Bernard Katz, quantal transmitter release and the foundations of presynaptic physiology.
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Action Potentials↗
Biomedical subjects
Haruo Kasai
Publications and source records attributed to Haruo Kasai.
Two-photon excitation imaging of exocytosis and endocytosis and determination of their spatial organization.
Two-photon excitation imaging is the least invasive optical approach to study living tissues. We have established two-photon extracellular polar-tracer (TEP) imaging with which it is possible to visualize and quantify all exocytic events in the plane of focus within secretory tissues. This technology also enables estimate of the precise diameters of vesicles independently of the spatial resolution of the optical microscope, and determination of the fusion pore dynamics at nanometer resolution using TEP-imaging based quantification (TEPIQ). TEP imaging has been applied to representative secretory glands, e.g., exocrine pancreas, endocrine pancreas, adrenal medulla and a pheochromocytoma cell line (PC12), and has revealed unexpected diversity in the spatial organization of exocytosis and endocytosis crucial for the physiology and pathology of secretory tissues and neurons. TEP imaging and TEPIQ analysis are powerful tools for elucidating the molecular and cellular mechanisms of exocytosis and certain related diseases, such as diabetes mellitus, and the development of new therapeutic agents and diagnostic tools.
Animals↗
Vacuolar sequential exocytosis of large dense-core vesicles in adrenal medulla.
Individual exocytic events in intact adrenal medulla were visualized by two-photon extracellular polar-tracer imaging. Exocytosis of chromaffin vesicles often occurred in a sequential manner, involving first vesicles located at the cell periphery and then those present deeper within the cytoplasm. Sequential exocytosis occurred preferentially at regions of the plasma membrane facing the intercellular space. The compound vesicles swelled to more than five times their original volume and formed vacuolar exocytic lumens as a result of expansion of intravesicular gels and their confinement within the lumen by the fusion pore and the narrow intercellular space. Such luminal swelling greatly promoted sequential exocytosis. The SNARE protein SNAP25 rapidly migrated from the plasma membrane to the membrane of fused vesicles. These data indicate that vesicles present deeper within the cytoplasm can be fusion ready like those at the cell periphery, and that swelling of exocytic lumens promotes assembly of the fusion machinery. We suggest the existence of two molecular configurations for fusion-ready states in Ca2+ -dependent exocytosis.
Adrenal Medulla↗
Hepatocyte nuclear factor-4alpha is essential for glucose-stimulated insulin secretion by pancreatic beta-cells.
Mutations in the hepatocyte nuclear factor (HNF)-4alpha gene cause a form of maturity-onset diabetes of the young (MODY1) that is characterized by impairment of glucose-stimulated insulin secretion by pancreatic beta-cells. HNF-4alpha, a transcription factor belonging to the nuclear receptor superfamily, is expressed in pancreatic islets as well as in the liver, kidney, and intestine. However, the role of HNF-4alpha in pancreatic beta-cell is unclear. To clarify the role of HNF-4alpha in beta-cells, we generated beta-cell-specific HNF-4alpha knock-out (betaHNF-4alphaKO) mice using the Cre-LoxP system. The betaHNF-4alphaKO mice exhibited impairment of glucose-stimulated insulin secretion, which is a characteristic of MODY1. Pancreatic islet morphology, beta-cell mass, and insulin content were normal in the HNF-4alpha mutant mice. Insulin secretion by betaHNF-4alphaKO islets and the intracellular calcium response were impaired after stimulation by glucose or sulfonylurea but were normal after stimulation with KCl or arginine. Both NAD(P)H generation and ATP content at high glucose concentrations were normal in the betaHNF-4alphaKO mice. Expression levels of Kir6.2 and SUR1 proteins in the betaHNF-4alphaKO mice were unchanged as compared with control mice. Patch clamp experiments revealed that the current density was significantly increased in betaHNF-4alphaKO mice compared with control mice. These results are suggestive of the dysfunction of K(ATP) channel activity in the pancreatic beta-cells of HNF-4alpha-deficient mice. Because the K(ATP) channel is important for proper insulin secretion in beta-cells, altered K(ATP) channel activity could be related to the impaired insulin secretion in the betaHNF-4alphaKO mice.
