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Rab蛋白调控GLUT4转运的确切步骤尚不清楚。为了解决这个问题，我们系统地研究了Rab蛋白在GLUT4运输中的作用，重点是Rab蛋白直接介导GLUT4存储囊泡(GSV)传递到PM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融合分析，我们证明了Rab10直接促进GSV转位到PM并对接Rab14通过含有转铁蛋白受体(TFR)的内体隔室将GLUT4传递到PM，而Rab4A、Rab4B和Rab8A通过内体系统循环GLUT4。肌球蛋白-Va通过与Rab10相互作用，定位外周招募的GSV以实现最终融合，从而与GSVs相关联。因此，多个Rab蛋白调节GLUT4的运输，其中Rab10与myosin-Va协调，介导胰岛素刺激的GSV转位到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，它们使用氧化磷酸化来提高其ATP水平，而这种能量产生是极化所必需的。在细胞极化后，肝细胞转变为更加依赖糖酵解来产生ATP。除了这种对氧化磷酸化作为极化培养中ATP产生的主要来源的中心依赖之外，其他几个代谢过程在极化过程中被重新编程。随着细胞极化，线粒体拉长，线粒体膜电位增加。此外，随着时间的推移，脂滴的丰度会降低。这些发现表明极化细胞依赖于脂肪酸氧化，这是由依托莫西对氧化的药物抑制所支持的。最后，自噬在细胞极化过程中被上调，抑制自噬会延缓细胞极化。综上所述，我们的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期间的能量产生。 有丝分裂结束时两个子细胞的物理分离，称为细胞分裂，涉及一条狭窄的、以微管为基础的细胞间桥的分裂，该桥连接在细胞分裂过程中产生的两个新生子细胞。我们一直在研究运输所需的内体分选复合体(ESCRT)-III复合体在这一过程中的作用。利用ESCRT-III在细胞分裂过程中的高分辨率定量成像，我们观察到ESCRT-III最初在中体暗区聚集，然后向外聚合到细胞分裂的部位。将这些观察结果与ESCRT-III复合体的已知生物物理性质结合起来，我们随后制定并测试了ESCRT介导的细胞动力学脱落的计算模型。在这个模型中，ESCRT-III形成一个裂变复合体，驱动细胞间细胞动桥的收缩和脱落。ESCRT-III裂变复合体的产生是Vps4使最初的ESCRT-III寡聚体断裂的结果，在中体暗区的边缘聚合。一旦形成，裂变复合体就会收缩到50纳米的自发直径，同时沿着细胞间桥滑动，远离中体暗区。滑动会继续，直到裂变复合体达到桥膜的最小弹性能，也就是裂变发生的地方。我们通过对膜弹性能的理论分析和对主要模型假设的实验验证，证实了该模型。 初级纤毛在感知和向细胞内传递信息方面起着重要作用。我们假设，与活动纤毛一样，初级纤毛也有可能形成粘连。为了验证这一点，我们研究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，我们观察到纤毛相互接触。使用细胞培养模型结合荧光细胞成像，我们证明了来自附近细胞的纤毛可以形成持久的、受调节的、依赖于糖蛋白的纤毛-纤毛粘连。此外，我们还发现了细胞控制黏附释放的证据。我们认为，就像由活动纤毛形成的接触一样，初级纤毛的粘连在功能上是相关的。这些结果还表明，哺乳动物的初级纤毛可能不仅仅是被动的、孤立的感受器。

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.