Biology subjects

Lerner, E.

Publications and source records attributed to Lerner, E..

At least 19 recordsLinked to original sources

bioRxiv2026

BLeaching In-cell Single-molecule burstS (BLISS) reveals a small dynamic fraction of HP1α clusters in undifferentiated embryonic stem cells

Confocal fluorescence microscopy measurements of dense cellular regions of interest (ROIs) in cells using fluorescence lifetime imaging microscopy (FLIM) provide detailed pixelated images. Yet, those pixels report ensemble- and time-averaged biomolecular data, due to the diffraction limit and acquisition times that are slower than typical biomolecular dynamics. The ability to acquire data on one biomolecule at a time within a given ROI can help recover some of the underlying biomolecular subpopulations that are otherwise masked out. Here, we present a simple approach to achieving single-biomolecule photon bursts in live cells, dubbed BLeaching In-cell Single-molecule burstS (BLISS), which does not necessarily require super-resolution modalities. Using BLISS, we show that mCherry-tagged heterochromatin protein 1 (mCherry-HP1) in chromocenters of undifferentiated mouse embryonic stem cells (ESCs), exhibit photon bursts with millisecond durations arising from dynamic clusters. Fluorescence lifetimes of these bursts are substantially lower than the pixel-wise averaged values observed in FLIM, attributed to higher density in HP1 clusters. These higher density clusters are observed primarily in undifferentiated ESCs, while two days post retinoic acid (RA)-induced differentiation (2d-RA), these bursts are rarely observed. Using BLISS, we also detect dynamic clusters of the chromocenter-associated protein, CENP-V, and the nucleolar protein, nucleolin; however, in both cases the fluorescence lifetimes of their clusters closely resemble the pixel-wise averaged values recovered from FLIM. In conclusion, we introduce BLISS as a method for revealing rare and dynamic subpopulation in dense ROIs that are otherwise hidden by averaging in diffraction-limited imaging techniques. Importantly, using BLISS we demonstrate that denser-than-average HP1 clusters are hyperdynamic.

biophysics↗

bioRxiv2024

TNFR2 loss leads to decreased TOX expression in T cells without affecting TIM3 and improves responses to tumor and chronic LCMV

Exhaustion represents a collection of programmed T cell differentiation states and an important mode of T cell dysfunction. T cell progression from progenitor to terminal exhaustion is associated with upregulation of the transcription factor TOX and expression of TIM3. Our understanding of factors regulating TOX expression and the transition from progenitor to terminal exhaustion, however, remains incomplete. We reveal here that T cell upregulation of tumor necrosis factor receptor type II (TNFR2) coincides with the gain of phenotypic markers and functions reflective of terminal exhaustion. Meanwhile, knocking out TNFR2 affords a novel population of T cells that express TIM3 but possess diminished TOX levels and functional characteristics of both progenitor and terminally exhausted cells. TIM3+ TNFR2 KO T cells exhibit reduced exhaustion transcriptional programs and enhanced AP1 pathway signatures. Finally, TNFR2 KO mice demonstrate improved T cell-dependent control of tumor and chronic lymphocytic choriomeningitis (cLCMV) infection, while pharmacologic antagonism of TNFR2 licenses responses to checkpoint blockade in multiple subcutaneous and intracranial tumor models.

immunology↗

bioRxiv2024

PEG-mCherry interactions beyond classical macromolecular crowding

The dense cellular environment influences bio-macromolecular structure, dynamics, interactions and function. Despite advancements in understanding protein-crowder interactions, predicting their precise effects on protein structure and function remains challenging. Here, we elucidate the effects of PEG-induced crowding on the fluorescent protein mCherry using molecular dynamics simulations and fluorescence-based experiments. We identify and characterize specific PEG-induced structural and dynamical changes in mCherry. Importantly, we find interactions in which PEG molecules wrap around specific surface-exposed residues in a binding mode previously observed in protein crystal structures. Fluorescence correlation spectroscopy experiments capture PEG-induced changes, including aggregation, suggesting a potential role for the specific PEG-mCherry interactions identified in simulations. Additionally, mCherry fluorescence lifetimes are influenced by PEG and not by the bulkier crowder dextran or by another linear polymer, polyvinyl alcohol, highlighting the importance of crowder-protein soft interactions. This work augments our understanding of macromolecular crowding effects on protein structure and dynamics.