ATP-Binding Cassette Transporters↗
Rapid glucose sensing by protein kinase A for insulin exocytosis in mouse pancreatic islets.
The role of protein kinase A (PKA) in insulin exocytosis was investigated with the use of two-photon excitation imaging of mouse islets of Langerhans. Inhibitors of PKA selectively reduced the number of exocytic events during the initial period (< 250 s) of the first phase of glucose-induced exocytosis (GIE), without affecting the second phase, in intact islets or small clusters of islet cells. The PKA inhibitors did not reduce the extent of the glucose-induced increase in [Ca(2+)](i). The actions of glucose and PKA in Ca(2+)-induced insulin exocytosis (CIE) triggered by photolysis of a caged-Ca(2+) compound, which resulted in large increases in [Ca(2+)](i) and thereby bypassed the ATP-sensitive K(+) channel-dependent mechanism of glucose sensing, were therefore studied. A high concentration (20 mM) of glucose potentiated CIE within 1 min, and this effect was blocked by inhibitors of PKA. This PKA-dependent action of glucose required glucose metabolism, given that increasing the intracellular concentration of cAMP by treatment with forskolin potentiated CIE only at the high glucose concentration. Finally, PKA appeared to reduce the frequency of 'kiss-and-run' exocytic events and to promote full-fusion events during GIE. These data indicate that a PKA-dependent mechanism of glucose sensing, which is operative even at the basal level of PKA activity, plays an important role specifically in the first phase of GIE, and they suggest that the action of PKA is mediated at the level of the fusion reaction.
Animals↗
Genetically encoded bright Ca2+ probe applicable for dynamic Ca2+ imaging of dendritic spines.
G-CaMP is a Ca2+ probe based on a single green fluorescent protein (GFP). G-CaMP shows a large fluorescence increase upon Ca2+ binding, but its fluorescence is dim and pH sensitive, similar to other single GFP-based probes. Here we report an improved G-CaMP, named G-CaMP1.6, which enables easier detection of intracellular Ca2+ signals. G-CaMP1.6 was approximately 40 times more fluorescent than G-CaMP, mainly due to an increase in quantum yield. Furthermore, compared with G-CaMP, G-CaMP1.6 had not only a lower pH sensitivity but also a higher selectivity for divalent cations having an ionic radius similar to Ca2+. Ca2+ sensitivity of G-CaMP1.6 (Kd = 146 nM, Hill coefficient = 3.8, Fmax/Fmin = 4.9) was slightly shifted toward higher affinity compared with that of G-CaMP. When expressed in mammalian cells, G-CaMP1.6 showed large fluorescence changes with drug applications. Notably, local Ca2+ changes in such tiny structures as dendritic spines of neurons were successfully observed with G-CaMP1.6, this being the first observation using a GFP-based probe. Additional mutations in Ca2+-binding sites of G-CaMP1.6 shifted the affinity for Ca2+ and reduced the Ca2+-buffering effect. G-CaMP1.6-CaM(E140K), which has a mutation in the Ca2+ binding site, is an improved probe with its increased brightness and reduced Ca2+-buffering capacity.
Animals↗
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.
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.
Anatomy, Cross-Sectional↗
Sequential compound exocytosis of large dense-core vesicles in PC12 cells studied with TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis.
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.
Anatomy, Cross-Sectional↗
Exocytosis and endocytosis of small vesicles in PC12 cells studied with TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis.
