RESUMO
Rab-GTPases and their interacting partners are key regulators of secretory vesicle trafficking, docking, and fusion to the plasma membrane in neurons and neuroendocrine cells. Where and how these proteins are positioned and organized with respect to the vesicle and plasma membrane are unknown. Here, we use correlative super-resolution light and platinum replica electron microscopy to map Rab-GTPases (Rab27a and Rab3a) and their effectors (Granuphilin-a, Rabphilin3a, and Rim2) at the nanoscale in 2D. Next, we apply a targetable genetically-encoded electron microscopy labeling method that uses histidine based affinity-tags and metal-binding gold-nanoparticles to determine the 3D axial location of these exocytic proteins and two SNARE proteins (Syntaxin1A and SNAP25) using electron tomography. Rab proteins are distributed across the entire surface and t-SNARE proteins at the base of docked vesicles. We propose that the circumferential distribution of Rabs and Rab-effectors could aid in the efficient transport, capture, docking, and rapid fusion of calcium-triggered exocytic vesicles in excitable cells.
Assuntos
Imagem Molecular/métodos , Células Neuroendócrinas/citologia , Vesículas Secretórias/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Carbocianinas/química , Células Cultivadas , Exocitose , Ouro , Células HeLa , Humanos , Imageamento Tridimensional , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Nanopartículas Metálicas/química , Microscopia/métodos , Células Neuroendócrinas/metabolismo , Células PC12 , Ratos , Proteínas SNARE/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteína Vermelha FluorescenteRESUMO
Clathrin-mediated endocytosis is the primary pathway for receptor and cargo internalization in eukaryotic cells. It is characterized by a polyhedral clathrin lattice that coats budding membranes. The mechanism and control of lattice assembly, curvature, and vesicle formation at the plasma membrane has been a matter of long-standing debate. Here, we use platinum replica and cryoelectron microscopy and tomography to present a structural framework of the pathway. We determine the shape and size parameters common to clathrin-mediated endocytosis. We show that clathrin sites maintain a constant surface area during curvature across multiple cell lines. Flat clathrin is present in all cells and spontaneously curves into coated pits without additional energy sources or recruited factors. Finally, we attribute curvature generation to loosely connected and pentagon-containing flat lattices that can rapidly curve when a flattening force is released. Together, these data present a universal mechanistic model of clathrin-mediated endocytosis.
