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Clathrin-dependent entry and vesicle-mediated exocytosis define insulin transcytosis across microvascular endothelial cells.

Azizi PM, Zyla RE, Guan S, Wang C, Liu J, Bolz SS, Heit B, Klip A, Lee WL - Mol. Biol. Cell (2014)

Bottom Line: Instead, insulin transcytosis was significantly inhibited by the clathrin-mediated endocytosis inhibitor Pitstop 2 or siRNA-mediated clathrin depletion.Accordingly, insulin internalized for 1 min in HAMECs colocalized with clathrin far more than with caveolin-1.This study constitutes the first evidence of vesicle-mediated insulin transcytosis and highlights that its initial uptake is clathrin dependent and caveolae independent.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Sciences, University of Toronto, Toronto, ON M5S 1A8, Canada Keenan Research Centre, St. Michael's Hospital, Toronto, ON M5B 1W8, Canada Programme in Cell Biology, Hospital for Sick Children, Toronto, ON M5G 0A4, Canada.

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Development of a novel single-cell assay to measure insulin transcytosis. (A) Schematic depicting the TIRF microscopy assay. A vesicle bearing fluorescent insulin is visualized as it enters the excitation zone of the endothelial cell and its signal is lost upon fusion with the basal plasmalemma. (B) Intensity profile of a tracked particle that undergoes exocytosis, causing a rapid loss of signal. (C) Intensity profile of a tracked particle undergoing Brownian diffusion (and photobleaching) but not exocytosis. (D) Varying the penetration depth of the TIRF laser does not affect detection events. (E) Transendothelial electrical resistance (TEER) drops after addition of histamine (2 mM) to top and bottom chambers of endothelial cells grown on Transwells (***p < 0.001 compared with initial time point) but (F) does not affect the average number of transcytosis events (data are normalized to control cells). (G) Addition of excess unlabeled insulin (50-fold) to the membrane-binding step essentially abrogates insulin-AF568 transcytosis, consistent with a receptor-mediated process. **p < 0.01 by one-sample t test; data are normalized to control cells. (H) Time course of insulin transcytosis in single cells using the TIRF assay.
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Figure 4: Development of a novel single-cell assay to measure insulin transcytosis. (A) Schematic depicting the TIRF microscopy assay. A vesicle bearing fluorescent insulin is visualized as it enters the excitation zone of the endothelial cell and its signal is lost upon fusion with the basal plasmalemma. (B) Intensity profile of a tracked particle that undergoes exocytosis, causing a rapid loss of signal. (C) Intensity profile of a tracked particle undergoing Brownian diffusion (and photobleaching) but not exocytosis. (D) Varying the penetration depth of the TIRF laser does not affect detection events. (E) Transendothelial electrical resistance (TEER) drops after addition of histamine (2 mM) to top and bottom chambers of endothelial cells grown on Transwells (***p < 0.001 compared with initial time point) but (F) does not affect the average number of transcytosis events (data are normalized to control cells). (G) Addition of excess unlabeled insulin (50-fold) to the membrane-binding step essentially abrogates insulin-AF568 transcytosis, consistent with a receptor-mediated process. **p < 0.01 by one-sample t test; data are normalized to control cells. (H) Time course of insulin transcytosis in single cells using the TIRF assay.

Mentions: The rapid appearance of insulin in the supernatant of microvascular endothelial cells shown in Figure 1 is consistent with the secretion of internalized insulin (as would be expected for its transcytosis). Mechanistic studies of insulin transcytosis have focused on the endothelium from large vessels, even though these cells are not the physiological route of insulin extravasation, and further have relied on Transwell assays, where insulin delivery from the upper to the lower chamber by transcytosis may be confounded by paracellular leak (Armstrong et al., 2012). To overcome this potential confounder, and, more important, to obtain information on the actual events of insulin secretion at the exit membrane, we used total internal reflection fluorescence (TIRF) microscopy (Figure 4A and Supplemental Movie S1). The assay was combined with the internalization of a pulse of fluorescently conjugated insulin, so that only insulin that binds and is taken up into the cell is then imaged at the ventral membrane. Thus this assay represents a direct assessment of insulin transcytosis. Briefly, a pulse of Alexa Fluor 568 (AF568)–tagged insulin was added to a confluent HAMEC monolayer at 4°C for 10 min, and then insulin-AF568 was washed off and the temperature shifted to 37°C. Immediately thereafter, the ventral membrane was imaged by TIRF microscopy. The live-cell videos were then analyzed in a blinded, automated manner as described in Materials and Methods to quantify the number of individual fusion events (vesicle exocytosis) evinced by the abrupt disappearance of individual fluorescent particles (Figure 4B) versus photobleaching of trafficked but nonexocytosed vesicles (Figure 4C).