We investigated exocytosis of PC12 cells using two-photon excitation imaging and extracellular polar tracers (TEP imaging) in the lateral membranes not facing the glass-cover slip. Upon photolysis of a caged Ca2+ compound, TEP imaging with FM1-43 (a polar membrane tracer) detected massive exocytosis of vesicles with a time constant of about 1 s. TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis revealed that the diameter of vesicles was small (55 nm). Extensive exocytosis of small vesicles (SVs) was shown to be mediated by the transient opening of a fusion pore with a diameter less than about 1.6 nm, and to be followed by direct ('kiss-and-run') endocytosis and translocation of the endocytic vesicles (EVs) deep into the cytoplasm. These processes were unaffected by GTP-gamma-S. In contrast, constitutive endocytic vesicles exhibited a diameter of 90 nm, took up molecules with a diameter of > 12 nm, and their formation was blocked by GTP-gamma-S. Electron-microscopic investigation with photoconversion of diaminobenzidine using FM1-43 confirmed an abundance of EVs with a diameter of 54 nm in stimulated cells. They rapidly translocated into the cytosol, and fused with endosomal organelles. The number of SV exocytosis events vastly exceeded the number of SVs morphologically docked at the plasma membrane. Simultaneous capacitance and FM1-43 measurements indicated that TEP imaging detected most SV exocytosis, and the fusion pore was closed within 2 s. Thus, we have, for the first time, directly visualized massive exocytosis of small vesicles in a non-synaptic preparation, and have revealed their fusion-pore mediated exocytosis and endocytosis.
Anatomy, Cross-Sectional↗
Spine-neck geometry determines NMDA receptor-dependent Ca2+ signaling in dendrites.
Increases in cytosolic Ca2+ concentration ([Ca2+]i) mediated by NMDA-sensitive glutamate receptors (NMDARs) are important for synaptic plasticity. We studied a wide variety of dendritic spines on rat CA1 pyramidal neurons in acute hippocampal slices. Two-photon uncaging and Ca2+ imaging revealed that NMDAR-mediated currents increased with spine-head volume and that even the smallest spines contained a significant number of NMDARs. The fate of Ca2+ that entered spine heads through NMDARs was governed by the shape (length and radius) of the spine neck. Larger spines had necks that permitted greater efflux of Ca2+ into the dendritic shaft, whereas smaller spines manifested a larger increase in [Ca2+]i within the spine compartment as a result of a smaller Ca2+ flux through the neck. Spine-neck geometry is thus an important determinant of spine Ca2+ signaling, allowing small spines to be the preferential sites for isolated induction of long-term potentiation.
Animals↗
Number and density of AMPA receptors in single synapses in immature cerebellum.
The number of ionotropic receptors in synapses is an essential factor for determining the efficacy of fast transmission. We estimated the number of functional AMPA receptors at single postsynaptic sites by a combination of two-photon uncaging of glutamate and the nonstationary fluctuation analysis in immature rat Purkinje cells (PCs), which receive a single type of excitatory input from climbing fibers. Areas of postsynaptic membrane specialization at the recorded synapses were measured by reconstruction of serial ultrathin sections. The number of functional AMPA receptors was proportional to the synaptic area with a density of approximately 1280 receptors/microm2. Moreover, highly sensitive freeze-fracture replica labeling revealed a homogeneous density of immunogold particles for AMPA receptors in synaptic sites (910 +/- 36 particles/microm2) and much lower density in extrasynaptic sites (19 +/- 2 particles/microm2) in the immature PCs. Our results indicate that in this developing synapse, the efficacy of transmission is determined by the synaptic area.
Animals↗
Rapid Ca2+-dependent increase in oxygen consumption by mitochondria in single mammalian central neurons.
Oxygen consumption increases within a fraction of a second after the onset of neuronal activity, a phenomenon referred to as the "initial dip" in functional imaging studies of the living brain. The cellular mechanism that underlies this rapid increase in oxygen consumption has remained unclear, however. We have now used two-photon excitation imaging to characterize rapid activity-dependent mitochondrial responses in single neurons. This approach allowed simultaneous multicolor imaging of individual mitochondria in single mouse Purkinje neurons in culture. Mitochondrial depolarization was induced immediately when the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) exceeded 15 microM and was associated with oxidation of mitochondrial NAD(P)H, suggesting that Ca(2+)-induced mitochondrial depolarization mediated by the Ca(2+) uniporter directly facilitated oxidation of NAD(P)H. With the use of a miniature oxygen electrode, we detected a burst of oxygen consumption within 0.2s after the onset of cell depolarization in single Purkinje neurons, and this rapid increase in oxygen consumption was dependent on the increase in [Ca(2+)](i). We have thus demonstrated a rapid Ca(2+)-dependent consumption of oxygen that is mediated by mitochondrial depolarization in mammalian central neurons. This process might function as a rapid feed-forward mechanism in homeostatic control of the cytosolic ATP concentration.
