Biology subjects

Veatch, S. L.

Publications and source records attributed to Veatch, S. L..

10 recordsLinked to original sources

bioRxiv2026

A single-molecule reporter of membrane-proximal actin detects rapid remodeling upon B cell receptor clustering

Membrane-proximal (MP) actin represents the subset of cortical f-actin localized within 10 nm of the plasma membrane. Here, we describe a family of single-molecule MP actin (SM-MPAct) probes that diffuse within the plasma membrane and are transiently immobilized through binding to f-actin, enabling the localization of MP actin in both time and space. These probes quantify aspects of MP actin structure, dynamics, and remodeling by analyzing probe positions using a combined single-particle tracking and correlation-function approach. This is demonstrated using chemical and physical perturbations of actin and actin-binding proteins, and by interrogating MP actin organization and dynamics in early B cell receptor (BCR) activation. Upon crosslinking of the IgM BCR, MP actin transiently remodels to increase the size of actin corals, facilitating the efficient assembly of BCR clusters and the local accumulation of MP actin. Notably, analogous remodeling is not detected in measurements using total f-actin probes, indicating that SM-MPAct is uniquely sensitive to the f-actin pool that regulates signaling processes at the plasma membrane. STATEMENT OF SIGNIFICANCEThe actin cortex provides mechanical stability to the plasma membrane and contributes to the organization and dynamics of plasma membrane components. This report presents single-molecule probes and analytical methods to characterize the density, mesh size, motion, and turnover dynamics of the portion of the actin mesh in direct contact with the plasma membrane, enabling quantitative studies of actin remodeling in plasma membrane processes. This general framework is demonstrated through quantification of actin remodeling during early B cell receptor signaling and could be applied to a broad range of cell processes.

biophysics↗

bioRxiv2024

PIP2 promotes the incorporation of CD43, PSGL-1 and CD44 into nascent HIV-1 particles

AbstractDeterminants regulating sorting of host transmembrane proteins at sites of enveloped virus assembly on the plasma membrane (PM) remain poorly understood. Here, we demonstrate for the first time that PM acidic phospholipid PIP2 regulates such sorting into an enveloped virus, HIV-1. Incorporation of CD43, PSGL-1, and CD44 into HIV-1 particles is known to have profound effects on viral spread; however, the mechanisms promoting their incorporation were unknown. We found that depletion of cellular PIP2 blocks the incorporation of CD43, PSGL-1, and CD44 into HIV-1 particles. Expansion microscopy revealed that PIP2 depletion diminishes nanoscale co-clustering between viral structural protein Gag and the three transmembrane proteins at PM and that Gag induces PIP2 enrichment around itself. CD43, PSGL-1, and CD44 also increased local PIP2 density, revealing their PIP2 affinity. Altogether, these results support a new mechanism where local enrichment of an acidic phospholipid drives co-clustering between viral structural and cellular transmembrane proteins, thereby modulating the content, and hence the fate, of progeny virus particles.

microbiology↗

bioRxiv2024

Prewetting couples membrane and protein phase transitions to greatly enhance coexistence in models and cells.

Biopolymers that separate into condensed and dilute phases in solution also prewet membranes when one or more components couple to membrane lipids. Here we demonstrate that this prewetting transition becomes exquisitely sensitive to lipid composition when membranes have compositions near the boundary of liquid-ordered/liquid-disordered phase coexistence. In simulation and reconstitution, we couple polyelectrolytes to membranes of saturated lipids, unsaturated lipids, and cholesterol, and find that the coexistence of prewet and dry surface phases is dramatically potentiated by proximity to the membrane phase transition. In cells, we employ an optogenetic tool to characterize prewetting both at the plasma membranes (PM) and the endoplasmic reticulum (ER), and find that prewetting is potentiated or inhibited by perturbations of membrane composition. Prewetting can also mediate membrane adhesion, with avidity dependent on membrane composition. This effect is demonstrated in cells through the potentiation or inhibition of ER-PM contact sites. The strong correspondence between results in simulation, reconstitution, and cells demonstrates a new role for membrane lipids in regulating the recruitment and assembly of soluble proteins.

biophysics↗

bioRxiv2023

TorsinA is essential for the timing and localization of neuronal nuclear pore complex biogenesis

Nuclear pore complexes (NPCs) regulate information transfer between the nucleus and cytoplasm. NPC defects are linked to several neurological diseases, but the processes governing NPC biogenesis and spatial organization are poorly understood. Here, we identify a temporal window of strongly upregulated NPC biogenesis during neuronal maturation. We demonstrate that the AAA+ protein torsinA, whose loss of function causes the neurodevelopmental movement disorder DYT-TOR1A (DYT1) dystonia, coordinates NPC spatial organization during this period without impacting total NPC density. Using a new mouse line in which endogenous Nup107 is Halo-Tagged, we find that torsinA is essential for correct localization of NPC formation. In the absence of torsinA, the inner nuclear membrane buds excessively at sites of mislocalized, nascent NPCs, and NPC assembly completion is delayed. Our work implies that NPC spatial organization and number are independently regulated and suggests that torsinA is critical for the normal localization and assembly kinetics of NPCs.

