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 endocytosis 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 endocytosis. To accomplish this, we developed a combination of high-throughput live cell imaging, super-resolution fluorescence imaging, and electron microscopy to directly visualize organelles. 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. 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 an effort to understand the assembly and curvature of single clathrin-coated endocytic structures, we 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. Conformational changes in CLC have been shown to regulate triskelia assembly in solution, yet the nature of these structural changes, and their effects on lattice growth, curvature, and endocytosis in cells are unclear. Here, we develop a correlative fluorescence resonance energy transfer (FRET) and platinum replica electron microscopy method, named FRET-CLEM. With FRET-CLEM, we measured conformational changes in proteins at thousands of individual morphologically distinct clathrin-coated structures across cell membranes. We find that the N-terminus of CLC moves away from the plasma membrane and triskelia vertex as lattices curve. Preventing this conformational switch with acute chemical tools inside cells increased clathrin structure sizes and inhibited endocytosis. Therefore, a specific conformational switch in CLC regulates lattice curvature and endocytosis in mammalian cells. 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. 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. Another endocytic and signaling organelle in human cells are caveolae. Caveolae are 80-nm diameter coated vesicles that invaginate into the cytosol from the plasma membrane. Their diverse functions span from endocytosis to signaling, regulating key cellular processes including lipid uptake, pathogen entry, and membrane tension. Caveolae undergo shape changes from flat to curved. We developed a correlative multi-color stimulated emission depletion (STED) fluorescence and platinum replica EM imaging (CLEM) method to image caveolae-associated proteins at caveolae of different shapes at the nanoscale. Caveolins and cavins were found at all caveolae, independent of their curvature. EHD2, a classic caveolar neck protein, was strongly detected at both curved and flat caveolae. Both pacsin2 and the regulator EHBP1 were found only at a subset of caveolae. Pacsin2 was localized primarily to areas surrounding flat caveolae, whereas EHBP1 was mostly detected at spheres. Contrary to classic models, dynamin was absent from caveolae and localized only to clathrin-coated structures. Cells lacking dynamin showed no substantial changes to caveolae, suggesting that dynamin is not directly involved in caveolae curvature. Together, we provide a mechanistic map for the molecular control of caveolae shape by eight of the major caveolae-associated coat and regulatory proteins. We propose a model where caveolins, cavins, and EHD2 assemble as a cohesive structural unit regulated by more intermittent associations with pacsin2 and EHBP1. These complexes can flatten and curve, capturing membrane to enable lipid and protein traffic and changes to the surface area of the cell.