biophysics↗

bioRxiv2024

Rapid and specific detection of single nanoparticles and viruses in microfluidic laminar flow via confocal fluorescence microscopy

Mainstream virus detection relies on the specific amplification of nucleic acids via polymerase chain reaction, a process that is slow and requires extensive laboratory expertise and equipment. Other modalities, such as antigen-based tests, allow much faster virus detection but have reduced sensitivity. In this study, we report the development of a flow virometer for the specific and rapid detection of single nanoparticles based on confocal microscopy. The combination of laminar flow and multiple dyes enable the detection of correlated fluorescence signals, providing information on nanoparticle volumes and specific chemical composition properties, such as viral envelope proteins. We evaluated and validated the assay using fluorescent beads and viruses, including SARS-CoV-2. Additionally, we demonstrate how hydrodynamic focusing enhances the assay sensitivity for detecting clinically-relevant virus loads. Based on our results, we envision the use of this technology for clinically relevant bio-nanoparticles, supported by the implementation of the assay in a portable and user-friendly setup. O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/573251v3_ufig1.gif" ALT="Figure 1"> View larger version (97K): org.highwire.dtl.DTLVardef@11781bdorg.highwire.dtl.DTLVardef@46d845org.highwire.dtl.DTLVardef@120d399org.highwire.dtl.DTLVardef@240022_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Multiparameter-based photosynthetic state transitions of single phytoplankton cells

Phytoplankton are a major source of primary production. Their photosynthetic fluorescence uniquely reports on their type, physiological state and response to environmental conditions. Changes in phytoplankton photophysiology are commonly monitored by bulk fluorescence spectroscopy, where gradual changes are reported in response to different perturbations such as light intensity changes. What is the meaning of such trends in bulk parameters if their values report ensemble averages of multiple unsynchronized cells? To answer this, we developed an experimental scheme that enables acquiring multiple fluorescence parameters, from multiple excitation sources and spectral bands. This enables tracking fluorescence intensities, brightnesses and their ratios, as well as mean photon nanotimes equivalent to mean fluorescence lifetimes, one cell at a time. We monitored three different phytoplankton species during diurnal cycles and in response to an abrupt increase in light intensity. Our results show that we can define specific subpopulations of fluorescence parameters for each of the phytoplankton species and in response to varying light conditions. Importantly, we identify the cells undergo well-defined transitions between these subpopulations that characterize the different light behaviors. The approach shown in this work will be useful in the exact characterization of phytoplankton cell states and parameter signatures in response to different changes these cells experience in marine environments, which will be useful in monitoring marine-related effects of global warming. Significance StatementUsing three representatives of red-linage phytoplankton we demonstrate distinct photophysiological behaviors at the single cell level. The results indicate cell wide coordination into discrete cell states. We test cell state transitions as a function of light acclimation during diurnal cycle and in response to large intensity increases, which stimulate distinct photoprotective response mechanisms. The analysis was made possible through the development of flow-based confocal detection at multiple excitation and emission wavelengths monitoring both pigment composition and photosynthetic performance. Our findings show that with enough simultaneously recorded parameters per each cell, the detection of multiple phytoplankton species at their distinct cell states is possible. This approach will be useful in examining the response of complex natural marine populations to environmental perturbations.

biophysics↗

bioRxiv2023

Single-molecule detection and super-resolution imaging with a portable and adaptable 3D-printed microscopy platform (Brick-MIC)

Over the past decades, single-molecule and super-resolution microscopy have advanced and represent essential tools for life science research. There is,however, a growing gap between the state-of-the-art and what is accessible to biologists, biochemists, medical researchers or labs with financial constraints. To bridge this gap, we introduce Brick-MIC, a versatile and affordable open-source 3D-printed micro-spectroscopy and imaging platform. Brick-MIC enables the integration of various fluorescence imaging techniques with single-molecule resolution within a single platform and exchange between different modalities within minutes. We here present variants of Brick-MIC that facilitate single-molecule fluorescence detection, fluorescence correlation spectroscopy and super-resolution imaging (STORM and PAINT). Detailed descriptions of the hardware and software components, as well as data analysis routines are provided, to allow non-optics specialist to operate their own Brick-MIC with minimal effort and investments. We foresee that our affordable, flexible, and open-source Brick-MIC platform will be a valuable tool for many laboratories worldwide.

