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.

Spatial and signaling overlap of growth factor receptor systems at clathrin-coated sites.

Cellular communication is regulated at the plasma membrane by the interactions of receptor, adhesion, signaling, and endocytic proteins. Yet, the composition and control of these complexes in response to external cues remain unclear. We use high-resolution and high-throughput fluorescence imaging to map the localization of growth factor receptors and related proteins at single clathrin-coated structures in human squamous HSC3 cells. We find distinct protein signatures between control cells and cells stimulated with growth factors. Clathrin sites at the plasma membrane are preloaded with some receptors but not others. Stimulation with epidermal growth factor induces capture and concentration of epidermal growth factor-, fibroblast growth factor-, and low-density lipoprotein-receptors (EGFR, FGFR1, and LDLR). Regulatory proteins including ubiquitin ligase Cbl, the scaffold Grb2, and the mechanoenzyme dynamin2 are also recruited. Disrupting FGFR or EGFR activity with drugs prevents the recruitment of both EGFR and FGFR1. EGF was able to activate FGFR1 phosphorylation. Our data reveals novel co-clustering and activation of receptors and regulatory factors at clathrin-coated sites in response to stimulation by a single growth factor, EGF or FGF. This behavior integrates growth factor signaling and allows for complex responses to extracellular cues and drugs at the plasma membrane of human cells.

Crosstalk of growth factor receptors at plasma membrane clathrin-coated sites.

Cellular communication is regulated at the plasma membrane by the interactions of receptor, adhesion, signaling, exocytic, and endocytic proteins. Yet, the composition and control of these nanoscale complexes in response to external cues remain unclear. Here, we use high-resolution and high-throughput fluorescence imaging to map the localization of growth factor receptors and related proteins at single clathrin-coated structures across the plasma membrane of human squamous HSC3 cells. We find distinct protein signatures between control cells and cells stimulated with ligands. Clathrin sites at the plasma membrane are preloaded with some receptors but not others. Stimulation with epidermal growth factor induces a capture and concentration of epidermal growth factor-, fibroblast growth factor-, and low-density lipoprotein-receptors (EGFR, FGFR, and LDLR). Regulatory proteins including ubiquitin ligase Cbl, the scaffold Grb2, and the mechanoenzyme dynamin2 are also recruited. Disrupting FGFR or EGFR individually with drugs prevents the recruitment of both EGFR and FGFR. Our data reveals novel crosstalk between multiple unrelated receptors and regulatory factors at clathrin-coated sites in response to stimulation by a single growth factor, EGF. This behavior integrates growth factor signaling and allows for complex responses to extracellular cues and drugs at the plasma membrane of human cells.

Clathrin mediates membrane fission and budding by constricting membrane pores.

Membrane budding, which underlies fundamental processes like endocytosis, intracellular trafficking, and viral infection, is thought to involve membrane coat-forming proteins, including the most observed clathrin, to form Ω-shape profiles and helix-forming proteins like dynamin to constrict Ω-profiles' pores and thus mediate fission. Challenging this fundamental concept, we report that polymerized clathrin is required for Ω-profiles' pore closure and that clathrin around Ω-profiles' base/pore region mediates pore constriction/closure in neuroendocrine chromaffin cells. Mathematical modeling suggests that clathrin polymerization at Ω-profiles' base/pore region generates forces from its intrinsically curved shape to constrict/close the pore. This new fission function may exert broader impacts than clathrin's well-known coat-forming function during clathrin (coat)-dependent endocytosis, because it underlies not only clathrin (coat)-dependent endocytosis, but also diverse endocytic modes, including ultrafast, fast, slow, bulk, and overshoot endocytosis previously considered clathrin (coat)-independent in chromaffin cells. It mediates kiss-and-run fusion (fusion pore closure) previously considered bona fide clathrin-independent, and limits the vesicular content release rate. Furthermore, analogous to results in chromaffin cells, we found that clathrin is essential for fast and slow endocytosis at hippocampal synapses where clathrin was previously considered dispensable, suggesting clathrin in mediating synaptic vesicle endocytosis and fission. These results suggest that clathrin and likely other intrinsically curved coat proteins are a new class of fission proteins underlying vesicle budding and fusion. The half-a-century concept and studies that attribute vesicle-coat contents' function to Ω-profile formation and classify budding as coat-protein (e.g., clathrin)-dependent or -independent may need to be re-defined and re-examined by considering clathrin's pivotal role in pore constriction/closure.

