) by high-performance liquid chromatography/high-resolution mass spectrometry (HPLC/HRMS). RESULTS Both core and intact Polar lipids can be analyzed simultaneously by HESI with good sensitivity (sub ng to 100 ng) and separation using a semi-bore diol column by normal-phase chromatography. The core lipids eluted firstly to separate archeaol, then glycerol dibiphytanyl glycerol tetraethers (GDGTs), followed by the Polar lipids with glycosides and glycophosphates. The relative GDGT composition is identical between HESI and APCI methods. CONCLUSIONS The simultaneous analysis has the benefit of minimizing sample amount and elution solvent as well as preparation work. The method can also be applied to a compound class fractionation for compound-specific carbon and hydrogen isotope analysis.
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Simultaneous total analysis of core and Polar Membrane lipids in archaea by high-performance liquid chromatography/high-resolution mass spectrometry coupled with heated electrospray ionization
Rapid communications in mass spectrometry : RCM, 2019Co-Authors: Shunsuke Horai, Noriaki Yamauchi, Hiroshi NaraokaAbstract:RATIONALE Archaea have characteristic Membrane lipids including diether and/or tetraether isoprenoidal core lipids with various Polar head groups. Since the Polar group is removed soon after the end of archaeal activity, the occurrences of core and Polar lipids are regarded as dead and active signals, respectively. The core and Polar lipids have generally been analyzed separately using atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI), respectively, coupled with mass spectrometry. METHODS In this study, simultaneous analyses of core and Polar archaeal lipids have been examined using heated electrospray ionization (HESI) by high-performance liquid chromatography/high-resolution mass spectrometry (HPLC/HRMS). RESULTS Both core and intact Polar lipids can be analyzed simultaneously by HESI with good sensitivity (sub ng to 100 ng) and separation using a semi-bore diol column by normal-phase chromatography. The core lipids eluted firstly to separate archeaol, then glycerol dibiphytanyl glycerol tetraethers (GDGTs), followed by the Polar lipids with glycosides and glycophosphates. The relative GDGT composition is identical between HESI and APCI methods. CONCLUSIONS The simultaneous analysis has the benefit of minimizing sample amount and elution solvent as well as preparation work. The method can also be applied to a compound class fractionation for compound-specific carbon and hydrogen isotope analysis.
Takuya Kishimoto - One of the best experts on this subject based on the ideXlab platform.
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Sequential compound exocytosis of large dense‐core vesicles in PC12 cells studied with TEPIQ (two‐photon extracellular Polar‐tracer imaging‐based quantification) analysis
The Journal of Physiology, 2005Co-Authors: Takuya Kishimoto, Ting-ting Liu, Hiroyasu Hatakeyama, Tomomi Nemoto, Noriko Takahashi, Haruo KasaiAbstract:We investigated exocytosis of PC12 cells using two-photon excitation imaging and extracellular Polar tracers (TEP imaging) at the basal region of PC12 cells adjacent to the glass cover slip. TEPIQ (two-photon extracellular Polar-tracer imaging-based quantification) analysis revealed that most exocytosis was mediated by large dense-core vesicles (LVs) with a mean diameter of 220 nm, and that exocytosis of LVs occurred slowly with a mean latency of ∼7 s even though exocytosis was induced with large increases in cytosolic Ca2+ concentration by uncaging of a caged-Ca2+ compound. We also found that 97% of exocytic LVs remained poised at the plasma Membrane, 72% maintained their fusion pores in an open conformation for more than 30 s, and 76% triggered sequential compound exocytosis of vesicles that were located deeper in the cytosol. Sequential compound exocytosis by PC12 cells was confirmed by electron microscopic investigation with photoconversion of diaminobenzidine by FM1-43 (a Polar Membrane tracer). Our data suggest that pre-stimulus docking of LVs to the plasma Membrane does not necessarily hasten the fusion reaction, while docking and resulting stability of exocytic LVs facilitates sequential compound exocytosis, and thereby allowing mobilization of deep vesicles.
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Sequential compound exocytosis of large dense-core vesicles in PC12 cells studied with TEPIQ (two-photon extracellular Polar-tracer imaging-based quantification) analysis.
The Journal of physiology, 2005Co-Authors: Takuya Kishimoto, Ting-ting Liu, Hiroyasu Hatakeyama, Tomomi Nemoto, Noriko Takahashi, Haruo KasaiAbstract:We investigated exocytosis of PC12 cells using two-photon excitation imaging and extracellular Polar tracers (TEP imaging) at the basal region of PC12 cells adjacent to the glass cover slip. TEPIQ (two-photon extracellular Polar-tracer imaging-based quantification) analysis revealed that most exocytosis was mediated by large dense-core vesicles (LVs) with a mean diameter of 220 nm, and that exocytosis of LVs occurred slowly with a mean latency of approximately 7 s even though exocytosis was induced with large increases in cytosolic Ca2+ concentration by uncaging of a caged-Ca2+ compound. We also found that 97% of exocytic LVs remained poised at the plasma Membrane, 72% maintained their fusion pores in an open conformation for more than 30 s, and 76% triggered sequential compound exocytosis of vesicles that were located deeper in the cytosol. Sequential compound exocytosis by PC12 cells was confirmed by electron microscopic investigation with photoconversion of diaminobenzidine by FM1-43 (a Polar Membrane tracer). Our data suggest that pre-stimulus docking of LVs to the plasma Membrane does not necessarily hasten the fusion reaction, while docking and resulting stability of exocytic LVs facilitates sequential compound exocytosis, and thereby allowing mobilization of deep vesicles.
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A new quantitative (two-photon extracellular Polar-tracer imaging-based quantification (TEPIQ)) analysis for diameters of exocytic vesicles and its application to mouse pancreatic islets.
The Journal of physiology, 2005Co-Authors: Haruo Kasai, Takuya Kishimoto, Ting-ting Liu, Hiroyasu Hatakeyama, Tomomi Nemoto, Noriko TakahashiAbstract:We have developed an imaging approach to estimate the diameter of exocytic vesicles that are smaller than the resolution of an optical microscope and present within intact tissue. This approach is based on two-photon excitation imaging of Polar tracers in the extracellular medium, is designated TEPIQ (two-photon extracellular Polar-tracer imaging-based quantification), and has three variants. TEPIQ analysis of DeltaV measures vesicle volume with a fluid-phase tracer, sulforhodamine B (SRB). TEPIQ analysis of DeltaS determines vesicle surface area with a Polar Membrane tracer, FM1-43. TEPIQ analysis of DeltaV/DeltaS estimates vesicle diameter from the SRB/FM1-43 fluorescence ratio. TEPIQ analysis is insensitive to microscope settings because the same setup is used for calibration and actual experiments. We tested the validity of TEPIQ with glucose-induced exocytosis from beta-cells within pancreatic islets. The three TEPIQ variants yielded estimates for the mean diameter of exocytic vesicles of between 340 and 390 nm, consistent with the size of insulin granules. TEPIQ analysis relies on the combination of two-photon excitation imaging, the narrow intercellular spaces of intact tissue, and the presence of diffusible Polar tracers in the extracellular medium. It allows quantitative imaging of exocytosis within secretory organs, yielding estimates of vesicle diameter with nanometer resolution.