Localization And Dynamics Of Intracellular Organelles

细胞器生物学部分，由Jennifer Lippincott-Schwartz领导，研究亚细胞细胞器（即内质网、核膜、高尔基体、核内体和溶酶体）行为的机制，包括它们的生物发生、维持、膜分选特性和细胞内动力学。目前的研究主要集中在：1)表征膜进出细胞器的途径及其在细胞器生物发生和维持中的作用；2)定义有丝分裂细胞器拆卸和重组的途径和机制；3)了解参与蛋白质转运的外壳蛋白复合物的作用和调控；4)开发工具来分析活细胞内的细胞器动力学和蛋白质运输；5)细胞内脂筏运动的特征及其在协调信号反应和产生细胞极性中的作用。为了确定高尔基体是否含有稳定成分，我们使用荧光光漂白技术研究了gfp标记的不同类别的高尔基蛋白（包括酶、基质蛋白、外壳蛋白和流动蛋白）与高尔基膜的结合方式。我们发现所有种类的高尔基成分都与这个细胞器有短暂的联系，这与稳定的细胞器模型的预测相反。酶和流动组分通过进出内质网的膜运输途径不断进出高尔基体，而高尔基基质蛋白和包覆体在膜和细胞质之间不断快速交换。当BFA处理和使用在GTPase周期的不同阶段被阻断的Arf1和Sar1突变体结构体抑制er到高尔基体的运输时，高尔基体结构被分解，没有留下残留的高尔基体元件。这些结果表明高尔基体是一个动态稳态系统，其维持依赖于内质网的持续输入，并受Sar1-COPII和Arf1-coatomer系统的活性调节。在有丝分裂过程中，许多膜结合细胞器，包括内质网和线粒体，基本上保持完整。然而，高尔基体是可逆分解的。高尔基体在有丝分裂过程中解体的确切原因尚不清楚，但普遍认为这是在子细胞之间等量分配高尔基体膜所必需的。我们已经通过使用gtp锁定的Arf1突变体或药物H89抑制高尔基体的有丝分裂分解来验证这一假设。在这些细胞中，尽管存在完整的高尔基体，染色体凝聚、纺锤体形成、核膜破裂和染色体在中期板上的排列都正常发生。引人注目的是，高尔基堆积仍然与中心粒紧密相关，并且在细胞分裂时在子细胞之间均匀分布。因此，数据表明，高尔基分解对于确保子细胞之间的高尔基分解是不必要的。此外，他们证明在没有高尔基分解的情况下，细胞仍然可以通过有丝分裂进行。然而，当高尔基体在有丝分裂过程中未能解体时，染色体不能正确分离，细胞质分裂往往不完整。这种表型可以通过在H89处理之前用BFA处理细胞来挽救，BFA可以从高尔基体中释放Arf1调节的外周蛋白。考虑到高尔基体上存在大量Arf1调节的外周蛋白，这些蛋白在细胞质分裂中具有已知的核功能和作用，我们目前正在研究是否有丝分裂的高尔基体分解依赖于Arf1失活，有助于从高尔基体中释放Arf1调节的蛋白，使其在有丝分裂的染色体分离和细胞质分裂中发挥作用。高尔基膜包衣和脱包衣的COPI和Arf1细胞胞质外壳蛋白的分析，可逆地结合到膜上，在分泌途径的膜运输中起核心作用。我们使用gfp标记的COPI和Arf1嵌合体来表征COPI的膜包衣/脱包衣周期以及gtp结合蛋白Arf1对其的调控。通过低温阻断囊泡形成，我们发现COPI开/关膜的循环以及Arf1对其的调控可以与囊泡形成分离。这表明来自生产性囊泡出芽的反馈对COPI脱膜不是必需的。基于这些结果和对COPI和Arf1膜结合/释放动力学的分析，我们提出Arf1和COPI的反复结合和随机释放周期有助于产生和维持分化的？膜域。这些结构域将比膜上单个COPI复合物的37秒寿命更长，并且将允许转运中间体以不同于COPI膜解离的时间尺度出芽。脂筏标记物在细胞表面和高尔基体之间的快速循环我们使用延时荧光显微镜和选择性光漂白技术来跟踪脂筏标记物的细胞内行程，包括CD59、GPI、GFP和霍乱和志贺毒素的脂质结合B亚基（分别为CTxB和STxB）。我们的数据表明，筏标记连续循环之间的PM和高尔基体。GPI-GFP以及一定比例的CTxB和STxB被排除在网格蛋白包被的坑中，独立于网格蛋白相互作用的内吞机制和实验5到达高尔基体。与网格蛋白无关的高尔基体通路紧随其后的筏标记物对胆固醇消耗和20℃敏感。这一途径可能对高尔基体中蛋白质机制和鞘糖脂的高尔基体回收很重要，鞘糖脂参与蛋白质的分类和运输。我们目前正在研究它在筏相关信号分子的分类和细胞极性的产生中的作用。

The section on organelle biology, led by Jennifer Lippincott-Schwartz, investigates the mechanisms underlying the behavior of subcellular organelles (i.e., ER, nuclear envelope, Golgi apparatus, endosomes and lysosomes), including their biogenesis, maintenance, membrane sorting properties and dynamics within cells. Current studies are aimed at: 1) characterizing the pathways for membrane transport into and out of organelles, and their role in organelle biogenesis and maintenance; 2) defining the pathways and machinery underlying mitotic organelle disassembly and reassembly; 3) understanding the role and regulation of coat protein complexes involved in protein trafficking; 4) developing tools to analyze organelle dynamics and protein transport within live cells; and 5) characterizing lipid raft movement within cells, and its role in coordinating signaling responses and in generating cell polarity. Evidence that all Golgi proteins associate with the Golgi transiently and depend on the integrity of ER export for correct targetting To determine whether the Golgi apparatus contains stable components, we used fluorescence photobleaching techniques to investigate the manner in which GFP-tagged members of different classes of Golgi proteins, including enzymes, matrix proteins, coat proteins and itinerant proteins, associate with Golgi membranes. We found that all classes of Golgi components are transiently associated with this organelle, contrary to the prediction of a stable organelle model. Enzymes and itinerant components were found to continuously exit and re enter the Golgi apparatus by membrane trafficking pathways to and from the ER, while Golgi matrix proteins and coatomer underwent constant, rapid exchange between membrane and cytoplasm. When ER-to-Golgi transport was inhibited by BFA treatment and the use of mutant constructs of Arf1 and Sar1 that are blocked at different stages of the GTPase cycle, Golgi structure disassembled leaving no residual Golgi elements. These results reveal that the Golgi apparatus is a dynamic steady-state system, whose maintenance depends on continual input from the ER and is regulated by the activities of the