Protein Kinase C-Dependent Supply of Secretory Granules to the Plasma Membrane

Tsuboi, Takashi; Kikuta, Toshiteru; Warashina, Akira; Terakawa, Susumu

doi:10.1006/bbrc.2001.4603

Cited by 26 publications

(22 citation statements)

References 32 publications

(27 reference statements)

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“…The ratio images were used to calculate [Ca 2ϩ ] off line according to established methods (42). TIRF Microscopy-To assess translocation of PKC␤II⅐EGFP, we employed a TIRF (also known as evanescent wave microscopy) microscope similar to that described previously by Tsuboi et al (32)(33)(34). The incident light for total internal reflection illumination was introduced from the objective lens (Olympus, numerical aperture ϭ 1.65, 100X magnification) through a single mode optical fiber and two illumination lenses.…”

Section: Methodsmentioning

confidence: 99%

“…Retranslocation to the cytosol was clearly evident in almost half (three of seven) of the cells examined. To provide greater temporal and spatial resolution we next employed TIRF microscopy (31)(32)(33)(34). This technique involves the generation of a thin (Ͻ100 nm) field of fluorescence at the surface of the coverslip and thus at the surface of an attached cell.…”

Section: Responses Of Pkc␤ii⅐egfp Distribution To Elevated [Glucose] mentioning

confidence: 99%

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Dynamics of Glucose-induced Membrane Recruitment of Protein Kinase C βII in Living Pancreatic Islet β-Cells

Pinton¹,

Tsuboi²,

Ainscow³

et al. 2002

Journal of Biological Chemistry

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The mechanisms by which glucose may affect protein kinase C (PKC) activity in the pancreatic islet ␤-cell are presently unclear. By developing adenovirally expressed chimeras encoding fusion proteins between green fluorescent protein and conventional (␤II), novel (␦), or atypical () PKCs, we show that glucose selectively alters the subcellular localization of these enzymes dynamically in primary islet and MIN6 ␤-cells.

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Section: Methodsmentioning

confidence: 99%

Section: Responses Of Pkc␤ii⅐egfp Distribution To Elevated [Glucose] mentioning

confidence: 99%

Dynamics of Glucose-induced Membrane Recruitment of Protein Kinase C βII in Living Pancreatic Islet β-Cells

Pinton¹,

Tsuboi²,

Ainscow³

et al. 2002

Journal of Biological Chemistry

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“…Imaging Systems-For most fluorescence observations, we employed a total internal reflection fluorescence microscope (TIRFM or evanescent field microscope) described previously by Tsuboi et al (7). The incident light for evanescent illumination was introduced from the objective lens (NA ϭ 1.45, 60ϫ magnification) installed on an inverted microscope (IX70, Olympus, Tokyo, Japan).…”

Section: Methodsmentioning

confidence: 99%

Sweeping Model of Dynamin Activity

Tsuboi

Terakawa

Scalettar

et al. 2002

Journal of Biological Chemistry

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Vesicle recycling through exocytosis and endocytosis is mediated by a coordinated cascade of protein-protein interactions. Previously, exocytosis and endocytosis were studied separately so that the coupling between them was understood only indirectly. We focused on the coupling of these processes by observing the secretory vesicle marker synaptobrevin and the endocytotic vesicle marker dynamin I tagged with green and red fluorescent proteins under an evanescent wave microscope in pheochromocytoma cells. In control cells, many synaptobrevin-expressing vesicles were found as fluorescent spots near the plasma membrane. Upon electrical stimulation, many of these vesicles showed an exocytotic response as a transient increase in fluorescence intensity followed by their disappearance. In contrast, fluorescent dynamin appeared as clusters increasing slowly in number upon stimulation. The clusters of fluorescent dynamin moved around beneath the plasma membrane for a significant distance. Simultaneous observations of green fluorescent dynamin and red fluorescent synaptobrevin indicated that more than 70% of the exocytotic responses of synaptobrevin had no immediate dynamin counterpart at the same site. From these findings it was concluded that dynamin-mediated recycling is not directly coupled to exocytosis but rather completed by a scanning movement of dynamin for the sites of invaginating membrane destined to endocytosis.Exocytosis and endocytosis are linked and regulated coordinately by a cascade of protein-protein interactions (1) to ensure the highly complex spatial and temporal patterns of membrane recycling. Previous studies focused mainly on the last step of exocytosis and inferred the kinetics of endocytosis only indirectly (2-5). In the present study, using the green fluorescent protein (GFP) 1 technique, we have focused on the coupling of exocytosis and endocytosis. We observed the vesicle-associated membrane protein, which is also referred to as synaptobrevin (Syb), and the vesicle-producing protein, dynamin, simultaneously under an evanescent field microscope (6 -8). The fluorescence imaging restricted to the plasma membrane allowed us to capture the exact moment and the site of exocytosis and compare them with the foci of endocytotic activity. In many cases, dynamin clusters appeared in the void space between the sites of exocytotic responses, and then they moved around continuously beneath the membrane, as if they were searching or scanning for the proper site of membrane retrieval. Here, based on these observations, we will propose a novel hypothesis, "sweeping model of dynamin," for an efficient retrieval of superfluous membranes by endocytosis. EXPERIMENTAL PROCEDURESExpression Vector and Transfection-A construct of Syb fused with enhanced GFP (EGFP) was produced by subcloning the rat Syb cDNA as a HindIII/BamHI fragment into pEGFP C1 (CLONTECH, Tokyo, Japan). A construct of Syb fused with DsRed was made by shuffling the Syb cDNA from EGFP fusion construct into DsRed C1 (CLONTECH). Constructs of dynamin ...

