Abstract
In adipocytes, vesicles containing glucose transporter-4 (GLUT4) redistribute from intracellular stores to the cell periphery in response to insulin stimulation. Vesicles then fuse with the plasma membrane, facilitating glucose transport into the cell. To gain insight into the details of microtubule involvement, we examined the spatial organization and dynamics of microtubules in relation to GLUT4 vesicle trafficking in living 3T3-L1 adipocytes using total internal reflection fluorescence (TIRF) microscopy. Insulin stimulated an increase in microtubule density and curvature within the TIRF-illuminated region of the cell. The high degree of curvature and abrupt displacements of microtubules indicate that substantial forces act on microtubules. The time course of the microtubule density increase precedes that of the increase in intensity of fluorescently-tagged GLUT4 in this same region of the cell. In addition, portions of the microtubules are highly curved and are pulled closer to the cell cortex, as confirmed by Parallax microscopy. Microtubule disruption delayed and modestly reduced GLUT4 accumulation at the plasma membrane. Quantitative analysis revealed that fusions of GLUT4-containing vesicles with the plasma membrane, detected using insulin-regulated aminopeptidase with a pH-sensitive GFP tag (pHluorin), preferentially occur near microtubules. Interestingly, long-distance vesicle movement along microtubules visible at the cell surface prior to fusion does not appear to account for this proximity. We conclude that microtubules may be important in providing spatial information for GLUT4 vesicle fusion.
Highlights
glucose transporter-4 (GLUT4) is a facilitative glucose transporter important in the uptake of glucose into fat and muscle tissue in response to insulin [1,2]
To gain insight into the molecular role of microtubules in GLUT4 exocytosis, we examined the arrangement and dynamics of microtubules in 3T3-L1 adipocytes using live-cell total internal reflection fluorescence (TIRF) imaging
We show that insulin increases the density and dynamics of microtubules at the cell surface
Summary
GLUT4 is a facilitative glucose transporter important in the uptake of glucose into fat and muscle tissue in response to insulin [1,2]. Because of the importance of GLUT4 in maintaining blood glucose homeostasis, its intracellular localization and plasma membrane insertion are highly regulated. The intracellular pool of GLUT4 is dynamic, and the basal distribution reflects fast endocytosis from the plasma membrane [5,6,7] and slow exocytosis of GLUT4containing vesicles [5,6,7,8]. This results in a low level of plasma membrane-inserted GLUT4 in the basal state. Insulin binding to the insulin receptor begins a series of signaling events, which culminate in a substantial increase in the rate of exocytosis [5,6,7,8]
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