Rapid three-dimensional imaging of individual insulin release events by Nipkow disc confocal microscopy

2006 ◽  
Vol 34 (5) ◽  
pp. 675-678 ◽  
Author(s):  
G.A. Rutter ◽  
M.K. Loder ◽  
M.A. Ravier
Keyword(s):  
Confocal Microscopy ◽  
Cell Surface ◽  
Spatial Organization ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Three Dimensional ◽  
Confocal Imaging ◽  
Β Cells ◽  
Large Dense Core Vesicles ◽  
Cell Depolarization

Minute-to-minute control of the release of insulin by pancreatic β-cells in response to glucose or other stimuli requires the precise delivery of large dense-core vesicles to the plasma membrane and regulated exocytosis. At present, the precise spatial organization at the cell surface and the nature of these events (‘transient’ versus ‘full fusion’) are debated. In order to monitor secretory events simultaneously over most of the surface of clusters of single MIN6 β-cells, we have expressed recombinant neuropeptide Y-Venus (an enhanced and vesicle-targeted form of yellow fluorescent protein) as an insulin surrogate. Individual exocytotic events were monitored using Nipkow spinning disc confocal microscopy, with acquisition of a three-dimensional complete image (eight to twelve confocal slices) in <1 s, in response to cell depolarization. Corroborating earlier studies using TIRF (total internal reflection fluorescence) microscopy, this approach indicates that events occur with roughly equal probability over the entire cell surface, with only minimal clustering in individual areas, and provides no evidence for multiple events at the same site. Nipkow disc confocal imaging may thus provide a useful tool to determine whether event types occur at different sites at the cell surface and to explore the role of endocytic proteins including dynamin-1 and -2 in terminating individual exocytotic events.

scholarly journals Tight junction targeting and intracellular trafficking of occludin in polarized epithelial cells

2007 ◽  
Vol 293 (5) ◽  
pp. C1717-C1726 ◽  
Author(s):  
Veedamali S. Subramanian ◽  
Jonathan S. Marchant ◽  
Dongmei Ye ◽  
Thomas Y. Ma ◽  
Hamid M. Said
Keyword(s):  
Cell Surface ◽  
Intracellular Trafficking ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Yellow Fluorescent Protein ◽  
Live Cell ◽  
Confocal Imaging ◽  
Dependent Manner ◽  
Epithelial Cell Lines ◽  
Heterogenous Population

Occludin, a transmembrane (TM)-spanning protein, is an integral component of the tight junctional (TJ) complexes that regulate epithelial integrity and paracellular barrier function. However, the molecular determinants that dictate occludin targeting and delivery to the TJs remain unclear. Here, using live cell imaging of yellow fluorescent protein-labeled occludin fragments, we resolved the intracellular trafficking of occludin-fusion proteins in polarized Madin-Darby canine kidney and Caco-2 cells to delineate the regions within the occludin polypeptide that are important for occludin targeting to the TJs. Live cell confocal imaging showed that complete or partial truncation of the COOH-terminal tail of the occludin polypeptide did not prevent occludin targeting to the TJs in epithelial cell lines. Progressive truncations into the COOH-terminal tail decreased the efficiency of occludin expression; after the removal of the regions proximal to the fourth transmembrane domain (TM4), the efficiency of expression increased. However, further deletions into the TM4 abolished TJ targeting, which resulted in constructs that were retained intracellularly within the endoplasmic reticulum. The full-length occludin polypeptide trafficked to the cell surface within a heterogenous population of intracellular vesicles that delivered occludin to the plasma membrane in a microtubule- and temperature-dependent manner. In contrast, the steady-state localization of occludin at the cell surface was dependent on intact microfilaments but not microtubules.

scholarly journals Glucose-dependent blood flow dynamics in murine pancreatic islets in vivo

2010 ◽  
Vol 298 (4) ◽  
pp. E807-E814 ◽  
Author(s):  
Lara R. Nyman ◽  
Eric Ford ◽  
Alvin C. Powers ◽  
David W. Piston
Keyword(s):  
Blood Flow ◽  
Blood Glucose ◽  
Pancreatic Islets ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Cell Types ◽  
Β Cells ◽  
Islet Blood Flow ◽  
Significant Difference

