Biology subjects

Hinshaw, J. E.

Publications and source records attributed to Hinshaw, J. E..

6 recordsLinked to original sources

bioRxiv2024

Cryo-electron tomography pipeline for plasma membranes.

Cryo-electron tomography (cryoET) provides sub-nanometer protein structure within the dense cellular environment. Existing sample preparation methods are insufficient at accessing the plasma membrane and its associated proteins. Here, we present a correlative cryo-electron tomography pipeline optimally suited to image large ultra-thin areas of isolated basal and apical plasma membranes. The pipeline allows for angstrom-scale structure determination with sub-tomogram averaging and employs a genetically-encodable rapid chemically-induced electron microscopy visible tag for marking specific proteins within the complex cell environment. The pipeline provides fast, efficient, distributable, low-cost sample preparation and enables targeted structural studies of identified proteins at the plasma membrane of cells.

cell biology↗

bioRxiv2022

Molecular mechanics underlying flat-to-round membrane budding in live secretory cells

Membrane budding entails forces to transform flat membrane into vesicles essential for cell survival. Accumulated studies have identified coat-proteins (e.g., clathrin) as potential budding factors. However, forces mediating many non-coated membrane buddings remain unclear. By visualizing proteins in mediating endocytic budding in live neuroendocrine cells, performing in vitro protein reconstitution and physical modelling, we discovered how non-coated-membrane budding is mediated: actin filaments and dynamin generate a pulling force transforming flat membrane into {Lambda}-shape; subsequently, dynamin helices surround and constrict {Lambda}-profiles base, transforming {Lambda}- to {Omega}-profile, and then constrict {Omega}-profiles pore, converting {Omega}-profiles to vesicles. These mechanisms control budding speed, vesicle size and number, generating diverse endocytic modes differing in these parameters. Their impact is widespread beyond secretory cells, as the unexpectedly powerful functions of dynamin and actin, previously thought to mediate fission and overcome tension, respectively, may contribute to many dynamin/actin-dependent non- coated-membrane buddings, coated-membrane buddings, and other membrane remodelling processes.

cell biology↗

bioRxiv2021

Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding

Transmembrane {beta}-barrel proteins are folded into the outer membrane (OM) of Gram-negative bacteria by the {beta}-barrel assembly machine (BAM) via an unexplained process that occurs without known external energy sources. Here we used single-particle cryo-EM to visualize the folding dynamics of a model {beta}-barrel protein (EspP) by BAM. We found that BAM binds the highly conserved "{beta}-signal" motif of EspP to correctly orient {beta}-strands in the OM during folding. We also found that the folding of EspP proceeds via remarkable "hybrid-barrel" intermediates in which membrane integrated {beta}-sheets are attached to the essential BAM subunit, BamA. The structures show an unprecedented deflection of the membrane surrounding the EspP intermediates and suggest that {beta}-sheets progressively fold towards BamA to form a {beta}-barrel. Along with in vivo experiments that tracked {beta}-barrel folding while the OM tension was modified, our results support a model in which BAM harnesses OM elasticity to accelerate {beta}-barrel folding.

biochemistry↗

bioRxiv2020

The structure and spontaneous curvature of clathrin lattices at the plasma membrane.

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 longstanding debate. Here, we use platinum replica and cryo electron microscopy and tomography to present a global 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 new universal mechanistic model of clathrin-mediated endocytosis.

cell biology↗

bioRxiv2020

Time-resolved cryoEM using Spotiton

We present an approach for preparing cryoEM grids to study short-lived molecular states. Using piezo electric dispensing, two independent streams of ~50 pL sample drops are deposited within 10 ms of each other onto a nanowire EM grid surface, and the mixing reaction stops when the grid is vitrified in liquid ethane, on the order of ~100 ms later. We demonstrate the utility of this approach for four biological systems where short-lived states are of high interest.

biophysics↗

bioRxiv2019

The mechanisms of dynamin-actin interaction

Cell-cell fusion is an indispensable process in the conception, development and physiology of multicellular organisms. Here we demonstrate a direct and noncanonical role for dynamin, best known as a fission GTPase in endocytosis, in cell-cell fusion. Our genetic and cell biological analyses show that dynamin colocalizes within the F-actin-enriched podosome-like structures at the fusogenic synapse, which is required for generating invasive membrane protrusions and myoblast fusion in vivo, in an endocytosis-independent manner. Biochemical, negative stain EM and cryo-electron tomography (cryo-ET) analyses revealed that dynamin forms helices that directly bundles actin filaments by capturing multiple actin filaments at their outer rim via interactions with dynamins proline-rich domain. GTP hydrolysis by dynamin triggers disassembly of the dynamin helix, exposes the sides of the actin filaments, promotes dynamic Arp2/3-mediated branched actin polymerization, and generates a mechanically stiff actin network. Thus, dynamin functions as a unique actin-bundling protein that enhances mechanical force generation by the F-actin network in a GTPase-dependent manner. Our findings have universal implications for understanding dynamin-actin interactions in various cellular processes beyond cell-cell fusion.

cell biology↗