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Vol. 12, Issue 1, 129-141, January 2001
and
Program in Molecular Medicine and Departments of *Cell
Biology, A novel imaging technology, high-speed microscopy, has been used to
visualize the process of GLUT4 translocation in response to insulin in
single 3T3-L1 adipocytes. A key advantage of this technology is that it
requires extremely low light exposure times, allowing the
quasi-continuous capture of information over 20-30 min without
photobleaching or photodamage. The half-time for the accumulation of
GLUT4-eGFP (enhanced green fluorescent protein) at the plasma membrane
in a single cell was found to be of 5-7 min at 37°C. This half-time
is substantially longer than that of exocytic vesicle fusion in
neuroendocrine cells, suggesting that additional regulatory mechanisms
are involved in the stimulation of GLUT4 translocation by insulin.
Analysis of four-dimensional images (3-D over time) revealed that, in
response to insulin, GLUT4-eGFP-enriched vesicles rapidly travel from
the juxtanuclear region to the plasma membrane. In nontransfected
adipocytes, impairment of microtubule and actin filament function
inhibited insulin-stimulated glucose transport by 70 and 50%,
respectively. When both filament systems were impaired
insulin-stimulated glucose transport was completely inhibited. Taken
together, the data suggest that the regulation of long-range motility
of GLUT4-containing vesicles through the interaction with microtubule-
and actin-based cytoskeletal networks plays an important role in the
overall effect of insulin on GLUT4 translocation.
Biochemistry and Molecular Biology, and
Physiology, University of Massachusetts Medical School,
Worcester, Massachusetts 01605
Online version of this article contains video
material for Figures 1,2,5, and 9. Online version available at
www.molbiolcell.org.
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