Pancreatic islets are highly vascularized and arranged so that regions containing β-cells are distinct from those containing other cell types. Although islet blood flow has been studied extensively, little is known about the dynamics of islet blood flow during hypoglycemia or hyperglycemia. To investigate changes in islet blood flow as a function of blood glucose level, we clamped blood glucose sequentially at hyperglycemic (∼300 mg/dl or 16.8 mM) and hypoglycemic (∼50 mg/dl or 2.8 mM) levels while simultaneously imaging intraislet blood flow in mouse models that express green fluorescent protein in the β-cells or yellow fluorescent protein in the α-cells. Using line scanning confocal microscopy, in vivo blood flow was assayed after intravenous injection of fluorescent dextran or sulforhodamine-labeled red blood cells. Regardless of the sequence of hypoglycemia and hyperglycemia, islet blood flow is faster during hyperglycemia, and apparent blood volume is greater during hyperglycemia than during hypoglycemia. However, there is no change in the order of perfusion of different islet endocrine cell types in hypoglycemia compared with hyperglycemia, with the islet core of β-cells usually perfused first. In contrast to the results in islets, there was no significant difference in flow rate in the exocrine pancreas during hyperglycemia compared with hypoglycemia. These results indicate that glucose differentially regulates blood flow in the pancreatic islet vasculature independently of blood flow in the rest of the pancreas.

Apical membrane targeting and trafficking of the human proton-coupled transporter in polarized epithelia

2008 ◽  
Vol 294 (1) ◽  
pp. C233-C240 ◽  
Author(s):  
Veedamali S. Subramanian ◽  
Jonathan S. Marchant ◽  
Hamid M. Said
Keyword(s):  
Cell Surface ◽  
Structure Function ◽  
Fluorescent Protein ◽  
Apical Membrane ◽  
Yellow Fluorescent Protein ◽  
Apical Cell ◽  
Membrane Targeting ◽  
Acid Uptake ◽  
Conserved Sequence ◽  
Polarized Cells

The human proton-coupled folate transporter (hPCFT) is a recently discovered intestinal transporter involved in folate uptake in epithelia (and possibly other cells). Little is currently known about the structure-function relationship of the different domains of this transporter, particularly which regions are important for substrate transport as well as targeting of the transporter to the apical cell surface of polarized cells. Here we have investigated the role of the COOH-terminal domain and a well-conserved sequence separating transmembrane (TM) domains TM2 and TM3 (DXXGRR; amino acids 109–114) speculated by others to be important for transport function. Using live cell imaging approaches, we show that 1) an hPCFT-yellow fluorescent protein construct is functionally expressed at the apical membrane domain and is localized differentially to the human reduced folate carrier; 2) the predicted cytoplasmic COOH-terminal region of hPCFT is not essential for apical targeting or transporter functionality; 3) mutations that ablate a consensus β-turn sequence separating predicted TM2 and TM3 abolished apical [3H]folic acid uptake as a consequence of endoplasmic reticulum retention of mutant, likely misfolded, transporters; and 4) cell surface delivery of hPCFT is disrupted by microtubule depolymerization or by overexpression of the dynactin complex dynamitin (p50). For the first time, our data present information regarding structure-function and membrane targeting of the hPCFT polypeptide, as well as the mechanisms that control its steady-state expression in polarized cells.

G-Protein-Dependent Cell Surface Dynamics of the Human Serotonin1AReceptor Tagged to Yellow Fluorescent Protein†

Biochemistry ◽  
2004 ◽  
Vol 43 (50) ◽  
pp. 15852-15862 ◽  
Author(s):  
Thomas J. Pucadyil ◽  
Shanti Kalipatnapu ◽  
Kaleeckal G. Harikumar ◽  
Nandini Rangaraj ◽  
Sadashiva S. Karnik ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Surface Dynamics ◽  
Dependent Cell

Identification of the roles of conserved charged residues in the extracellular domain of an epithelial sodium channel (ENaC) subunit by alanine mutagenesis

2011 ◽  
Vol 300 (4) ◽  
pp. F887-F897 ◽  
Author(s):  
Oded Edelheit ◽  
Israel Hanukoglu ◽  
Nathan Dascal ◽  
Aaron Hanukoglu
Keyword(s):  
Cell Surface ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Xenopus Laevis Oocytes ◽  
Epithelial Sodium Channels ◽  
Charged Residues ◽  
Inhibition Control ◽  
Two Parameters