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“…Mobility of membrane-proximal DCGs in hippocampal neurons could be facilitated by remodeling (depolymerization) or mobility of actin filaments, heterogeneity in the structure of the cortex, or the relatively small size of neuronal DCGs compared with chromaffin DCGs (Plattner et al, 1997;Chuang et al, 1999;Loerke et al, 2002). In contrast, with rare exceptions (Oheim et al, 1999), most TIRFM data suggest that the actin cortex is a major barrier to DCGs in chromaffin cells and PC12 cells, revealing that essentially all membrane-proximal DCGs in resting chromaffin cells and PC12 cells are nearly immobile (Oheim et al, 1998;Steyer and Almers, 1999;Johns et al, 2001;Tsuboi et al, 2001;Desnos et al, 2003).…”

Section: Cortical Actin Is Not a Major Barrier To Dcg Transport And Ementioning

confidence: 99%

Mechanisms of Transport and Exocytosis of Dense-Core Granules Containing Tissue Plasminogen Activator in Developing Hippocampal Neurons

Silverman¹,

Johnson²,

Gurkins³

et al. 2005

J. Neurosci.

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Dense-core granules (DCGs) are organelles found in specialized secretory cells, including neuroendocrine cells and neurons. Neuronal DCGs facilitate many critical processes, including the transport and secretion of proteins involved in learning, and yet their transport and exocytosis are poorly understood. We have used wide-field and total internal reflection fluorescence microscopy, in conjunction with transport theory, to visualize the transport and exocytosis of DCGs containing a tissue plasminogen activator-green fluorescent protein hybrid in cell bodies, neurites, and growth cones of developing hippocampal neurons and to quantify the roles that diffusion, directed motion, and immobility play in these processes. Our results demonstrate that shorter-ranged transport of DCGs near sites of exocytosis in hippocampal neurons and neuroendocrine cells differs markedly. Specifically, the immobile fraction of DCGs within growth cones and near the plasma membrane of hippocampal neurons is small and relatively unaltered by actin disruption, unlike in neuroendocrine cells. Moreover, transport of DCGs in these domains of hippocampal neurons is unusually heterogeneous, being significantly rapid and directed as well as slow and diffusive. Our results also demonstrate that exocytosis is preceded by substantial movement and heterogeneous transport; this movement may facilitate delivery of DCG cargo in hippocampal neurons, given the relatively low abundance of neuronal DCGs. In addition, the extensive mobility of DCGs in hippocampal neurons argues strongly against the hypothesis that cortical actin is a major barrier to membrane-proximal DCGs in these cells. Instead, our results suggest that extended release of DCG cargo from hippocampal neurons arises from heterogeneity in DCG mobility.

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Protein Kinase C-Dependent Supply of Secretory Granules to the Plasma Membrane

Cited by 26 publications

References 32 publications

Dynamics of Glucose-induced Membrane Recruitment of Protein Kinase C βII in Living Pancreatic Islet β-Cells

Dynamics of Glucose-induced Membrane Recruitment of Protein Kinase C βII in Living Pancreatic Islet β-Cells

Sweeping Model of Dynamin Activity

Mechanisms of Transport and Exocytosis of Dense-Core Granules Containing Tissue Plasminogen Activator in Developing Hippocampal Neurons

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