Animals↗
Two-photon microscopic analysis of acetylcholine-induced mucus secretion in guinea pig nasal glands.
The spatiotemporal changes in intracellular free Ca(2+) concentration ([Ca(2+)](i)) as well as fluid secretion and exocytosis induced by acetylcholine (ACh) in intact acini of guinea pig nasal glands were investigated by two-photon excitation imaging. Cross-sectional images of acini loaded with the fluorescent Ca(2+) indicator fura-2 revealed that the ACh-evoked increase in [Ca(2+)](i) was immediate and spread from the apical region (the secretory pole) of acinar cells to the basal region. Immersion of acini in a solution containing a fluorescent polar tracer, sulforhodamine B (SRB), revealed that fluid secretion, detected as a rapid disappearance of SRB fluorescence from the extracellular space, occurred exclusively in the luminal region and was accompanied by a reduction in acinar cell volume. Individual exocytic events were also visualized with SRB as the formation of Omega-shaped profiles at the apical membrane. In contrast to the rapidity of fluid secretion, exocytosis of secretory granules occurred with a delay of approximately 70s relative to the increase in [Ca(2+)](i). Exocytic events also occurred deep within the cytoplasm in a sequential manner with the latency of secondary exocytosis being greatly reduced compared with that of primary exocytosis. The delay in sequential compound exocytosis relative to fluid secretion may be important for release of the viscous contents of secretory granules into the nasal cavity.
Acetylcholine↗
The HNF-1 target collectrin controls insulin exocytosis by SNARE complex formation.
Defective glucose-stimulated insulin secretion is the main cause of hyperglycemia in type 2 diabetes mellitus. Mutations in HNF-1alpha cause a monogenic form of type 2 diabetes, maturity-onset diabetes of the young (MODY), characterized by impaired insulin secretion. Here we report that collectrin, a recently cloned kidney-specific gene of unknown function, is a target of HNF-1alpha in pancreatic beta cells. Expression of collectrin was decreased in the islets of HNF-1alpha (-/-) mice, but was increased in obese hyperglycemic mice. Overexpression of collectrin in rat insulinoma INS-1 cells or in the beta cells of transgenic mice enhanced glucose-stimulated insulin exocytosis, without affecting Ca(2+) influx. Conversely, suppression of collectrin attenuated insulin secretion. Collectrin bound to SNARE complexes by interacting with snapin, a SNAP-25 binding protein, and facilitated SNARE complex formation. Therefore, collectrin is a regulator of SNARE complex function, which thereby controls insulin exocytosis.
Animals↗
Rab27a mediates the tight docking of insulin granules onto the plasma membrane during glucose stimulation.
The monomeric small GTPase Rab27a is specifically localized on both secretory granules and lysosome-related organelles. Although natural mutations of the Rab27a gene in human Griscelli syndrome and in ashen mice cause partial albinism and immunodeficiency reflecting the dysfunction of lysosome-related organelles, phenotypes resulting from the defective exocytosis of secretory granules have not been reported. To explore the roles of Rab27a in secretory granules, we analyzed insulin secretion profiles in ashen mice. Ashen mice showed glucose intolerance after a glucose load without signs of insulin resistance in peripheral tissues or insulin deficiency in the pancreas. Insulin secretion from isolated islets was decreased specifically in response to high glucose concentrations but not other nonphysiological secretagogues such as high K+ concentrations, forskolin, or phorbol ester. Neither the intracellular Ca2+ concentration nor the dynamics of fusion pore opening after glucose stimulation were altered. There were, however, marked reductions in the exocytosis from insulin granules predocked on the plasma membrane and in the replenishment of docked granules during glucose stimulation. These results provide the first genetic evidence to our knowledge for the role of Rab27a in the exocytosis of secretory granules and suggest that the Rab27a/effector system mediates glucose-specific signals for the exocytosis of insulin granules in pancreatic beta cells.