Epithelial sodium channels (ENaC) are composed of three homologous subunits whose extracellular domains (ECD) form a funnel that directs ions from the lumen into the pore of ENaC. To examine the roles of conserved charged residues (Asp, Glu, Arg, and Lys) on ECD, we mutated 16 residues in human α-ENaC to alanine. The modified cRNAs were expressed in Xenopus laevis oocytes together with wild-type β- and γ-ENaC. The effect of each mutation was examined on three parameters: amiloride-sensitive Na+ conductance (assayed by the two-electrode voltage-clamp method), Na+-dependent self-inhibition of ENaC, and oocyte cell surface expression of ENaC (quantitated by confocal microscopy of yellow fluorescent protein linked to γ-ENaC). Mutation of 13 of 16 residues reduced the ENaC Na+ conductance (to 40–80% of WT). Mutation of only six residues showed a significant effect on the Na+ self-inhibition time constant (τ). All 16 mutants showed a strong correlation between ENaC activity and oocyte surface expression ( r = 0.62). Exclusion of four mutants showing the greatest effect on self-inhibition kinetics (Glu250 and Arg350 with τ = ∼30% of WT, and Asp393 and Glu530 with τ = ∼170% of WT) increased the correlation to r = 0.87. In the ASIC1 homotrimeric model, the homologs of α-ENaC Asp400 and Asp446 are exposed on the protein surface far from the other two chains. The mutations of these two residues showed the strongest effect on cell surface expression but had no effect on self-inhibition. Control mutations to a homologous charged residue (e.g., Asp to Glu) did not significantly affect ENaC activity. Changes in the two parameters, Na+ self-inhibition and oocyte surface expression level, accounted for the magnitude of reduction in ENaC activity as a result of the mutation to Ala. These results establish that while some conserved charged residues are part of the structure responsible for Na+ self-inhibition, most are essential for transport to the oocyte cell surface.

scholarly journals Dopamine D1 receptor distribution in Sf9 cells imaged by confocal microscopy: a quantitative evaluation.

1995 ◽  
Vol 43 (5) ◽  
pp. 497-506 ◽  
Author(s):  
J E Trogadis ◽  
G Y Ng ◽  
B F O'Dowd ◽  
S R George ◽  
J K Stevens
Keyword(s):  
Confocal Microscopy ◽  
Cell Surface ◽  
Radioligand Binding ◽  
Three Dimensional ◽  
Dopamine D1 Receptor ◽  
D1 Receptor ◽  
Receptor Internalization ◽  
D1 Receptors ◽  
Sf9 Cells ◽  
Receptor Distribution

A c-myc epitope-tagged human dopamine D1 receptor (c-myc D1 receptor) was expressed in Sf9 cells and its cellular distribution under basal conditions and after exposure to the agonist dopamine was examined. In the basal state, immunofluorescently labeled c-myc D1 receptors imaged by confocal microscopy appeared as a bright ring of label predominantly on the cell surface, and to a lesser extent as intracellular clusters of label. This pattern of receptor distribution was confirmed by radioligand-binding assays on plasma membrane and light membrane fractions using the D1 receptor-antagonist [3H]-SCH-23390. After exposure to dopamine, c-myc D1 receptors were redistributed on the cell surface, changing from a continuous ring to a discontinuous pattern of label. Analysis of fluorescence intensity and three-dimensional computer reconstruction of labeled receptors revealed a 30% decrease in surface labeling with no decrease in total number of receptors confirmed by radioligand-binding analysis. These findings constituted the first direct evidence of agonist-induced D1 receptor internalization. The results showed that the combination of confocal microscopy and three-dimensional reconstruction can be used to visualize and assess receptor distribution in Sf9 cells.

Imaging pancreatic β-cells in the intact pancreas

2006 ◽  
Vol 290 (5) ◽  
pp. E1041-E1047 ◽  
Author(s):  
Manami Hara ◽  
Restituto F. Dizon ◽  
Benjamin S. Glick ◽  
Catherine S. Lee ◽  
Klaus H. Kaestner ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Confocal Imaging ◽  
Β Cells ◽  
Β Cell ◽  
Disease States ◽  
Adult Mice ◽  
And Function

We have developed a method to visualize fluorescent protein-labeled β-cells in the intact pancreas through combined reflection and confocal imaging. This method provides a 3-D view of the β-cells in situ. Imaging of the pancreas from mouse insulin I promoter (MIP)-green (GFP) and red fluorescent protein (RFP) transgenic mice shows that islets, β-cell clusters, and single β-cells are not evenly distributed but are aligned along the large blood vessels. We also observe the solitary β-cells in both fetal and adult mice and along the pancreatic and common bile ducts. We have imaged the developing endocrine cells in the embryos using neurogenin-3 (Ngn3)-GFP mice crossed with MIP-RFP mice. The dual-color-coded pancreas from embryos (E15.5) shows a large number of green Ngn3-expressing proendocrine cells with a smaller number of red β-cells. The imaging technique that we have developed, coupled with the transgenic mice in which β-cells and β-cell progenitors are labeled with different fluorescent proteins, will be useful for studying pancreatic development and function in normal and disease states.

