Biology subjects

Taraska, J. W.

Publications and source records attributed to Taraska, J. W..

At least 19 recordsLinked to original sources

bioRxiv2026

Clathrin recruitment and assembly of phosphorylated B cell receptor is impaired in human diffuse large B cell lymphoma

The B cell receptor (BCR) drives the differentiation of naive B cells into activated plasma cells that produce antibodies. The BCR consists of a plasma membrane-bound surface immunoglobulin (sIg) that binds antigens, and two coreceptor subunits CD79A and CD79B. The intracellular domains of CD79A and CD79B initiate immune signaling and endocytosis of the receptor in healthy B cells. In diffuse large B cell lymphoma (DLBCL), an aggressive form of human blood cancer, the activated B cell like (ABC) subtype exhibits constitutive signaling that drives survival and proliferation. Here, in a model of human ABC DLBCL, we determine the localization of BCR components relative to plasma membrane structures using correlative super-resolution light and platinum replica transmission electron microscopy. We find that spontaneous clusters of the surface Immunoglobulin common to ABC lymphoma localize to smooth raised membrane domains. These structures are involved in the endocytosis of large receptor clusters. Surprisingly, activated phosphorylated CD79A (pCD79A) shows little colocalization with clathrin on or off smooth raised membranes. Furthermore, pCD79A spatially segregates away from both the slg and CD79B subunits of the BCR complex. A similar distribution was found for downstream phosphorylated Src family kinases. We propose that a disengagement of pCD79A away from sIg and CD79B allows the receptor to evade down-regulation through endocytosis and maintains signaling at the plasma membrane. This mechanism could drive aberrant signaling and the proliferation of lymphomas leading to disease.

cell biology↗

bioRxiv2026

APOE4 and INPP5D converge on membrane mechanics to regulate endocytosis in human astrocytes

Disrupted endocytosis is an early feature of Alzheimers disease (AD), but how genetic risk factors functionally impact this pathway remains unclear. Using isogenic human iPSC-derived astrocytes, we show that the AD risk variant, APOE4, impairs clathrin-mediated membrane curvature, clathrin-mediated endocytosis, and alters plasma membrane lipid saturation and tension. Compared to APOE3 astrocytes, APOE4 cells display an accumulation of flat clathrin structures, reduced maturation of clathrin-coated pits, decreased early endosomes, and reduced endocytic uptake. We then identify the AD risk gene INPP5D as a modifier that restores early endocytosis in APOE4 astrocytes by promoting clathrin curvature and maturation through a mechanism distinct from membrane tension regulation. Beyond effects on trafficking, INPP5D overexpression also reduces lipid droplet accumulation and attenuates inflammatory signaling, linking membrane dynamics to disease-associated astrocytic phenotypes. Together, these findings establish altered membrane mechanics as a proximal consequence of the APOE4 variant and identify endocytosis as a common node linking AD risk genes.

cell biology↗

bioRxiv2025

Dynamics of the formation of flat clathrin lattices in response to growth factor stimulus

Clathrin assemblies on the cell membrane are critical for endocytosis and signal transduction in cells. Specifically, {Omega}-shaped clathrin assemblies function as the coat of endocytic vesicles, while flat clathrin assemblies, also known as flat clathrin lattices, serve as signaling hubs for various signaling pathways. Multiple flat clathrin lattices exist on the cell membrane, and these lattices grow after epidermal growth factor stimulation (EGF) and then return to baseline. In this work, we used a particle-based model to simulate the assembly and disassembly of flat clathrin lattices to capture these dynamics. We found that the formation of flat clathrin lattices is highly dynamic, that is, cluster number, size and dwelling time often change even in the absence of any stimulus. Moreover, these key features are affected by adaptor protein 2 (AP-2) number, clathrin-clathrin binding rate, and clathrin diffusion coefficient. Specifically, an increase in AP-2 number leads to the transition from no cluster, short-lasting multiple small clusters, to a long-lasting single giant cluster. An increased clathrin-clathrin binding rate or decreased clathrin diffusion coefficient both result in an increased cluster number, reduced cluster size, and shortened dwelling time. Furthermore, we also predicted that under EGF stimulation, simultaneous changes in the AP-2 number, the clathrin-clathrin binding rate, and the clathrin diffusion coefficient can reproduce the experimentally observed trend of FCLs: an increase in cluster number and size in the first 30 minutes, followed by a decrease after 30 minutes. These findings reveal kinetic mechanisms underlying the formation of multiple FCLs and how EGF regulates FCL dynamics.

biophysics↗

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↗

bioRxiv2024

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. SignificanceClassically, receptor pathways including epidermal growth factor receptor and fibroblast growth factor receptor were thought of as independent systems. Yet, the plasma membrane is a complex environment where proteins interact, cluster, signal, and associate with organelles. For example, after EGF activation, EGFR is captured at sites on the inner plasma membrane coated with the protein clathrin. This causes clathrin to grow flat across the adherent membrane. Here, we observe co-capture along with EGFR of the related receptor FGFR and unrelated LDLR by clathrin after EGF stimulation. This is specific as other receptors are unaffected. Thus, separate but specific receptor systems co-assemble and signal to each other at nanoscale zones on the plasma membrane organized by clathrin. This provides new avenues for treating diseases like cancer.