Cryo-EM structures of membrane-bound dynamin in a post-hydrolysis state primed for membrane fission.

Dynamin assembles as a helical polymer at the neck of budding endocytic vesicles, constricting the underlying membrane as it progresses through the GTPase cycle to sever vesicles from the plasma membrane. Although atomic models of the dynamin helical polymer bound to guanosine triphosphate (GTP) analogs define earlier stages of membrane constriction, there are no atomic models of the assembled state post-GTP hydrolysis. Here, we used cryo-EM methods to determine atomic structures of the dynamin helical polymer assembled on lipid tubules, akin to necks of budding endocytic vesicles, in a guanosine diphosphate (GDP)-bound, super-constricted state. In this state, dynamin is assembled as a 2-start helix with an inner lumen of 3.4 nm, primed for spontaneous fission. Additionally, by cryo-electron tomography, we trapped dynamin helical assemblies within HeLa cells using the GTPase-defective dynamin K44A mutant and observed diverse dynamin helices, demonstrating that dynamin can accommodate a range of assembled complexes in cells that likely precede membrane fission.

Adhesion energy controls lipid binding-mediated endocytosis.

Several bacterial toxins and viruses can deform membranes through multivalent binding to lipids for clathrin-independent endocytosis. However, it remains unclear, how membrane deformation and endocytic internalization are mechanistically linked. Here we show that many lipid-binding virions induce membrane deformation and clathrin-independent endocytosis, suggesting a common mechanism based on multivalent lipid binding by globular particles. We create a synthetic cellular system consisting of a lipid-anchored receptor in the form of GPI-anchored anti-GFP nanobodies and a multivalent globular binder exposing 180 regularly-spaced GFP molecules on its surface. We show that these globular, 40 nm diameter, particles bind to cells expressing the receptor, deform the plasma membrane upon adhesion and become endocytosed in a clathrin-independent manner. We explore the role of the membrane adhesion energy in endocytosis by using receptors with affinities varying over 7 orders of magnitude. Using this system, we find that once a threshold in adhesion energy is overcome to allow for membrane deformation, endocytosis occurs reliably. Multivalent, binding-induced membrane deformation by globular binders is thus sufficient for internalization to occur and we suggest it is the common, purely biophysical mechanism for lipid-binding mediated endocytosis of toxins and pathogens.

Local monomer levels and established filaments potentiate non-muscle myosin 2 assembly.

The ability to dynamically assemble contractile networks is required throughout cell physiology, yet direct biophysical mechanisms regulating non-muscle myosin 2 filament assembly in living cells are lacking. Here, we use a suite of dynamic, quantitative imaging approaches to identify deterministic factors that drive myosin filament appearance and amplification. We find that actin dynamics regulate myosin assembly, but that the static actin architecture plays a less clear role. Instead, remodeling of actin networks modulates the local myosin monomer levels and facilitates assembly through myosin:myosin-driven interactions. Using optogenetically controlled myosin, we demonstrate that locally concentrating myosin is sufficient to both form filaments and jump-start filament amplification and partitioning. By counting myosin monomers within filaments, we demonstrate a myosin-facilitated assembly process that establishes filament stacks prior to partitioning into clusters that feed higher-order networks. Together, these findings establish the biophysical mechanisms regulating the assembly of non-muscle contractile structures that are ubiquitous throughout cell biology.

Adhesion energy controls lipid binding-mediated endocytosis.

Several bacterial toxins and viruses can deform membranes through multivalent binding to lipids for clathrin-independent endocytosis. However, it remains unclear, how membrane deformation and endocytic internalization are mechanistically linked. Here we show that many lipid-binding virions induce membrane deformation and clathrin-independent endocytosis, suggesting a common mechanism based on multivalent lipid binding by globular particles. We create a synthetic cellular system consisting of a lipid-anchored receptor in the form of GPI-anchored anti-GFP nanobodies and a multivalent globular binder exposing 180 regularly-spaced GFP molecules on its surface. We show that these globular, 40 nm diameter, particles bind to cells expressing the receptor, deform the plasma membrane upon adhesion and become endocytosed in a clathrin-independent manner. We explore the role of the membrane adhesion energy in endocytosis by using receptors with affinities varying over 7 orders of magnitude. Using this system, we find that once a threshold in adhesion energy is overcome to allow for membrane deformation, endocytosis occurs reliably. Multivalent, binding-induced membrane deformation by globular binders is thus sufficient for internalization to occur and we suggest it is the common, purely biophysical mechanism for lipid-binding mediated endocytosis of toxins and pathogens.