biophysics↗

bioRxiv2023

FRET-sensitized acceptor emission localization (FRETsael) - nanometer localization of biomolecular interactions using fluorescence lifetime imaging

Super-resolution light microscopy techniques facilitate the observation of nanometer-size biomolecules, which are 1-2 orders of magnitude smaller than the diffraction limit of light. Using super-resolution microscopy techniques, it is possible to observe fluorescence from two biomolecules in close proximity, however not necessarily in direct interaction. Using FRET-sensitized acceptor emission localization (FRETsael), we localize biomolecular interactions exhibiting FRET with nanometer accuracy, from two-color fluorescence lifetime imaging data. The concepts of FRETsael were tested first against simulations, in which the recovered localization accuracy is 20-30 nm for true-positive detections of FRET pairs. Further analyses of the simulation results report the conditions in which true-positive rates are maximal. We then show the capabilities of FRETsael on simulated samples of Actin-Vinculin and ER-ribosomes interactions, as well as on experimental samples of actin-myosin two-color confocal imaging. Conclusively, the FRETsael approach paves the way towards studying biomolecular interactions with improved spatial resolution from laser scanning confocal two-color fluorescence lifetime imaging. SignificanceFRET is used in fluorescence microscopy to report whether dye-labeled biomolecules of choice are close within distances of 10 nm or less, hence typical interaction distances. However, in many cases, using FRET imaging for the study of biomolecular interactions is difficult due to the high density of dye-labeled biomolecules and due to the existence of unbound dye-labeled biomolecules. In addition, the resolution of localizing molecules using light microscopy is diffraction limited. This work presents FRETsael, a new approach for localizing interacting biomolecules undergoing FRET, with improved resolution of 20-30 nm for confocal microscopy using search algorithms for local extrema in contribution to FRET using two-channel fluorescence intensity and lifetime data.

biophysics↗

bioRxiv2023

Intracranial tumors elicit systemic sympathetic hyperactivity that limits immunotherapeutic responses

Intracranial tumors present unique challenges for immunotherapy. These can include both local and systemic modes of immune suppression whose mechanistic underpinnings are incompletely understood. Here, we reveal that tumors harbored intracranially elicit systemic increases to circulating catecholamine levels, with the resultant chronic sympathetic hyperactivity driving T cell dysfunction and limiting immunotherapeutic success. Conversely, treatment with {beta}-adrenergic blockade increases NF-{kappa}B activity in immune cells, restores T cell polyfunctionality, modifies the tumor microenvironment, and licenses immune-based therapies in murine models of glioblastoma (GBM) to extend survival. Extended survival is also observed in GBM patients having received {beta}-adrenergic blockade, as well as in patients with melanoma and lung cancer brain metastases who received {beta}-blockade alongside concomitant immune checkpoint inhibition. While {beta}-blockade also impacts outcomes in the setting of extracranial disease, the benefits are especially pronounced in patients harboring intracranial disease burdens. These data suggest that sympathetic hyperactivity facilitates systemic immune dysfunction in the setting of intracranial tumors, specifically and advance a role for {beta}-adrenergic blockade in licensing immunotherapeutic responses within the intracranial compartment.

immunology↗

bioRxiv2023

Dissecting Mechanisms of Ligand Binding and Conformational Changes in the Glutamine-Binding Protein

