Architecture and control of exocytosis and endocytosis in excitable cells

National Heart, Lung, And Blood Institute

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Abstract

Dozens of proteins control the docking and fusion of exocytic vesicles in mammalian 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, dynamics, and mechanisms of these proteins have not yet been determined. These data are key to understanding how proteins regulate membrane trafficking in healthy cells and malfunction in diseases. Thus, we aimed to map key proteins that act during exocytosis. To accomplish this, we developed a combination of high-throughput live cell imaging, super-resolution fluorescence imaging, and electron microscopy to directly visualize organelles. Through this multi-modal approach the location, dynamics, occupancy, and mechanisms of individual proteins were determined at specific populations of vesicles in cells. This allowed us to determine the fundamental organization of exocytic vesicles and how specific molecular components responsible for trafficking, capture, and fusion assemble together and function in time and space at the plasma membrane of cells. Specifically, we developed a universal map of proteins that control exocytosis and provide a network level analysis of vesicle fusion events with TIRF microscopy and image analysis. We were able to identify unique classes of key regulatory molecules that strongly associate with the vast majority of exocytic vesicles in both cultured neuroendocrine PC12 chromaffin and INS1 beta cells. Proteins we identified were Rabs and their effectors, SNARE proteins, SNARE modulators, and BAR-domain proteins, and mechanoenzymes including dynamin. The nanoscale distribution of key exocytotic proteins on vesicles are mostly unknow. How proteins assemble together on an organelle regulates the activity of the organelle. To determine how proteins are arranged on exocytic vesicles, we developed a three part light, super-resolution, and electron microscopy correlative imaging method. With this method we mapped how the Rab proteins Rab27a and Rab3a, Rab partners including Rabphillin, Granuphilin, and RIM, assemble on docked vesicles at the plasma membrane. We found that these proteins assembled around the entire vesicle membrane and are not layered. To further map these proteins in 3D, we used a semi-synthetic nanogold labeling method to attach gold nanoparticles to histidine-tagged fusion proteins. With this method, we could located proteins at the nanoscale in platinum replica electron tomograms. Again, we found that Rab proteins and their binding partners globally distribute around docked vesicles and SNARE proteins assemble at the base of vesicles. These data provide a physical view of how proteins direct exocytic vesicle fusion at the plasma membrane of mammalian cells. The primary mechanism of uptake in mammalian cells is clathrin-mediated endocytosis. To determine the nanoscale structure of clathrin-coated endocytic sites in living cells, we used a super-resolution correlative light and electron microscopy imaging (CLEM) method. This allowed us to image the nanometer-scale location of proteins in the context of their local cellular environment. Specifically, we imaged the plasma membrane of cells with 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 summary, these studies allow us to build structural models for how proteins are organized at single organelles to regulate endocytosis, a key process for all eukaryotic cells. In an effort to better understand the assembly and curvature of single clathrin-coated endocytic structures, we next studied the specific geometric transitions in the clathrin-coat with high resolution 3D and cryogenic electron tomography. To accomplish this, platinum-replica EM was used to track and examine in detail how the lattices assemble and dynamically rearrange across 8 different common cells lines. These studies showed that the clathrin lattice is capable of assembling first as a flat lattice that then can spontaneously curve into a dome without added energy or factors. These structural transitions drive coat assembly to re-shape a transport vesicle and control cellular signaling pathways. Cryogenic electron tomography showed that flat lattices are disordered and contain many pentagons. We propose that the ordering of this lattice drives spontaneous curvature. Furthermore, we propose that flat lattices are held flat with adhesion forces to the extracellular matrix. The release of these forces and physical properties of the membrane are needed to initiate curvature. This is a new updated mechanistic model of clathrin lattice curvature. Endocytosis is responsible for the capture of many important receptors and cargos into cells. In B cells of the immune system, B cell receptors (BCR) bind, gather, and then internalize antigens. This is required for antigen processing and adaptive immunity. We examined the clustering and endocytosis of activated BCRs with light and electron microscopy. First, we found that increasing concentrations of antigen caused progressively larger receptor clusters at the plasma membrane. When these clusters were small, we found that single clathrin coated pits associate with the clusters and were required for internalization. As the receptor clusters increased in size beyond the size of a single coated pit, we discovered that BCR clusters were internalized by large invaginations of the plasma membrane. These large smooth membrane invaginations were coated in clathrin structures and colocalized with ligand-bound receptors. With these data we describe a new form of endocytosis responsible for the capture and processing of large antigen clusters into immune B cells. This process is key to a robust immune response in humans and is likely disturbed in disease. One of the major signaling pathways in human cells needed for growth, differentiation, and homeostasis is the epidermal growth factor receptor pathway. Here, extracellular growth factor ligands activate plasma membrane receptor kinases to generate intracellular signals that modulate gene expression programs. EGFR is a common target for cancer therapy. The amount of active EGFR on the cells surface is through to be controlled by uptake of the receptor by clathrin mediated endocytosis. We discovered that activation of the EGFR receptor causes a massive increase in the number and size of flat clathrin lattices in the plasma membrane. These flat clathrin lattices cluster the EGFR receptor and contain the Beta V integrin. Manipulation of EGFR, SRC kinase, and the integrin by drugs or knockdown prevented the formation of flat clathrin lattices needed for signaling of the receptor. Thus, we propose that flat clathrin latices act as singling hubs to co-cluster and control these three interconnected signaling systems at the plasma membrane of human cells.