Local Monomer Levels and Established Filaments Potentiate Non-Muscle Myosin 2 Assembly.

The ability to dynamically assemble contractile networks is required throughout cell physiology, yet the biophysical mechanisms regulating non-muscle myosin 2 filament assembly in living cells are lacking. Here we use a suite of dynamic, quantitative imaging approaches to identify deterministic factors that drive myosin filament appearance and amplification. We find that actin dynamics regulate myosin assembly, but that the actin architecture plays a minimal direct role. Instead, remodeling of actin networks modulates the local myosin monomer levels and facilitates assembly through myosin:myosin driven interactions. Using optogenetically controlled myosin, we demonstrate that locally concentrating myosin is sufficient to both form filaments and jump-start filament amplification and partitioning. By counting myosin monomers within filaments, we demonstrate a myosin-facilitated assembly process that establishes sub-resolution filament stacks prior to partitioning into clusters that feed higher-order networks. Together these findings establish the biophysical mechanisms regulating the assembly of non-muscle contractile structures that are ubiquitous throughout cell biology.

A conformational switch in clathrin light chain regulates lattice structure and endocytosis at the plasma membrane of mammalian cells.

Conformational changes in endocytic proteins are regulators of clathrin-mediated endocytosis. Three clathrin heavy chains associated with clathrin light chains (CLC) assemble into triskelia that link into a geometric lattice that curves to drive endocytosis. Structural changes in CLC have been shown to regulate triskelia assembly in solution, yet the nature of these changes, and their effects on lattice growth, curvature, and endocytosis in cells are unknown. Here, we develop a new correlative fluorescence resonance energy transfer (FRET) and platinum replica electron microscopy method, named FRET-CLEM. With FRET-CLEM, we measure conformational changes in clathrin at thousands of individual morphologically distinct clathrin-coated structures. We discover that the N-terminus of CLC repositions away from the plasma membrane and triskelia vertex as coats curve. Preventing this conformational switch with chemical tools increases lattice sizes and inhibits endocytosis. Thus, a specific conformational switch in the light chain regulates lattice curvature and endocytosis in mammalian cells.

B cell receptor (BCR) endocytosis.

The B cell receptor (BCR) interacts with foreign antigens to mediate B cell activation and secretion of antibodies. B cell activation begins with initiation of signaling pathways, such as NFAT, NF-κB, and MAPK, and endocytosis of the BCR-antigen complex. Many studies have investigated the signaling pathways associated with BCR activation, and this work has led to significant advances in drug therapies to treat cancer and autoimmune diseases that are linked to aberrant BCR signaling. Less is known, however, about the mechanisms of BCR endocytosis and the role endocytosis plays in B cell pathogenesis. This chapter will review key characteristics of the BCR, including a review of signaling pathways, and endocytic mechanisms associated with the activated BCR. We will also review recent studies investigating the role of BCR endocytosis disease pathogenesis.

Three-dimensional structured illumination microscopy with enhanced axial resolution.

The axial resolution of three-dimensional structured illumination microscopy (3D SIM) is limited to ∼300 nm. Here we present two distinct, complementary methods to improve axial resolution in 3D SIM with minimal or no modification to the optical system. We show that placing a mirror directly opposite the sample enables four-beam interference with higher spatial frequency content than 3D SIM illumination, offering near-isotropic imaging with ∼120-nm lateral and 160-nm axial resolution. We also developed a deep learning method achieving ∼120-nm isotropic resolution. This method can be combined with denoising to facilitate volumetric imaging spanning dozens of timepoints. We demonstrate the potential of these advances by imaging a variety of cellular samples, delineating the nanoscale distribution of vimentin and microtubule filaments, observing the relative positions of caveolar coat proteins and lysosomal markers and visualizing cytoskeletal dynamics within T cells in the early stages of immune synapse formation.