cell biology↗

bioRxiv2023

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.

biophysics↗

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

Three-dimensional structured illumination microscopy with enhanced axial resolution

We present two distinct, complementary methods for improving axial resolution in three-dimensional structured illumination microscopy (3D SIM) with minimal or no modification to the optical system. First, we show that placing a mirror directly opposite the sample enables 4-beam interference with higher spatial frequency content than 3D SIM illumination, offering near-isotropic imaging with [~]120 nm lateral and 160 nm axial resolution. Second, we develop an improved deep learning method that can be directly applied to 3D SIM data, obviating the need for additional hardware. This procedure results in [~]120 nm isotropic resolution and can be combined with denoising to facilitate volumetric imaging spanning dozens of time points. 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 rich cytoskeletal dynamics within T-cells in the early stages of immune synapse formation.

cell biology↗

bioRxiv2022

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

Caveolae are small coated inner plasma membrane invaginations found in many cell types. Their diverse functions span from endocytosis to signaling, regulating key cellular processes including lipid uptake, pathogen entry, and membrane tension. Caveolae undergo shape changes from flat to curved. It is unclear which proteins regulate this process. To address this gap, we studied the shapes of caveolae with platinum replica electron microscopy in six common cell types. Next, we developed a correlative multi-color stimulated emission depletion (STED) fluorescence and platinum replica EM imaging (CLEM) method to image caveolae-associated proteins at caveolae of different shapes at the nanoscale. Caveolins and cavins were found at all caveolae, independent of their curvature. EHD2, a classic caveolar neck protein, was strongly detected at both curved and flat caveolae. Both pacsin2 and the regulator EHBP1 were found only at a subset of caveolae. Pacsin2 was localized primarily to areas surrounding flat caveolae, whereas EHBP1 was mostly detected at spheres. Contrary to classic models, dynamin was absent from caveolae and localized only to clathrin-coated structures. Cells lacking dynamin showed no substantial changes to caveolae, suggesting that dynamin is not directly involved in caveolae curvature. Together, we provide a mechanistic map for the molecular control of caveolae shape by eight of the major caveolae-associated coat and regulatory proteins. We propose a model where caveolins, cavins, and EHD2 assemble as a cohesive structural unit regulated by more intermittent associations with pacsin2 and EHBP1. These complexes can flatten and curve, capturing membrane to enable lipid traffic and changes to the surface area of the cell.

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↗

bioRxiv2021

Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin mediated endocytosis

As the principal internalization mechanism in mammalian cells, clathrin-mediated endocytosis (CME) is critical for cellular signal transduction, receptor recycling, and membrane homeostasis. Acute depletion of cholesterol disrupts CME, motivating analysis of CME dynamics in the context of disrupted cholesterol synthesis, sterol specificity, mechanisms involved, and relevance to disease pathology. Using genome-edited cell lines, we demonstrate that inhibition of post-squalene cholesterol biosynthesis as observed in inborn errors of cholesterol metabolism, results in striking immobilization of CME and impaired transferrin uptake. Imaging of membrane bending dynamics and CME pit ultrastructure revealed prolonged clathrin pit lifetimes and accumulation of shallow clathrin-coated structures that scaled with diminishing sterol abundance. Moreover, fibroblasts derived from Smith-Lemli-Opitz syndrome subjects displayed reduced CME function. We conclude that sterols lower the energetic costs of membrane bending during pit formation and vesicular scission during CME and suggest reduced CME contributes to cellular phenotypes observed within disorders of cholesterol metabolism.

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

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↗

bioRxiv2018

Local Protein Dynamics During Microvesicle Exocytosis in Neuroendocrine Cells

Calcium triggered exocytosis is key to many physiological processes, including neurotransmitter and hormone release by neurons and endocrine cells. Dozens of proteins regulate exocytosis, yet the temporal and spatial dynamics of these factors during vesicle fusion remain unclear. Here we use total internal reflection fluorescence microscopy to visualize local protein dynamics at single sites of exocytosis of small synaptic-like microvesicles in live cultured neuroendocrine PC12 cells. We employ two-color imaging to simultaneously observe membrane fusion (using vesicular acetylcholine transporter (VAChT) tagged to pHluorin) and the dynamics of associated proteins at the moments surrounding exocytosis. Our experiments show that many proteins, including the SNAREs syntaxin1 and VAMP2, the SNARE modulator tomosyn, and Rab proteins, are pre-clustered at fusion sites and rapidly lost at fusion. The ATPase NSF is locally recruited at fusion. Interestingly, the endocytic BAR domain-containing proteins amphiphysin1, syndapin2, and endophilins are dynamically recruited to fusion sites, and slow the loss of vesicle membrane-bound cargo from fusion sites. A similar effect on vesicle membrane protein dynamics was seen with the over-expression of the GTPases dynamin1 and dynamin2. These results suggest that proteins involved in classical clathrin-mediated endocytosis can regulate exocytosis of synaptic-like microvesicles. Our findings provide insights into the dynamics, assembly, and mechanistic roles of many key factors of exocytosis and endocytosis at single sites of microvesicle fusion in live cells.

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↗