Abstract
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.
Highlights
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
We localize the proteins Rab27a, Rab3a, Granuphilin-a, Rabphilin3a, and Rim[2] on identified dense-core vesicles (DCVs), and quantify their nanometer-scale positions across entire vesicle populations docked to the plasma membranes
The plasma membrane that remains contains bound organelles including cytoskeletal filaments, membrane proteins, endocytic, exocytic vesicles, and unknown or unidentified objects[27]. When these living membranes are rapidly fixed, stabilized, dried, and coated with a thin layer of platinum and carbon, a high-contrast image of the membrane replica can be acquired with transmission electron microscopy—a method commonly called platinum replica electron microscopy (PREM)[29,30]
Summary
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. In the cell’s cytoplasm, activated Rabs bind to membranous organelles and recruit effector and adaptor proteins These modular multi-protein complexes dynamically associate with motors and SNAREs to coordinate vesicle movement, tethering, docking, and fusion[1,2,3]. Work from the past several decades has identified the specific Rab-GTPases Rab27a and Rab3a and their effectors Granuphilina, Rabphilin3a, and Rim[2] as regulators of dense-core vesicle tethering and docking in neurons, Chromaffin, INS-1, and PC12 cells[10,11,12,13]. Gold-labeled proteins were imaged with platinum replica EM tomography to pinpoint their 3D location with nanoscale precision These data provide a new comprehensive view of proteins assembled in and around docked exocytic vesicles at the plasma membrane of an excitable cell. Because vesicles can dock and fuse within milliseconds[24,25], this physical orientation and lack of clustering, layering, or reorganization of Rabs during docking could allow for the extremely rapid attachment and near-instantaneous fusion of vesicles at the plasma membrane in excitable cells[26]
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