scholarly journals Real-time single cell analysis of Smac/DIABLO release during apoptosis

2003 ◽  
Vol 162 (6) ◽  
pp. 1031-1043 ◽  
Author(s):  
Markus Rehm ◽  
Heiko Düßmann ◽  
Jochen H.M. Prehn
Keyword(s):  
Single Cell ◽  
Real Time ◽  
Fluorescent Protein ◽  
Single Cell Analysis ◽  
Release Kinetics ◽  
Yellow Fluorescent Protein ◽  
Confocal Imaging ◽  
Significant Difference ◽  
Cyt C ◽  
Mcf 7

We examined the temporal and causal relationship between Smac/DIABLO release, cytochrome c (cyt-c) release, and caspase activation at the single cell level during apoptosis. Cells treated with the broad-spectrum caspase inhibitor z-VAD-fmk, caspase-3 (Casp-3)–deficient MCF-7 cells, as well as Bax-deficient DU-145 cells released Smac/DIABLO and cyt-c in response to proapoptotic agents. Real-time confocal imaging of MCF-7 cells stably expressing Smac/DIABLO-yellow fluorescent protein (YFP) revealed that the average duration of Smac/DIABLO-YFP release was greater than that of cyt-c-green fluorescent protein (GFP). However, there was no significant difference in the time to the onset of release, and both cyt-c-GFP and Smac/DIABLO-YFP release coincided with mitochondrial membrane potential depolarization. We also observed no significant differences in the Smac/DIABLO-YFP release kinetics when z-VAD-fmk–sensitive caspases were inhibited or Casp-3 was reintroduced. Simultaneous measurement of DEVDase activation and Smac/DIABLO-YFP release demonstrated that DEVDase activation occurred within 10 min of release, even in the absence of Casp-3.

scholarly journals Dual-Reporter β-Cell-Specific Male Transgenic Rats for the Analysis of β-Cell Functional Mass and Enrichment by Flow Cytometry

Endocrinology ◽  
2015 ◽  
Vol 157 (3) ◽  
pp. 1299-1306 ◽  
Author(s):  
Julien Ghislain ◽  
Ghislaine Fontés ◽  
Caroline Tremblay ◽  
Melkam A. Kebede ◽  
Vincent Poitout
Keyword(s):  
Insulin Secretion ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Yellow Fluorescent Protein ◽  
Cell Mass ◽  
Renilla Luciferase ◽  
Diabetes Research ◽  
Β Cells ◽  
Transgenic Rats ◽  
Β Cell

Abstract Mouse β-cell-specific reporter lines have played a key role in diabetes research. Although the rat provides several advantages, its use has lagged behind the mouse due to the relative paucity of genetic models. In this report we describe the generation and characterization of transgenic rats expressing a Renilla luciferase (RLuc)-enhanced yellow fluorescent protein (YFP) fusion under control of a 9-kb genomic fragment from the rat ins2 gene (RIP7-RLuc-YFP). Analysis of RLuc luminescence and YFP fluorescence revealed that reporter expression is restricted to β-cells in the adult rat. Physiological characteristics including body weight, fat and lean mass, fasting and fed glucose levels, glucose and insulin tolerance, and β-cell mass were similar between two RIP7-RLuc-YFP lines and wild-type littermates. Glucose-induced insulin secretion in isolated islets was indistinguishable from controls in one of the lines, whereas surprisingly, insulin secretion was defective in the second line. Consequently, subsequent studies were limited to the former line. We asked whether transgene activity was responsive to glucose as shown previously for the ins2 gene. Exposing islets ex vivo to high glucose (16.7 mM) or in vivo infusion of glucose for 24 hours increased luciferase activity in islets, whereas the fraction of YFP-positive β-cells after glucose infusion was unchanged. Finally, we showed that fluorescence-activated cell sorting of YFP-positive islet cells can be used to enrich for β-cells. Overall, this transgenic line will enable for the first time the application of both fluorescence and bioluminescence/luminescence-based approaches for the study of rat β-cells.

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