Organization and Dynamics of Endomembrane Pathways and Organelles

Rab蛋白是胰岛素刺激的GLUT4转运到质膜（PM）的重要调节因子，但特定Rab蛋白调节GLUT4转运的确切步骤尚不清楚。为了解决这一问题，我们系统地研究了Rab蛋白在GLUT4运输中的作用，重点研究了Rab蛋白直接介导GLUT4储存囊泡（GSV）向PM的传递。利用双色全内反射荧光（TIRF）显微镜和胰岛素反应性氨基肽酶(IRAP)-pHluorin融合实验，我们证明Rab10直接促进GSV转运到PM并在PM上对接。Rab14通过含有转铁蛋白受体（TfR）的内体室介导GLUT4递送至PM，而Rab4A、Rab4B和Rab8A通过内体系统回收GLUT4。Myosin-Va通过与Rab10相互作用与GSVs相关联，定位外周募集的GSVs最终融合。因此，多种Rab蛋白调节GLUT4的运输，其中Rab10与肌球蛋白- va协调介导胰岛素刺激的GSV转运到PM的最后步骤。

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.