scholarly journals ELKS, a Protein Structurally Related to the Active Zone-associated Protein CAST, Is Expressed in Pancreatic β Cells and Functions in Insulin Exocytosis: Interaction of ELKS with Exocytotic Machinery Analyzed by Total Internal Reflection Fluorescence Microscopy

2005 ◽  
Vol 16 (7) ◽  
pp. 3289-3300 ◽  
Author(s):  
Mica Ohara-Imaizumi ◽  
Toshihisa Ohtsuka ◽  
Satsuki Matsushima ◽  
Yoshihiro Akimoto ◽  
Chiyono Nishiwaki ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Microscopy ◽  
Active Zone ◽  
Total Internal Reflection ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Insulin Granules ◽  
Insulin Exocytosis

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitters. Here, we demonstrate the expression and function of ELKS, a protein structurally related to the CAZ protein CAST, in insulin exocytosis. The results of confocal and immunoelectron microscopic analysis showed that ELKS is present in pancreatic β cells and is localized close to insulin granules docked on the plasma membrane-facing blood vessels. Total internal reflection fluorescence microscopy imaging in insulin-producing clonal cells revealed that the ELKS clusters are less dense and unevenly distributed than syntaxin 1 clusters, which are enriched in the plasma membrane. Most of the ELKS clusters were on the docking sites of insulin granules that were colocalized with syntaxin 1 clusters. Total internal reflection fluorescence images of single-granule motion showed that the fusion events of insulin granules mostly occurred on the ELKS cluster, where repeated fusion was sometimes observed. When the Bassoon-binding region of ELKS was introduced into the cells, the docking and fusion of insulin granules were markedly reduced. Moreover, attenuation of ELKS expression by small interfering RNA reduced the glucose-evoked insulin release. These data suggest that the CAZ-related protein ELKS functions in insulin exocytosis from pancreatic β cells.

scholarly journals The Rab27a effector exophilin7 promotes fusion of secretory granules that have not been docked to the plasma membrane

2013 ◽  
Vol 24 (3) ◽  
pp. 319-330 ◽  
Author(s):  
Hao Wang ◽  
Ray Ishizaki ◽  
Jun Xu ◽  
Kazuo Kasai ◽  
Eri Kobayashi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Knockout Mice ◽  
Secretory Granules ◽  
Small Gtpase ◽  
Membrane Phospholipids ◽  
Β Cells ◽  
Β Cell ◽  
Pancreatic Β Cells ◽  
Insulin Granules ◽  
Insulin Exocytosis

Granuphilin, an effector of the small GTPase Rab27a, mediates the stable attachment (docking) of insulin granules to the plasma membrane and inhibits subsequent fusion of docked granules, possibly through interaction with a fusion-inhibitory Munc18-1/syntaxin complex. However, phenotypes of insulin exocytosis differ considerably between Rab27a- and granuphilin-deficient pancreatic β cells, suggesting that other Rab27a effectors function in those cells. We found that one of the putative Rab27a effector family proteins, exophilin7/JFC1/Slp1, is expressed in β cells; however, unlike granuphilin, exophilin7 overexpressed in the β-cell line MIN6 failed to show granule-docking or fusion-inhibitory activity. Furthermore, exophilin7 has no affinities to either Munc18-1 or Munc18-1–interacting syntaxin-1a, in contrast to granuphilin. Although β cells of exophilin7-knockout mice show no apparent abnormalities in intracellular distribution or in ordinary glucose-induced exocytosis of insulin granules, they do show impaired fusion in response to some stronger stimuli, specifically from granules that have not been docked to the plasma membrane. Exophilin7 appears to mediate the fusion of undocked granules through the affinity of its C2A domain toward the plasma membrane phospholipids. These findings indicate that the two Rab27a effectors, granuphilin and exophilin7, differentially regulate the exocytosis of either stably or minimally docked granules, respectively.

scholarly journals Imaging Constitutive Exocytosis with Total Internal Reflection Fluorescence Microscopy

2000 ◽  
Vol 149 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Jan Schmoranzer ◽  
Mark Goulian ◽  
Dan Axelrod ◽  
Sanford M. Simon
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Fluorescence Microscopy ◽  
Total Internal Reflection ◽  
Fluorescent Protein ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Green Fluorescent ◽  
Kinetics Of ◽  
Flattening Process

Total internal reflection fluorescence microscopy has been applied to image the final stage of constitutive exocytosis, which is the fusion of single post-Golgi carriers with the plasma membrane. The use of a membrane protein tagged with green fluorescent protein allowed the kinetics of fusion to be followed with a time resolution of 30 frames/s. Quantitative analysis allowed carriers undergoing fusion to be easily distinguished from carriers moving perpendicularly to the plasma membrane. The flattening of the carriers into the plasma membrane is seen as a simultaneous rise in the total, peak, and width of the fluorescence intensity. The duration of this flattening process depends on the size of the carriers, distinguishing small spherical from large tubular carriers. The spread of the membrane protein into the plasma membrane upon fusion is diffusive. Mapping many fusion sites of a single cell reveals that there are no preferred sites for constitutive exocytosis in this system.

