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. To address this issue, we systematically investigated 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 facilitates GSV translocation to and docking at the PM. Rab14 was shown to mediate 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 was shown to associate 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. Cell polarization requires increased cellular energy and metabolic output, but how these energetic demands are met by polarizing cells is unclear. To address these issues, we investigated the roles of mitochondrial bioenergetics and autophagy during cell polarization of hepatocytes cultured in a collagen sandwich system. We found that as the hepatocytes begin to polarize, they use oxidative phosphorylation to raise their ATP levels, and this energy production is required for polarization. After the cells are polarized, the hepatocytes shift to become more dependent on glycolysis to produce ATP. Along with this central reliance on oxidative phosphorylation as the main source of ATP production in polarizing cultures, several other metabolic processes are reprogrammed during the time course of polarization. As the cells polarize, mitochondria elongate and mitochondrial membrane potential increases. In addition, lipid droplet abundance decreases over time. These findings suggest that polarizing cells are reliant on fatty acid oxidation, which is supported by pharmacologic inhibition of β-oxidation by etomoxir. Finally, autophagy is up-regulated during cell polarization, with inhibition of autophagy retarding cell polarization. Taken together, our results describe a metabolic shift involving a number of coordinated metabolic pathways that ultimately serve to increase energy production during cell polarization. The physical separation of two daughter cells at the end of mitosis, known as cytokinetic abscission, involves cleavage of a narrow, microtubule-based, intercellular bridge that connects two nascent daughter cells arising during cell division. We have been investigating the role of the endosomal sorting complex required for transport (ESCRT)-III complex in this process. Using high resolution, quantitative imaging of ESCRT-III during cytokinetic abscission, we observed that ESCRT-III initially assembles at the midbody dark zone and then polymerizes outward to the site of cytokinetic abscission. Integrating these observations with the known biophysical properties of ESCRT-III complexes, we then formulated and tested a computational model for ESCRT-mediated cytokinetic abscission. In this model, ESCRT-III forms a fission complex that drives constriction and abscission of the intercellular cytokinetic bridge. The ESCRT-III fission complex arises as a result of VPS4-enabled breakage of the initial ESCRT-III oligomer, polymerizing at the edge of the midbody dark zone. Once formed, the fission complex constricts to its spontaneous diameter of 50 nm, while sliding along the intercellular bridge away from the midbody dark zone. Sliding continues until the fission complex reaches the minimal elastic energy of the bridge membrane, which is where fission occurs. We substantiated this model by theoretical analysis of the membrane elastic energy and by experimental verification of the major model assumptions. Primary cilia have major roles in sensing and transmitting information into cells. We hypothesized that like motile cilia, primary cilia have the potential to form adhesions. To test this, we examined cilia in two different tissues: photoreceptors in the retina and cholangiocytes in liver. In both of these environments we observed cilia form contacts with each other. Using a cell culture model combined with fluorescent cell imaging we demonstrated that cilia from nearby cells could form persistent, regulated, glycoprotein dependent, cilia-cilia adhesions. In addition, we found evidence for cellular control of adhesion release. We suggest that like the contacts made by motile cilia, adhesion of primary cilia is functionally relevant. These results also suggest that mammalian primary cilia may be more than passive, solitary receivers.

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Guo, Ming; Ehrlicher, Allen J; Jensen, Mikkel H et al. (2014) Probing the stochastic, motor-driven properties of the cytoplasm using force spectrum microscopy. Cell 158:822-32
Burnette, Dylan T; Shao, Lin; Ott, Carolyn et al. (2014) A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells. J Cell Biol 205:83-96
Grove, Joe; Metcalf, Daniel J; Knight, Alex E et al. (2014) Flat Clathrin Lattices: stable features of the plasma membrane. Mol Biol Cell :
Van Engelenburg, Schuyler B; Shtengel, Gleb; Sengupta, Prabuddha et al. (2014) Distribution of ESCRT machinery at HIV assembly sites reveals virus scaffolding of ESCRT subunits. Science 343:653-6
Homolya, Laszlo; Fu, Dong; Sengupta, Prabuddha et al. (2014) LKB1/AMPK and PKA control ABCB11 trafficking and polarization in hepatocytes. PLoS One 9:e91921
Lippincott-Schwartz, Jennifer (2014) Foreword. Histochem Cell Biol 142:1-2
Shcherbakova, Daria M; Sengupta, Prabuddha; Lippincott-Schwartz, Jennifer et al. (2014) Photocontrollable fluorescent proteins for superresolution imaging. Annu Rev Biophys 43:303-29
Satpute-Krishnan, Prasanna; Ajinkya, Monica; Bhat, Savithri et al. (2014) ER stress-induced clearance of misfolded GPI-anchored proteins via the secretory pathway. Cell 158:522-33
Chen, Antony K; Sengupta, Prabuddha; Waki, Kayoko et al. (2014) MicroRNA binding to the HIV-1 Gag protein inhibits Gag assembly and virus production. Proc Natl Acad Sci U S A 111:E2676-83
Sengupta, Prabuddha; van Engelenburg, Schuyler B; Lippincott-Schwartz, Jennifer (2014) Superresolution imaging of biological systems using photoactivated localization microscopy. Chem Rev 114:3189-202

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