Albinism↗
Structural basis of long-term potentiation in single dendritic spines.
Dendritic spines of pyramidal neurons in the cerebral cortex undergo activity-dependent structural remodelling that has been proposed to be a cellular basis of learning and memory. How structural remodelling supports synaptic plasticity, such as long-term potentiation, and whether such plasticity is input-specific at the level of the individual spine has remained unknown. We investigated the structural basis of long-term potentiation using two-photon photolysis of caged glutamate at single spines of hippocampal CA1 pyramidal neurons. Here we show that repetitive quantum-like photorelease (uncaging) of glutamate induces a rapid and selective enlargement of stimulated spines that is transient in large mushroom spines but persistent in small spines. Spine enlargement is associated with an increase in AMPA-receptor-mediated currents at the stimulated synapse and is dependent on NMDA receptors, calmodulin and actin polymerization. Long-lasting spine enlargement also requires Ca2+/calmodulin-dependent protein kinase II. Our results thus indicate that spines individually follow Hebb's postulate for learning. They further suggest that small spines are preferential sites for long-term potentiation induction, whereas large spines might represent physical traces of long-term memory.
Actins↗
Stabilization of exocytosis by dynamic F-actin coating of zymogen granules in pancreatic acini.
Reorganization of F-actin in the apical region of mouse pancreatic acinar cells during Ca(2+)-dependent exocytosis of zymogen granules was investigated by two-photon excitation microscopy with intact acini. Granules were rapidly coated with F-actin in response to either agonist stimulation or photolysis of a caged-Ca(2+) compound. Such F-actin coating occurred exclusively at the surface of granules undergoing exocytosis and was prevented either by latrunculin-A, which inhibits actin polymerization, or by Clostridium botulinum exoenzyme C3, which inhibits the small GTPase Rho. Latrunculin-A or exoenzyme C3 also triggered the formation of vacuoles in acinar cells, a characteristic of acute pancreatitis. Stimulation of acini with high concentrations of cholecystokinin, which cause acute pancreatitis in mice, also impaired the F-actin coating of granules and induced vacuole formation. Latrunculin-A reduced the latency to exocytosis but did not affect the total number of exocytic events, suggesting that F-actin slows and further stabilizes exocytosis by facilitating F-actin coating. Rho-dependent F-actin coating of granule membranes thus stabilizes exocytic structures and is necessary for physiological progression of sequetial compound exocytosis in the exocrine pancreas and for prevention of acute pancreatitis.
Actins↗
Sequential exocytosis of insulin granules is associated with redistribution of SNAP25.
We have investigated sequential exocytosis in beta cells of intact pancreatic islets with the use of two-photon excitation imaging of a polar fluorescent tracer, sulforhodamine B, and a fusion protein comprising enhanced cyan fluorescent protein (ECFP) and the SNARE protein SNAP25 (synaptosome-associated protein of 25 kD) transfected with an adenoviral vector. Sequential exocytosis was found to account for <10% of exocytic events in beta cells stimulated either with glucose under various conditions or by photolysis of a caged-Ca2+ compound. Multigranular exocytosis, in which granule-to-granule fusion occurs before exocytosis, was rarely found. We detected redistribution of ECFP-SNAP25 from the plasma membrane into the membrane of the fused granule occurred in a large proportion (54%) of sequential exocytic events but in only a small fraction (5%) of solitary fusion events. Removal of cholesterol in the plasma membrane by methyl-beta-cyclodextrin facilitated both redistribution of ECFP-SNAP25 and sequential exocytosis by threefold. These observations support the hypothesis that SNAP25 is a plasma membrane factor that is responsible for sequential exocytosis.
Animals↗