Generation of nanoscopic membrane curvature for membrane trafficking.

Curved membranes are key features of intracellular organelles, and their generation involves dynamic protein complexes. Here we describe the fundamental mechanisms such as the hydrophobic insertion, scaffolding and crowding mechanisms these proteins use to produce membrane curvatures and complex shapes required to form intracellular organelles and vesicular structures involved in endocytosis and secretion. For each mechanism, we discuss its cellular functions as well as the underlying physical principles and the specific membrane properties required for the mechanism to be feasible. We propose that the integration of individual mechanisms into a highly controlled, robust process of curvature generation often relies on the assembly of proteins into coats. How cells unify and organize the curvature-generating factors at the nanoscale is presented for three ubiquitous coats central for membrane trafficking in eukaryotes: clathrin-coated pits, caveolae, and COPI and COPII coats. The emerging theme is that these coats arrange and coordinate curvature-generating factors in time and space to dynamically shape membranes to accomplish membrane trafficking within cells.

The molecular organization of differentially curved caveolae indicates bendable structural units at the plasma membrane.

Caveolae are small coated plasma membrane invaginations with diverse functions. Caveolae undergo curvature changes. Yet, it is unclear which proteins regulate this process. To address this gap, we develop a correlative stimulated emission depletion (STED) fluorescence and platinum replica electron microscopy imaging (CLEM) method to image proteins at single caveolae. Caveolins and cavins are found at all caveolae, independent of curvature. EHD2 is detected at both low and highly curved caveolae. Pacsin2 associates with low curved caveolae and EHBP1 with mostly highly curved caveolae. Dynamin is absent from caveolae. Cells lacking dynamin show no substantial changes to caveolae, suggesting that dynamin is not directly involved in caveolae curvature. We propose a model where caveolins, cavins, and EHD2 assemble as a cohesive structural unit regulated by intermittent associations with pacsin2 and EHBP1. These coats can flatten and curve to enable lipid traffic, signaling, and changes to the surface area of the cell.

Genome-wide CRISPR screen identified a role for commander complex mediated ITGB1 recycling in basal insulin secretion.

OBJECTIVES: Pancreatic beta cells secrete insulin postprandially and during fasting to maintain glucose homeostasis. Although glucose-stimulated insulin secretion (GSIS) has been extensively studied, much less is known about basal insulin secretion. Here, we performed a genome-wide CRISPR/Cas9 knockout screen to identify novel regulators of insulin secretion. METHODS: To identify genes that cell autonomously regulate insulin secretion, we engineered a Cas9-expressing MIN6 subclone that permits irreversible fluorescence labeling of exocytic insulin granules. Using a fluorescence-activated cell sorting assay of exocytosis in low glucose and high glucose conditions in individual cells, we performed a genome-wide CRISPR/Cas9 knockout screen. RESULTS: We identified several members of the COMMD family, a conserved family of proteins with central roles in intracellular membrane trafficking, as positive regulators of basal insulin secretion, but not GSIS. Mechanistically, we show that the Commander complex promotes insulin granules docking in basal state. This is mediated, at least in part, by its function in ITGB1 recycling. Defective ITGB1 recycling reduces its membrane distribution, the number of focal adhesions and cortical ELKS-containing complexes. CONCLUSIONS: We demonstrated a previously unknown function of the Commander complex in basal insulin secretion. We showed that by ITGB1 recycling, Commander complex increases cortical adhesions, which enhances the assembly of the ELKS-containing complexes. The resulting increase in the number of insulin granules near the plasma membrane strengthens basal insulin secretion.

Localizing Proteins on Single Trafficking Organelles in 3D with Semisynthetic Gold Labeling and Platinum Replica Electron Microscopy.

The three-dimensional structures of organelles can be visualized at high resolutions using electron microscopy and tomography. Combining genetically encoded tags with tomography enables the specific targeting and detection of identified proteins inside cells. Here, we describe a method for attaching metal-binding gold nanoparticles to proteins genetically tagged with hexa-histidine sequences. We apply this strategy to visualize the position of intracellular proteins on single organelles in unroofed cells with platinum replica electron microscopy at the nanoscale in three dimensions. We have found that this combined method can label and localize proteins with isotropic high precision to generate quantitative maps of protein positions in and around trafficking organelles at the inner plasma membrane of mammalian cells.