The glutamin-binding protein GlnBP is part of an ATP-binding cassette transporter system in E. coli and uses two well-characterized conformational states, an open ligand-free and a closed-liganded state, to facilitate active amino-acid uptake. Existing literature on its ligand binding mechanism lacked sufficient evidence to univocally assign the kinetic type of binding mechanism for GlnBP: ligand binding prior to conformational change, i.e., an induced fit or the conformational selection, in which the ligand binds the matching conformation from a pre-existing ensemble. Since such mechanistic questions are relevant for our fundamental understanding of how this and other biomacromolecules regulate cellular processes, we here revisit the question for GlnBP. We present a biochemical and biophysical analysis using a combination of calorimetry, single-molecule and surface-plasmon resonance spectroscopy and molecular dynamics simulations. We found that both apo- and holo-GlnBP show no detectable exchange between open and (semi-)closed conformations on timescales between 100 ns and 10 ms and that ligand binding and conformational changes in GlnBP are correlated. A global analysis of our experimental results suggests that the conformational selection model is only compatible with GlnBP for the extreme scenario of very fast conformational exchange between the open and closed states on timescales <100 ns. In contrast all data remains compatible with an induced-fit mechanism, where the ligand binds GlnBP prior to conformational rearrangements. Importantly, our work demonstrates that it is an intricate task to identify the type of kinetic binding mechanism and that this requires not only a sufficient set of data, but also an integrative experimental and theoretical framework to address the question. Based on this concept, we propose that various protein systems, for which so far only insufficient kinetic data are available, should be revisited.

biophysics↗

bioRxiv2023

Fluorescent protein lifetimes report increased local densities and phases of nuclear condensates during embryonic stem cell differentiation

Fluorescent proteins (FP) are frequently used for studying proteins inside cells. In advanced fluorescence microscopy, FPs can report on additional intracellular variables. One variable is the local density near FPs, which can be useful in studying densities within cellular bio-condensates. Here, we show that a reduction in fluorescence lifetimes of common monomeric FPs reports increased levels of local densities. We demonstrate the use of this fluorescence-based variable to report the distribution of local densities within heterochromatin protein 1 (HP1) in mouse embryonic stem cells (ESCs), before and after early differentiation. We find that local densities within HP1 condensates in pluripotent ESCs are heterogeneous and cannot be explained by a single liquid phase. Early differentiation, however, induces a change towards a more homogeneous distribution of local densities, which can be explained as a liquid-like phase. In conclusion, we provide a fluorescence-based method to report increased local densities and apply it to distinguish between homogeneous and heterogeneous local densities within bio-condensates.

biophysics↗

bioRxiv2022

The structural basis for the self-inhibition of DNA binding by apo-σ70

The initiation of transcription in Escherichia coli (E. coli) is facilitated by promoter specificity factors, also known as {sigma} factors, which may bind a promoter only as part of a complex with RNA polymerase (RNAP). By performing in vitro cross-linking mass spectrometry (CL-MS) of apo-{sigma}70, we reveal structural features suggesting a compact conformation compared to the known RNAP-bound extended conformation. Then, we validate the existence of the compact conformation using in vivo CL-MS by identifying cross-links similar to those found in vitro, which deviate from the extended conformation only during the stationary phase of bacterial growth. Conclusively, we provide information in support of a compact conformation of apo-{sigma}70 that exists in live cells, which might represent a transcriptionally-inactive form that can be activated upon binding to RNAP.

biophysics↗

bioRxiv2022

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Single-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in live cells. We performed a worldwide blind study involving 19 labs to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems that undergo distinct conformational changes, we obtained an uncertainty of the FRET efficiency of less than {+/-} 0.06, corresponding to an interdye distance precision of [≤] 0.2 nm and accuracy of [≤] 0.5 nm. We further discuss the limits for detecting distance fluctuations with sensitivity down to [lsim] 10% of the Forster distance and provide guidelines on how to detect potential dye perturbations. The ability of smFRET experiments to simultaneously measure distances and avoid averaging of conformational dynamics slower than the fluorescence lifetime is unique for dynamic structural biology.

biophysics↗

bioRxiv2022

Identification and Quantification of Within-Burst Dynamics in Singly-Labeled Single-Molecule Fluorescence Lifetime Experiments