scholarly journals Role of Microtubules in Fusion of Post-Golgi Vesicles to the Plasma Membrane

2003 ◽  
Vol 14 (4) ◽  
pp. 1558-1569 ◽  
Author(s):  
Jan Schmoranzer ◽  
Sanford M. Simon
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Microscopy ◽  
Actin Filaments ◽  
Total Internal Reflection ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Cell Periphery ◽  
Golgi Vesicles ◽  
Cytoskeletal Elements

Biosynthetic cargo is transported away from the Golgi in vesicles via microtubules. In the cell periphery the vesicles are believed to engage actin and then dock to fusion sites at the plasma membrane. Using dual-color total internal reflection fluorescence microscopy, we observed that microtubules extended within 100 nm of the plasma membrane and post-Golgi vesicles remained on microtubules up to the plasma membrane, even as fusion to the plasma membrane initiated. Disruption of microtubules eliminated the tubular shapes of the vesicles and altered the fusion events: vesicles required multiple fusions to deliver all of their membrane cargo to the plasma membrane. In contrast, the effects of disrupting actin on fusion behavior were subtle. We conclude that microtubules, rather than actin filaments, are the cytoskeletal elements on which post-Golgi vesicles are transported until they fuse to the plasma membrane.

scholarly journals Glinide, but Not Sulfonylurea, Can Evoke Insulin Exocytosis by Repetitive Stimulation: Imaging Analysis of Insulin Exocytosis by Secretagogue-Induced Repetitive Stimulations

2009 ◽  
Vol 2009 ◽  
pp. 1-6 ◽  
Author(s):  
Kyota Aoyagi ◽  
Mica Ohara-Imaizumi ◽  
Chiyono Nishiwaki ◽  
Yoko Nakamichi ◽  
Shinya Nagamatsu
Keyword(s):  
Plasma Membrane ◽  
Insulin Secretion ◽  
Total Internal Reflection ◽  
Repetitive Stimulation ◽  
Internal Reflection ◽  
Imaging Analysis ◽  
Tirf Microscopy ◽  
Insulin Granules ◽  
Insulin Exocytosis ◽  
Secondary Stimulation

To investigate the different effects between sulfonylurea (SU) and glinide drugs in insulin secretion, pancreaticβ-cells were repeatedly stimulated with SU (glimepiride) or glinide (mitiglinide). Total internal reflection fluorescent (TIRF) microscopy revealed that secondary stimulation with glimepiride, but not glucose and mitiglinide, failed to evoke fusions of insulin granules although primary stimulation with glucose, glimepiride, and mitiglinide induced equivalent numbers of exocytotic responses. Glimepiride, but not glucose and mitiglinide, induced abnormally sustained[Ca2+]ielevations and reductions of docked insulin granules on the plasma membrane. Our data suggest that the effect of glinide on insulin secretory mechanisms is similar to that of glucose.

scholarly journals Imaging analysis reveals mechanistic differences between first- and second-phase insulin exocytosis

2007 ◽  
Vol 177 (4) ◽  
pp. 695-705 ◽  
Author(s):  
Mica Ohara-Imaizumi ◽  
Tomonori Fujiwara ◽  
Yoko Nakamichi ◽  
Tadashi Okamura ◽  
Yoshihiro Akimoto ◽  
...  
Keyword(s):  
Total Internal Reflection ◽  
Second Phase ◽  
Imaging Analysis ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Biphasic Insulin ◽  
Insulin Granules ◽  
Biphasic Insulin Release ◽  
Two Phases ◽  
Insulin Exocytosis

The mechanism of glucose-induced biphasic insulin release is unknown. We used total internal reflection fluorescence (TIRF) imaging analysis to reveal the process of first- and second-phase insulin exocytosis in pancreatic β cells. This analysis showed that previously docked insulin granules fused at the site of syntaxin (Synt)1A clusters during the first phase; however, the newcomers fused during the second phase external to the Synt1A clusters. To reveal the function of Synt1A in phasic insulin exocytosis, we generated Synt1A-knockout (Synt1A−/−) mice. Synt1A−/− β cells showed fewer previously docked granules with no fusion during the first phase; second-phase fusion from newcomers was preserved. Rescue experiments restoring Synt1A expression demonstrated restoration of granule docking status and fusion events. Inhibition of other syntaxins, Synt3 and Synt4, did not affect second-phase insulin exocytosis. We conclude that the first phase is Synt1A dependent but the second phase is not. This indicates that the two phases of insulin exocytosis differ spatially and mechanistically.

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