Determining how myosin II affects GLUT4 docking and fusion to the plasma membrane in 3T3-L1 adipocytes

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Marc D. Bucciarelli (Creator)
The University of North Carolina at Greensboro (UNCG )
Web Site:
Yashomati Patel

Abstract: Myosin II is required for GLUT4 mediated glucose uptake in 3T3-L1 adipocytes. Previous studies from our lab have shown that myosin IIA and GLUT4 are stimulated to translocate to the plasma membrane from a perinuclear region upon insulin stimulation. We also know that upon insulin stimulation, myosin IIA colocalizes with filamentous actin (F-actin) near the site of GLUT4 fusion and that myosin IIB is localized at the cell cortex and is unaffected by insulin stimulation. Our study aimed to determine the role of myosin II in GLUT4 docking and fusion to the plasma membrane in adipocytes and also to determine which myosin II isoform is involved in this process. Using fluorescence microscopy, we visualized the localizations of myosin IIA, GLUT4, syntaxin-4, and VAMP2 in adipocytes stimulated with insulin in the presence or absence of the myosin II specific inhibitor, blebbistatin. Our studies showed that by inhibiting myosin II, there was a 56% decrease in the localization of GLUT4 at the plasma membrane as well as a 31% decrease in the interaction between the GLUT4 vesicle and the membrane bound docking and fusion protein, syntaxin-4. We also showed that there was an accumulation of GLUT4 below the plasma membrane in cells treated with blebbistatin suggesting that inhibition of myosin II did not prevent insulin-stimulated GLUT4 translocation towards the plasma membrane but did prevent the GLUT4 vesicles from reaching and fusing with the plasma membrane. Since 3T3-L1 adipocytes express two myosin II isoforms, we wanted to determine the roles of each isoform at the cell cortex. Using a plasma lawn assay and fluorescence microscopy we examined the localizations of myosin IIA, myosin IIB, GLUT4, and myosin Va at the plasma membrane. Our studies demonstrated that the level of myosin IIA at the plasma membrane decreased by 53% in the presence of blebbistatin while the level of myosin IIB was not affected. However, myosin IIA and GLUT4 had a distinct punctate staining pattern that showed a clear separation between myosin IIA and GLUT4. Immunostaining for myosin IIB and GLUT4 showed that myosin IIB remained in a dense lawn across the plasma membrane while the localization of GLUT4 at the plasma membrane decreased in adipocytes treated with blebbistatin. The studies described here demonstrate that GLUT4 was prevented from fusing with the plasma membrane while the localization of myosin IIA at the plasma membrane was also affected when myosin II was inhibited. This study suggests that myosin II is required for the GLUT4 vesicle to access the plasma membrane. The information gained from this study will help to further our understanding of GLUT4 vesicle trafficking. [This abstract has been edited to remove characters that will not display in this system. Please see the PDF for the full abstract.]

Additional Information

Language: English
Date: 2019
Glucose metabolism, GLUT4, Myosin II
Cell membranes
Fat cells

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