Architecture and control of exocytosis and endocytosis in excitable cells

数十种蛋白质控制着人类细胞中囊泡的对接和融合。许多这些蛋白质的身份和作用已经通过遗传学、生物化学和电生理学的结合来确定。然而，这些蛋白质的空间组织、异质性、调控和动力学尚未确定。找到这种组织是理解蛋白质如何在健康细胞中调节这些膜运输系统以及在疾病中可能发生故障的关键。因此，我们的目标是绘制在胞吐过程中起作用的关键蛋白。为了实现这一目标，我们开发了高通量活细胞成像，超分辨率荧光成像和电子显微镜的组合。通过这种多模态方法，在细胞中特定的囊泡群中确定了单个蛋白质的位置、动态和占用，并与组织质膜的潜在细胞结构进行了比较。这使我们能够确定胞外囊泡系统的基本组织，以及负责囊泡运输、捕获和融合的特定分子成分如何在时间和空间上聚集在一起并发挥作用。

Dozens of proteins control the docking and fusion of exocytic vesicles in human cells. The identity and roles of many of these proteins have been assigned through a combination of genetics, biochemistry, and electrophysiology. However, the spatial organization, heterogeneity, regulation, and dynamics of these proteins have yet to be determined. Finding this organization is key to understanding how proteins regulate these membrane trafficking systems in healthy cells and might malfunction in disease. Thus, we aimed to map key proteins proposed to act during exocytosis. To accomplish this, we developed a combination of high-throughput live cell imaging, super-resolution fluorescence imaging, and electron microscopy. Through this multi-modal approach the location, dynamics, and occupancy of individual proteins were determined at specific populations of vesicles in cells and compared to the underlying cellular architecture that organizes the plasma membrane. This allowed us to determine the fundamental organization of the exocytic vesicle system and how specific molecular components responsible for vesicle trafficking, capture, and fusion assemble together and function in time and space. Specifically, with TIRF microscopy and high-throughput image analysis we developed a universal map of the proteins that control exocytosis and provide a global network level analysis of vesicle fusion events. We were able to identify unique classes of key regulatory molecules that strongly associate with the vast majority of exocytic vesicles in both cultured neuorendocrine PC12 chromaffin and INS1 beta cells. Proteins we identified were Rabs and Rab effectors, SNARE proteins, SNARE modulators, and BAR-domain proteins, and mechanoenzymes including Dynamin. To determine the local dynamics of these molecules at single sites of exocytosis we imaged signal changes of proteins at the exact moment of fusion in live cells. In these studies we discovered an unexpected recruitment of several important endocytic proteins to sites of both synaptic-like microvesicle fusion and large dense-core granule fusion. These molecules include the regulatory proteins dynamin, amphiphysin, syndapin, and endophilin. We further discovered that mutations of several of these proteins altered the kinetics of vesicle membrane protein release into the plasma membrane. Our hypothesis is that these proteins (dynamin, syndapin, amphiphysin, and endophilin) regulate the dilation or permeability of the fusion pore to control the amount of membrane-bound cargo released during single exocytic fusion events. This would allow excitable cells to modulate the amount of material released during even single exocytic fusion events. With live cell imaging we discovered that after exocytosis vesicle material is captured on a dense network of pre-formed clathrin-coated structures following exocytosis. Despite the identification of many components of clathrin-mediated endocytosis, a structural understanding of how these molecules come together to build and retrieve material from the plasma membrane during endocytosis is incomplete. In this aim we determined the nanoscale structure of clathrin-coated vesicles responsible for endocytosis in mammalian cells. By understanding how proteins that have been functionally linked to endocytosis assemble together at the nanoscale we can place decades of biochemistry, cell biology, and genetics into a physical model of membrane retrieval. To determine the nanoscale structure of endocytic sites, we developed a super-resolution correlative light and electron microscopy imaging (CLEM) method. This allows us to image the nanometer-scale location of proteins in the context of their local cellular environment. Specifically, we succeeded in developing a robust pipeline for imaging the plasma membrane of cells with 2D super-resolution localization microscopy and transmission electron microscopy (TEM) of platinum replicas. In these studies we imaged the position of endocytic proteins at single clathrin-coated structures. We localized 19 other endocytic proteins (amphiphysin1, AP2, 2-arrestin, CALM, clathrin, DAB2, dynamin2, EPS15, epsin1, epsin2, FCHO2, HIP1R, intersectin, NECAP, SNX9, stonin2, syndapin2, transferrin receptor, VAMP2) on thousands of individual clathrin structures, generating a comprehensive molecular architecture of endocytosis with nano-precision in human Hela cells. From this work, we discovered that endocytic proteins distribute into distinct spatial zones (rings) in relation to the edge of the clathrin lattice. The presence or concentrations of specific proteins within these rings change at distinct stages of organelle development. We propose that endocytosis is driven by the recruitment, reorganization, and loss of proteins within these partitioned nanoscale zones. In total, these studies are allowing us to build structural models for how proteins are organized at single organelles to regulate endocytosis, a key process for all eukaryotic cells. Clathrin-coated pits dynamically assemble and disassemble at the membrane of mammalian cells. Models of how clathrin coats curve have been controversial. Specifically, some models propose that clathrin can only grow as a curved lattice while others propose that clathrin first assembles as a fully formed flat lattice that later curves into a sphere. We tested these models in two collaborative projects by imaging growing clathrin structures with both live cell polarized total internal reflection microscopy (pTIRF) and super-resolution EM correlative imaging. In these studies we watched single pits curve at the plasma membrane and related these observations to structures observed in EM. Our work demonstrated that the pathway for curvature is heterogenous. First, many clathrin sites form as small curved structures, others grow partially flat and then begin to curve, and some grow to their full size and bend. Thus, cells control the pathway of membrane curvature with multiple mechanisms of curvature generation in the lattice. In an effort to better understand how cells specifically regulate the assembly and curvature of single clathrin-coated endocytic structures we have been examining the geometric structural transitions in the clathrin-coat with high resolution 3D EM microscopy. To accomplish this, platinum-replica EM has been used to track and examine in detail how the lattices assemble and dynamically rearrange in living cells. These studies are showing that the clathrin lattice is dynamic and capable of assembling first as a flat lattice that then uses geometric transitions in the individual clathrin subunits to drive curvature. These structural transitions drive coat reassembly to re-shape a transport vesicle and control cellular signaling pathways. Because of the above findings, it was clear that even in a single cell the pathway of clathrin assembly is mixed and highly dynamic. To test if specific cell types have unique clathrin assembly pathways we again collaborated to explore how the endocytic system changed when stem cells were differentiated int