cell biology↗

bioRxiv2022

Measuring the co-localization and dynamics of mobile proteins in live cells undergoing signaling responses

ii.Summary/AbstractSingle molecule imaging in live cells enables the study of protein interactions and dynamics as they participate in signaling processes. When combined with fluorophores that stochastically transition between fluorescent and reversible dark states, as in super-resolution localization imaging, labeled molecules can be visualized in single cells over time. This improvement in sampling enables the study of extended cellular responses at the resolution of single molecule localization. This chapter provides optimized experimental and analytical methods used to quantify protein interactions and dynamics within the membranes of adhered live cells. Importantly, the use of pair-correlation functions resolved in both space and time allows researchers to probe interactions between proteins on biologically relevant distance and time-scales, even though fluorescence localization methods typically require long times to assemble well-sampled reconstructed images. We describe an application of this approach to measure protein interactions in B cell receptor signaling and include sample analysis code for post-processing of imaging data. These methods are quantitative, sensitive, and broadly applicable to a range of signaling systems.

biophysics↗

bioRxiv2022

Chemical potential measurements constrain models of cholesterol-phosphatidylcholine interactions

Bilayer membranes composed of cholesterol and phospholipids exhibit diverse forms of non-ideal mixing. In particular, many previous studies document macroscopic liquid-liquid phase separation as well as nanometer-scale heterogeneity in membranes of phosphatidylcholine (PC) lipids and cholesterol. Here, we present experimental measurements of cholesterol chemical potential (c) in binary membranes containing dioleoyl PC (DOPC), 1-palmitoyl-2-oleoyl PC (POPC), or dipalmitoyl PC (DPPC), and in ternary membranes of DOPC and DPPC, adapting a calibrated experimental protocol developed to measure c in cells (Ayuyan and Cohen, Biophys. J. 114:904-918). c is the thermodynamic quantity that dictates the availability of cholesterol to bind other factors, and notably must be equal between coexisting phases of a phase-separated mixture. It is simply related to concentration under conditions of ideal mixing but is found to be far from ideal for the majority of lipid mixtures investigated. Here we perform experimental measurements of c, constraining thermodynamic models of membrane interactions. Our measurements are consistent with models involving cholesterol-phospholipid complexes, but only if complexes are more weakly bound than has been assumed in previous reports. Experimental measurements are also well described by regular solution theory and lattice models with pairwise interactions between components. We find that c can vary by ~1.5 kBT at constant cholesterol mole-fraction implying a more than five-fold change in its availability for binding receptors and other reactions. These findings reinforce that c depends on membrane composition overall, suggesting avenues for cells to alter the availability of cholesterol without varying cholesterol concentration. SIGNIFICANCEThe chemical potential of cholesterol (c) reflects its availability to interact with other molecules. In a complex mixtures this chemical potential can vary dramatically even at fixed cholesterol concentration. In this report, we present measurements of c in bilayer membranes composed of mixtures of cholesterol with one or two phospholipids. We find that c in these mixtures depends strongly on the phospholipids that are present, with activity varying by a factor of more than five at fixed cholesterol concentration. This suggests that the availability of cholesterol in biological membranes could be tuned without altering cholesterol concentration directly, by adjusting the concentration of other lipid or protein components.

biophysics↗

bioRxiv2022

A method to estimate the effective point spread function of static single molecule localization microscopy images

Single molecule localization microscopy (SMLM) permits the visualization of cellular structures an order of magnitude smaller than the diffraction limit of visible light, and an accurate, objective evaluation of the resolution of an SMLM dataset is an essential aspect of the image processing and analysis pipeline. Here we present a simple method that uses the pair autocorrelation function evaluated both in space and time to measure the time-interval dependent effective point spread function of SMLM images of static samples. Using this approach, we demonstrate that experimentally obtained images typically have effective point spread functions that are broader than expected from the localization precision alone, due to additional uncertainty arising from factors such as drift and drift correction algorithms. The method is demonstrated on simulated localizations, DNA origami rulers, and cellular structures labelled by dye-conjugated antibodies, DNA-PAINT, or fluorescent fusion proteins. STATEMENT OF SIGNIFICANCESingle molecule localization microscopy (SMLM) is a class of imaging methods that resolve fluorescently labeled structures beyond the optical resolution limit of visible light. SMLM detects stochastically blinking labels over time, and localizes each blink with precision of order 10 nm. The effective resolution depends on factors such as signal-to-noise ratio, localization algorithm, and several post-processing steps such as stage drift correction. We present a method to evaluate this effective resolution by taking advantage of temporal correlations of fluorophore blinking to separate the distribution of pairs of localizations from the same molecule from those from different molecules. The method is robust on useful timescales for a range of SMLM probes.

