Biology subjects

Sochacki, K. A.

Publications and source records attributed to Sochacki, K. A..

10 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↗

bioRxiv2023

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.

cell biology↗

bioRxiv2022

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

The conformations of endocytic proteins and their interactions are key regulators of clathrin-mediated endocytosis. Three clathrin light chains (CLC), along with three clathrin heavy chains, assemble to form single clathrin triskelia that link into a geometric lattice that curves to drive endocytosis. 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 measure 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.

cell biology↗

bioRxiv2020

Dual clathrin and adhesion signaling systems regulate growth factor receptor activation

The crosstalk between growth factor and adhesion receptors is key for cell growth and migration. In pathological settings, these receptors are drivers of cancer. Yet, how growth and adhesion signals are spatially organized and integrated is poorly understood. Here we use quantitative fluorescence and electron microscopy to reveal a mechanism where flat clathrin lattices partition and activate growth factor signals via a coordinated response that involves crosstalk between epidermal growth factor receptor (EGFR) and the adhesion receptor {beta}5-integrin. We show that ligand-activated EGFR, Grb2, Src, and {beta}5-integrin are captured by clathrin coated-structures at the plasma membrane. Clathrin structures dramatically grow in response to ligand activation into large flat plaques and provide a signaling platform that link EGFR and {beta}5-integrin through Src-mediated phosphorylation. Disrupting this EGFR/Src/{beta}5-integrin axis prevents both clathrin plaque growth and receptor signaling. Our study reveals a reciprocal regulation of clathrin lattices and two different receptor systems to enhance cell growth factor signaling. These findings have broad implications for the control of growth factor receptors, mechanotransduction, and endocytosis.

cell biology↗

bioRxiv2020

The nanoscale anatomy of exocytic dense-core vesicles in neuroendocrine cells

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 develop a targetable genetically-encoded electron microscopy labeling method that uses histidine based affinity-tags and metal-binding gold-nanoparticles to determine the axial location of exocytic proteins using electron tomography. Our data show that Rab-GTPases and their effectors are distributed across the entire surface of individual docked vesicles. This circumferential distribution likely aids in the efficient transport, capture, docking, and rapid fusion of vesicles in excitable cells. The nanoscale molecular model of dense core vesicles generated from our methods reveals how key proteins assemble at the plasma membrane to regulate membrane trafficking and exocytosis.

cell biology↗

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↗

bioRxiv2018

Focal adhesion-generated cues in extracellular matrix regulate cell migration by local induction of clathrin-coated plaques

Clathrin is a unique scaffold protein, which forms polyhedral lattices with flat and curved morphology. The function of curved clathrin-coated pits in forming endocytic structures is well studied. On the contrary, the role of large flat clathrin arrays, called clathrin-coated plaques, remains ambiguous. Previous studies suggested an involvement of plaques in cell adhesion. However, the molecular origin leading to their formation and their precise functions remain to be determined. Here, we study the origin and function of clathrin-coated plaques during cell migration. We revealed that plaque formation is intimately linked to extracellular matrix (ECM) modification by focal adhesions (FAs). We show that in migrating cells, FAs digest the ECM creating extracellular topographical cues that dictate the future location of clathrin-coated plaques. We identify Eps15 and Eps15R as key regulators for the formation of clathrin-coated plaques at locally remodelled ECM sites. Using a genetic silencing approach to abrogate plaque formation and 3D-micropatterns to spatially control the location of clathrin-coated plaques, we could directly correlate cell migration directionality with the formation of clathrin-coated plaques and their ability to recognize extracellular topographical cues. We here define the molecular mechanism regulating the functional interplay between FAs and plaques and propose that clathrin-coated plaques act as regulators of cell migration promoting contact guidance-mediated collective migration in a cell-to-cell contact independent manner.

cell biology↗

bioRxiv2018

Structurally distinct endocytic pathways for B cell receptors in B lymphocytes

B lymphocytes play a critical role in adaptive immunity. Upon antigen binding, B cell receptors (BCR) cluster on the plasma membrane and are internalized by endocytosis. In this process, B cells capture diverse antigens in various contexts and concentrations. However, it is unclear whether the mechanism of BCR endocytosis changes in response to these factors. Here, we studied the mechanism of soluble antigen-induced BCR clustering and internalization in a cultured human B cell line using correlative super resolution fluorescence and platinum replica electron microscopy. First, by visualizing nanoscale BCR clusters, we provide direct evidence that BCR cluster size increases with F(ab)2 concentration. Next, we show that the physical mechanism of internalization switches in response to BCR cluster size. At low concentrations of antigen, B cells internalize small BCR clusters by classical clathrin-mediated endocytosis. At high antigen concentrations, when clusters size increases beyond the size of a single clathrin coated pit, B cells retrieve receptor clusters using large invaginations of the plasma membrane capped with clathrin. At these sites, we observed early and sustained recruitment of actin and an actin polymerizing protein FCHSD2. We further show that actin recruitment is required for the efficient generation of these novel endocytic carriers and for their capture into the cytosol. We propose that in B cells, the mechanism of endocytosis switches to accommodate large receptor clusters formed when cells encounter high concentrations of soluble antigen. This mechanism is regulated by the organization and dynamics of the cortical actin cytoskeleton.

cell biology↗

bioRxiv2017

Membrane bending begins at any stage of clathrin-coat assembly and defines endocytic dynamics

Summary ParagraphClathrin-mediated endocytosis internalizes membrane from the cell surface by reshaping flat regions of membrane into spherical vesicles(1, 2). The relationship between membrane bending and clathrin coatomer assembly has been inferred from electron microscopy and structural biology, without directly visualization of membrane bending dynamics (3-6). This has resulted in two distinct and opposing models for how clathrin bends membrane (7-10). Here, polarized Total Internal Reflection Fluorescence microscopy was improved and combined with electron microscopy, atomic force microscopy, and super-resolution imaging to measure membrane bending during endogenous clathrin and dynamin assembly in living cells. Surprisingly, and not predicted by either model, the timing of membrane bending was variable relative to clathrin assembly. Approximately half of the time, membrane bending occurs at the start of clathrin assembly, in the other half, the onset of membrane bending lags clathrin arrival, and occasionally completely assembled flat clathrin transitions into a pit. Importantly, once the membrane bends, the process proceeds to scission with similar timing. We conclude that the pathway of coatomer formation is versatile and can bend the membrane during or after the assembly of the clathrin lattice. These results highlight the heterogeneity in this fundamental biological process, and provide a more complete nanoscale view of membrane bending dynamics during endocytosis.

cell biology↗

bioRxiv2017

Flat-to-curved transition during clathrin-mediated endocytosis correlates with a change in clathrin-adaptor ratio and is regulated by membrane tension

Although essential for many cellular processes, the sequence of structural and molecular events during clathrin-mediated endocytosis remains elusive. While it was believed that clathrin-coated pits grow with a constant curvature, it was recently suggested that clathrin first assembles to form a flat structure and then bends while maintaining a constant surface area. Here, we combine correlative electron and light microscopy and mathematical modelling to quantify the sequence of ultrastructural rearrangements of the clathrin coat during endocytosis in mammalian cells. We confirm that clathrin-coated structures can initially grow flat and that lattice curvature does not show a direct correlation with clathrin coat assembly. We demonstrate that curvature begins when 70% of the final clathrin content is acquired. We find that this transition is marked by a change in the clathrin to clathrin-adaptor protein AP2 ratio and that membrane tension suppresses this transition. Our results support the model that mammalian cells dynamically regulate the flat-to-curved transition in clathrin-mediated endocytosis by both biochemical and mechanical factors.

cell biology↗