@article {47615, title = {Selective visualization of GLUT4 storage vesicles and associated Rab proteins using IRAP-pHluorin.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {1298}, year = {2015}, month = {2015}, pages = {173-9}, abstract = {

Fluorescence microscopy and fluorescent protein (FP)-tagged GLUT4 molecule have been great tools to characterize GLUT4 localization and dynamics inside the cell. However, it was difficult to distinguish GLUT4 storage vesicles (GSVs) from other intracellular compartments containing GLUT4 in live cells. Here, we describe the use of IRAP-pHluorin and total internal reflection fluorescence (TIRF) microscopy to selectively visualize GSVs and Rab proteins that associate with GSVs. This assay is also valuable to further defining GSV identity by unraveling other GSV-associated proteins.

}, keywords = {3T3-L1 Cells, Adipocytes, Animals, Cattle, Cell Membrane, Cystinyl Aminopeptidase, Exocytosis, Glucose Transporter Type 4, Green Fluorescent Proteins, Image Processing, Computer-Assisted, Insulin, Mice, Microscopy, Fluorescence, rab GTP-Binding Proteins, Transfection}, issn = {1940-6029}, doi = {10.1007/978-1-4939-2569-8_14}, author = {Chen, Yu and Lippincott-Schwartz, Jennifer} } @article {47638, title = {Insulin triggers surface-directed trafficking of sequestered GLUT4 storage vesicles marked by Rab10.}, journal = {Small GTPases}, volume = {4}, year = {2013}, month = {2013 Jul-Sep}, pages = {193-7}, abstract = {

Understanding how glucose transporter isoform 4 (GLUT4) redistributes to the plasma membrane during insulin stimulation is a major goal of glucose transporter research. GLUT4 molecules normally reside in numerous intracellular compartments, including specialized storage vesicles and early/recycling endosomes. It is unclear how these diverse compartments respond to insulin stimulation to deliver GLUT4 molecules to the plasma membrane. For example, do they fuse with each other first or remain as separate compartments with different trafficking characteristics? Our recent live cell imaging studies are helping to clarify these issues. Using Rab proteins as specific markers to distinguish between storage vesicles and endosomes containing GLUT4, we demonstrate that it is primarily internal GLUT4 storage vesicles (GSVs) marked by Rab10 that approach and fuse at the plasma membrane and GSVs don{\textquoteright}t interact with endosomes on their way to the plasma membrane. These new findings add strong support to the model that GSV release from intracellular retention plays a major role in supplying GLUT4 molecules onto the PM under insulin stimulation.

}, keywords = {Adipocytes, Animals, Cell Line, Cell Membrane, Endosomes, Glucose Transporter Type 4, Insulin, Mice, rab GTP-Binding Proteins, RNA Interference, RNA, Small Interfering}, issn = {2154-1256}, doi = {10.4161/sgtp.26471}, author = {Chen, Yu and Lippincott-Schwartz, Jennifer} } @article {47639, title = {Rab10 delivers GLUT4 storage vesicles to the plasma membrane.}, journal = {Communicative \& integrative biology}, volume = {6}, year = {2013}, month = {2013 May 1}, pages = {e23779}, abstract = {

The glucose transporter, GLUT4, redistributes to the plasma membrane (PM) upon insulin stimulation, but also recycles through endosomal compartments. Different Rab proteins control these transport itineraries of GLUT4. However, the specific roles played by different Rab proteins in GLUT4 trafficking has been difficult to assess, primarily due to the complexity of endomembrane organization and trafficking. To address this problem, we recently performed advanced live cell imaging using total internal reflection fluorescence (TIRF) microscopy, which images objects ~150 nm from the PM, directly visualizing GLUT4 trafficking in response to insulin stimulation. Using IRAP-pHluorin to selectively label GSVs undergoing PM fusion in response to insulin, we identified Rab10 as the only Rab protein that binds this compartment. Rab14 was found to label transferrin-positive, endosomal compartments containing GLUT4. These also could fuse with the PM in response to insulin, albeit more slowly. Several other Rab proteins, including Rab4A, 4B and 8A, were found to mediate GLUT4 intra-endosomal recycling, serving to internalize surface-bound GLUT4 into endosomal compartments for ultimate delivery to GSVs. Thus, multiple Rab proteins regulate the circulation of GLUT4 molecules within the endomembrane system, maintaining optimal insulin responsiveness within cells.

}, issn = {1942-0889}, doi = {10.4161/cib.23779}, author = {Chen, Yu and Lippincott-Schwartz, Jennifer} } @article {47647, title = {Rab10 and myosin-Va mediate insulin-stimulated GLUT4 storage vesicle translocation in adipocytes.}, journal = {The Journal of cell biology}, volume = {198}, year = {2012}, month = {2012 Aug 20}, pages = {545-60}, abstract = {

Rab proteins are important regulators of insulin-stimulated GLUT4 translocation to the plasma membrane (PM), but the precise steps in GLUT4 trafficking modulated by particular Rab proteins remain unclear. Here, we systematically investigate the involvement of Rab proteins in GLUT4 trafficking, focusing on Rab proteins directly mediating GLUT4 storage vesicle (GSV) delivery to the PM. Using dual-color total internal reflection fluorescence (TIRF) microscopy and an insulin-responsive aminopeptidase (IRAP)-pHluorin fusion assay, we demonstrated that Rab10 directly facilitated GSV translocation to and docking at the PM. Rab14 mediated GLUT4 delivery to the PM via endosomal compartments containing transferrin receptor (TfR), whereas Rab4A, Rab4B, and Rab8A recycled GLUT4 through the endosomal system. Myosin-Va associated with GSVs by interacting with Rab10, positioning peripherally recruited GSVs for ultimate fusion. Thus, multiple Rab proteins regulate the trafficking of GLUT4, with Rab10 coordinating with myosin-Va to mediate the final steps of insulin-stimulated GSV translocation to the PM.

}, keywords = {3T3-L1 Cells, Adipocytes, Animals, Cell Membrane, Cytoplasmic Vesicles, Glucose Transporter Type 4, Insulin, Mice, Myosin Heavy Chains, Myosin Type V, Protein Transport, rab GTP-Binding Proteins}, issn = {1540-8140}, doi = {10.1083/jcb.201111091}, author = {Chen, Yu and Wang, Yan and Zhang, Jinzhong and Deng, Yongqiang and Jiang, Li and Song, Eli and Wu, Xufeng S and Hammer, John A and Xu, Tao and Lippincott-Schwartz, Jennifer} }