Organization and Dynamics of Endomembrane Pathways and Organelles

Rab蛋白是胰岛素刺激的GLUT4转运至质膜（PM）的重要调节剂，但特定Rab蛋白调节的GLUT4转运的精确步骤仍不清楚。为了解决这个问题，我们系统地研究了Rab蛋白在GLUT4运输中的参与，重点是Rab蛋白直接介导GLUT4储存囊泡（GSV）递送到PM。使用双色全内反射荧光（TIRF）显微镜和胰岛素反应性氨肽酶（IRAP）-pHluorin融合试验，我们证明Rab10直接促进GSV易位和对接在PM。Rab14通过含有转铁蛋白受体（TfR）的内体隔室介导GLUT 4递送至PM，而Rab4A、Rab4B和Rab8A通过内体系统回收GLUT 4。肌球蛋白-Va显示通过与Rab10相互作用与GSV相关联，定位外周募集的GSV以最终融合。因此，多种Rab蛋白调节GLUT 4的运输，Rab 10与肌球蛋白-Va协调以介导胰岛素刺激的GSV易位至PM的最后步骤。 细胞极化需要增加细胞能量和代谢输出，但极化细胞如何满足这些能量需求尚不清楚。为了解决这些问题，我们研究了线粒体生物能量学和自噬在胶原蛋白夹心系统中培养的肝细胞极化过程中的作用。我们发现，当肝细胞开始极化时，它们利用氧化磷酸化来提高ATP水平，而这种能量的产生是极化所必需的。细胞极化后，肝细胞转变为更加依赖糖酵解产生ATP。沿着这种对氧化磷酸化的中心依赖，氧化磷酸化是极化培养物中ATP产生的主要来源，在极化的时间过程中，其他几种代谢过程被重新编程。随着细胞增殖，线粒体伸长，线粒体膜电位增加。此外，脂滴丰度随时间降低。这些研究结果表明，极化细胞是依赖于脂肪酸氧化，这是支持的药理学抑制氧化依托莫西。β最后，自噬在细胞极化过程中上调，抑制自噬延缓细胞极化。总之，我们的研究结果描述了一种代谢转变，涉及许多协调的代谢途径，最终有助于增加细胞极化过程中的能量产生。 在有丝分裂结束时两个子细胞的物理分离，称为细胞动力学分离，涉及一个狭窄的、基于微管的细胞间桥的分裂，该桥连接细胞分裂期间产生的两个新生子细胞。我们一直在研究转运所需的内体分选复合物（ESCRT）-III复合物在这一过程中的作用。使用高分辨率，定量成像的ESCRT-III在细胞动力学双分裂，我们观察到，ESCRT-III最初组装在中间体暗区，然后向外聚合的网站的细胞动力学双分裂。将这些观察结果与已知的ESCRT-III复合物的生物物理特性相结合，然后我们制定并测试了ESCRT介导的细胞动力学抑制的计算模型。在这个模型中，ESCRT-III形成一个分裂复合物，驱动细胞间细胞动力学桥的收缩和分裂。ESCRT-III裂变复合物的产生是由于VPS4使初始ESCRT-III寡聚体断裂，在中间体暗区的边缘聚合。一旦形成，裂变复合物收缩到50 nm的自发直径，同时沿着细胞间桥滑动离开中间体暗区。滑动继续，直到裂变复合物达到桥膜的最小弹性能量，这是裂变发生的地方。我们证实了这个模型的理论分析的膜弹性能和实验验证的主要模型假设。 初级纤毛在感知和向细胞传递信息方面起着重要作用。我们假设，像运动纤毛，初级纤毛有可能形成粘连。为了验证这一点，我们检查了两种不同组织中的纤毛：视网膜中的光感受器和肝脏中的胆管细胞。在这两种环境中，我们观察到纤毛彼此接触。使用细胞培养模型结合荧光细胞成像，我们证明，纤毛从附近的细胞可以形成持久的，调节，糖蛋白依赖，纤毛纤毛粘连。此外，我们发现了细胞控制粘附释放的证据。我们认为，像运动纤毛的接触，初级纤毛的粘附功能相关。这些结果也表明，哺乳动物的初级纤毛可能是被动的，孤独的接收器。

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.