biophysics↗

bioRxiv2021

A Mean Shift Algorithm for Drift Correction in Localization Microscopy

Single molecule localization microscopy (SMLM) techniques transcend the diffraction limit of visible light by localizing isolated emitters sampled stochastically. This time-lapse imaging necessitates long acquisition times, over which sample drift can become large relative to the localization precision. Here we present a novel, efficient, and robust method for estimating drift using a simple peak-finding algorithm based on mean shifts that is effective for SMLM in 2 or 3 dimensions.

biophysics↗

bioRxiv2019

SMAUG: Analyzing single-molecule tracks with nonparametric Bayesian statistics

Single-molecule fluorescence microscopy probes nanoscale, subcellular biology in real time. Existing methods for analyzing single-particle tracking data provide dynamical information, but can suffer from supervisory biases and high uncertainties. Here, we introduce a new approach to analyzing single-molecule trajectories: the Single-Molecule Analysis by Unsupervised Gibbs sampling (SMAUG) algorithm, which uses nonparametric Bayesian statistics to uncover the whole range of information contained within a single-particle trajectory (SPT) dataset. Even in complex systems where multiple biological states lead to a number of observed mobility states, SMAUG provides the number of mobility states, the average diffusion coefficient of single molecules in that state, the fraction of single molecules in that state, the localization noise, and the probability of transitioning between two different states. In this paper, we provide the theoretical background for the SMAUG analysis and then we validate the method using realistic simulations of SPT datasets as well as experiments on a controlled in vitro system. Finally, we demonstrate SMAUG on real experimental systems in both prokaryotes and eukaryotes to measure the motions of the regulatory protein TcpP in Vibrio cholerae and the dynamics of the B-cell receptor antigen response pathway in lymphocytes. Overall, SMAUG provides a mathematically rigorous approach to measuring the real-time dynamics of molecular interactions in living cells.\n\nStatement of SignificanceSuper-resolution microscopy allows researchers access to the motions of individual molecules inside living cells. However, due to experimental constraints and unknown interactions between molecules, rigorous conclusions cannot always be made from the resulting datasets when model fitting is used. SMAUG (Single-Molecule Analysis by Unsupervised Gibbs sampling) is an algorithm that uses Bayesian statistical methods to uncover the underlying behavior masked by noisy datasets. This paper outlines the theory behind the SMAUG approach, discusses its implementation, and then uses simulated data and simple experimental systems to show the efficacy of the SMAUG algorithm. Finally, this paper applies the SMAUG method to two model living cellular systems--one bacterial and one mammalian--and reports the dynamics of important membrane proteins to demonstrate the usefulness of SMAUG to a variety of systems.

biophysics↗

bioRxiv2017

Loss Of PTEN Promotes Formation Of Signaling-Specific Clathrin-Coated Pits

Defective endocytosis and vesicular trafficking of signaling receptors has recently emerged as a multifaceted hallmark of malignant cells. Clathrin-coated pits (CCPs), the fundamental unit of clathrin-mediated endocytosis, display highly heterogeneous dynamics on the plasma membrane where they can take from 20 seconds to over a minute to form cytosolic coated-vesicles. Despite the large number of cargo molecules that traffic through CCPs, it is not well understood whether signaling receptors activated in cancer, such as epidermal growth factor receptor (EGFR), are regulated through a specific subset of CCPs. The signaling lipid phosphatidylinositol (3,4,5)-triphosphate (PI(3,4,5)P3), which is dephosphorylated by phosphatase tensin homolog (PTEN), is a potent tumorigenic signaling lipid that is present in excess in many types of cancers. Using total internal reflection fluorescence microscopy and automated tracking and detection of CCPs, we find PTEN and EGF bound EGFR are enriched in a distinct subset of short-lived CCPs that corresponded with clathrin-dependent EGF-induced signaling. By deleting PTEN using CRISPR-Cas9 and reconstituting PTEN, we demonstrate that PTEN plays a role in the regulation of CCP dynamics; this appears to recapitulate CCP dynamics in highly metastatic PTEN-deleted cancer cells where we find a larger proportion of short-lived CCPs and higher initiation density compared to the normal cells. Furthermore, increased PI(3,4,5)P3 results in higher proportion of short-lived CCPs, an effect that recapitulates PTEN deletion. Our findings provide strong evidence for the existence of short-lived signaling-capable CCPs. Altogether, these findings demonstrate the importance of PTEN and PI(3,4,5)P3 in regulating CCP dynamics and assign a new function to PTEN as a modulator of signaling-capable CCPs.

cell biology↗