Sar1-COPII and Arf1-coatomer systems. Mitotic disassembly of the Golgi apparatus and its role in nuclear and cytoplasmic division During mitosis many membrane-bound organelles, including the ER and mitochondria, remain essentially intact. The Golgi apparatus, however, reversibly disassembles. Exactly why the Golgi disassembles during mitosis is not known, but is widely assumed to be required for partitioning Golgi membranes equivalently between daughter cells. We have tested this hypothesis by inhibiting mitotic breakdown of the Golgi apparatus using a GTP-locked Arf1 mutant or the drug H89. In these cells, chromosome condensation, spindle formation, nuclear envelope breakdown and chromosomes alignment at the metaphase plate all occurred normally despite the presence of an intact Golgi. Strikingly, Golgi stacks remained tightly associated with centrioles, and distributed equally between daughter cells at cytokinesis. The data indicate, therefore, that Golgi breakdown is not necessary for ensuring Golgi partitioning between daughter cells. Moreover, they demonstrate that the cell can still progress through mitosis in the absence of Golgi breakdown. However, when the Golgi failed to disassemble during mitosis, chromosomes didn't segregate properly and cytokinesis was often incomplete. This phenotype could be rescued by treating cells with BFA prior to H89 treatment, which released Arf1 regulated peripheral proteins from the Golgi. Given the presence of numerous Arf1-regulated peripheral proteins on the Golgi that have known nuclear functions and roles in cytokinesis, we are currently investigating whether mitotic Golgi disassembly, shown to be dependent on Arf1 inactivation, serves to release Arf1-regulated proteins from the Golgi, allowing them to function in mitotic chromosome segregation and cytokinesis. Analysis of Golgi membrane coating and uncoating by COPI and Arf1 Cytosolic coat proteins that bind reversibly to membranes play a central role in membrane transport within the secretory pathway. We have used GFP-tagged COPI and Arf1 chimeras to characterize the membrane coating/uncoating cycle of COPI and its regulation by the small GTP-binding protein, Arf1. Using low temperature to block vesicle formation, we show that cycling of COPI on/off membranes and its regulation by Arf1 can be uncoupled from vesicle formation. This demonstrates that feedback from productive vesicle budding is not necessary for COPI uncoating. Based on these results and an analysis of COPI and Arf1 membrane binding/release kinetics, we propose that repeated cycles of binding and stochastic release of Arf1 and COPI serve to generate and maintain differentiated ?coated? membrane domains. These domains would last longer than the 37 sec life-time of an individual COPI complex on membranes and would allow transport intermediates to bud on a different time-scale than COPI membrane dissociation. Rapid cycling of lipid raft markers between the cell surface and Golgi apparatus We have used time-lapse fluorescence microscopy and selective photobleaching techniques to follow the intracellular itineraries of lipid raft markers, including CD59, GPI GFP, and lipid binding B subunits of cholera and shiga toxins (CTxB and STxB, respectively). Our data show that the raft markers cycle continuously between the PM and Golgi apparatus. GPI-GFP, and a proportion of CTxB and STxB, were excluded from clathrin-coated pits and reached the Golgi apparatus independently of both clathrin-interacting endocytic machinery and rab 5. The clathrin-independent pathway to the Golgi followed by raft markers was sensitive to cholesterol depletion and to 20?C. This pathway is likely to be important for Golgi retrieval of protein machinery and glycosphingolipids that are involved in protein sorting and trafficking within the Golgi. We are currently investigating its role in sorting of raft-associated signaling molecules and in the generation of cell polarity.