Clathrin-dependent entry and vesicle-mediated exocytosis define insulin transcytosis across microvascular endothelial cells.

Azizi PM, Zyla RE, Guan S, Wang C, Liu J, Bolz SS, Heit B, Klip A, Lee WL - Mol. Biol. Cell (2014)

Development of a novel single-cell assay to measure insulin transcytosis. (A) Schematic depicting the TIRF microscopy assay. A vesicle bearing fluorescent insulin is visualized as it enters the excitation zone of the endothelial cell and its signal is lost upon fusion with the basal plasmalemma. (B) Intensity profile of a tracked particle that undergoes exocytosis, causing a rapid loss of signal. (C) Intensity profile of a tracked particle undergoing Brownian diffusion (and photobleaching) but not exocytosis. (D) Varying the penetration depth of the TIRF laser does not affect detection events. (E) Transendothelial electrical resistance (TEER) drops after addition of histamine (2 mM) to top and bottom chambers of endothelial cells grown on Transwells (***p < 0.001 compared with initial time point) but (F) does not affect the average number of transcytosis events (data are normalized to control cells). (G) Addition of excess unlabeled insulin (50-fold) to the membrane-binding step essentially abrogates insulin-AF568 transcytosis, consistent with a receptor-mediated process. **p < 0.01 by one-sample t test; data are normalized to control cells. (H) Time course of insulin transcytosis in single cells using the TIRF assay.
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Related In: Results  -  Collection

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Figure 4: Development of a novel single-cell assay to measure insulin transcytosis. (A) Schematic depicting the TIRF microscopy assay. A vesicle bearing fluorescent insulin is visualized as it enters the excitation zone of the endothelial cell and its signal is lost upon fusion with the basal plasmalemma. (B) Intensity profile of a tracked particle that undergoes exocytosis, causing a rapid loss of signal. (C) Intensity profile of a tracked particle undergoing Brownian diffusion (and photobleaching) but not exocytosis. (D) Varying the penetration depth of the TIRF laser does not affect detection events. (E) Transendothelial electrical resistance (TEER) drops after addition of histamine (2 mM) to top and bottom chambers of endothelial cells grown on Transwells (***p < 0.001 compared with initial time point) but (F) does not affect the average number of transcytosis events (data are normalized to control cells). (G) Addition of excess unlabeled insulin (50-fold) to the membrane-binding step essentially abrogates insulin-AF568 transcytosis, consistent with a receptor-mediated process. **p < 0.01 by one-sample t test; data are normalized to control cells. (H) Time course of insulin transcytosis in single cells using the TIRF assay.
Mentions: The rapid appearance of insulin in the supernatant of microvascular endothelial cells shown in Figure 1 is consistent with the secretion of internalized insulin (as would be expected for its transcytosis). Mechanistic studies of insulin transcytosis have focused on the endothelium from large vessels, even though these cells are not the physiological route of insulin extravasation, and further have relied on Transwell assays, where insulin delivery from the upper to the lower chamber by transcytosis may be confounded by paracellular leak (Armstrong et al., 2012). To overcome this potential confounder, and, more important, to obtain information on the actual events of insulin secretion at the exit membrane, we used total internal reflection fluorescence (TIRF) microscopy (Figure 4A and Supplemental Movie S1). The assay was combined with the internalization of a pulse of fluorescently conjugated insulin, so that only insulin that binds and is taken up into the cell is then imaged at the ventral membrane. Thus this assay represents a direct assessment of insulin transcytosis. Briefly, a pulse of Alexa Fluor 568 (AF568)–tagged insulin was added to a confluent HAMEC monolayer at 4°C for 10 min, and then insulin-AF568 was washed off and the temperature shifted to 37°C. Immediately thereafter, the ventral membrane was imaged by TIRF microscopy. The live-cell videos were then analyzed in a blinded, automated manner as described in Materials and Methods to quantify the number of individual fusion events (vesicle exocytosis) evinced by the abrupt disappearance of individual fluorescent particles (Figure 4B) versus photobleaching of trafficked but nonexocytosed vesicles (Figure 4C).

Bottom Line: Instead, insulin transcytosis was significantly inhibited by the clathrin-mediated endocytosis inhibitor Pitstop 2 or siRNA-mediated clathrin depletion.Accordingly, insulin internalized for 1 min in HAMECs colocalized with clathrin far more than with caveolin-1.This study constitutes the first evidence of vesicle-mediated insulin transcytosis and highlights that its initial uptake is clathrin dependent and caveolae independent.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Sciences, University of Toronto, Toronto, ON M5S 1A8, Canada Keenan Research Centre, St. Michael's Hospital, Toronto, ON M5B 1W8, Canada Programme in Cell Biology, Hospital for Sick Children, Toronto, ON M5G 0A4, Canada.

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Related in: MedlinePlus