Single-molecule spectroscopy has revolutionized molecular biophysics and provided means to probe how structural moieties within biomolecules spatially reorganize at different timescales. There are several single-molecule methodologies that probe local structural dynamics in the vicinity of a single dye-labeled residue, which rely on fluorescence lifetimes as readout. Nevertheless, an analytical framework to quantify dynamics in such single-molecule single-dye fluorescence bursts, at timescales of microseconds to milliseconds, has not yet been demonstrated. Here, we suggest an analytical framework for identifying and quantifying within-burst lifetime-based dynamics, such as conformational dynamics recorded in single-molecule photo-isomerization related fluorescence enhancement. After testing the capabilities of the analysis on simulations, we proceed to exhibit within-burst millisecond local structural dynamics in the unbound -synuclein monomer. The analytical framework provided in this work paves the way for extracting a full picture of the energy landscape for the coordinate probed by fluorescence-lifetime based single-molecule measurements.

biophysics↗

bioRxiv2021

Multi-parameter photon-by-photon hidden Markov modeling

Single-molecule Forster resonance energy transfer (smFRET) is a unique biophysical approach for studying conformational dynamics in biomacro-molecules. Photon-by-photon hidden Markov modelling (H2MM) is an analysis tool that can quantify FRET dynamics of single biomolecules, even if they occur on the sub-millisecond timescale. However, dye photophysical transitions intertwined with FRET dynamics may introduce artefacts. Here, we introduce multi-parameter H2MM (mpH2MM), which assists in identifying FRET dynamics based on simultaneous observation of multiple experimentally-derived parameters. We show the importance of using mpH2MM to decouple FRET dynamics caused by conformational changes from photophysical transitions in confocal-based smFRET measurements of a DNA hairpin, the maltose binding protein, MalE, and the type-III secretion system effector, YopO, from Yersinia species all exhibiting conformational dynamics ranging from the sub-second to microsecond timescales. Overall, we show that using mpH2MM facilitates the identification and quantification of biomolecular sub-populations and their origin.

biophysics↗

bioRxiv2020

The structural heterogeneity of α-synuclein is governed by several distinct subpopulations with interconversion times slower than milliseconds

The intrinsically disordered protein, -synuclein, implicated in synaptic vesicle homeostasis and neurotransmitter release, is also associated with several neurodegenerative diseases. The different roles of -synuclein are characterized by distinct structural states (membrane-bound, dimer, tetramer, oligomer, and fibril), which are originated from its various monomeric conformations. The pathological states, determined by the ensemble of -synuclein monomer conformations and dynamic pathways of interconversion between dominant states, remain elusive due to their transient nature. Here, we use inter-dye distance distributions from bulk time-resolved Forster resonance energy transfer as restraints in discrete molecular dynamics simulations to map the conformational space of the -synuclein monomer. We further confirm the generated conformational ensemble in orthogonal experiments utilizing far-UV circular dichroism and cross-linking mass spectrometry. Single-molecule protein-induced fluorescence enhancement measurements show that within this conformational ensemble, some of the conformations of -synuclein are surprisingly stable, exhibiting conformational transitions slower than milliseconds. Our comprehensive analysis of the conformational ensemble reveals essential structural properties and potential conformations that promote its various functions in membrane interaction or oligomer and fibril formation.

biophysics↗

bioRxiv2019

Abundant α-Synuclein compact dimers

Parkinsons disease is associated with the aggregation of the protein -synuclein. While -synuclein can exist in multiple oligomeric states, the dimer has been a subject of extensive debates. Here, using an array of biophysical approaches, we demonstrate that -synuclein in vitro exhibits primarily a monomer-dimer equilibrium in nanomolar concentrations and up to a few micromolars. We then use spatial information from hetero-isotopic cross-linking mass spectrometry experiments as restrains in discrete molecular dynamics simulations to obtain the ensemble structure of dimeric species. Out of eight structural sub-populations of dimers, we identify one that is compact, stable, abundant, and exhibits partially exposed {beta}-sheet structures. This compact dimer is the only one where the hydroxyls of tyrosine 39 are in proximity that may promote dityrosine covalent linkage upon hydroxyl radicalization, which is implicated in -synuclein amyloid fibrils. We propose that this -synuclein dimer features etiological relevance to Parkinsons disease.

biophysics↗

bioRxiv2019

High-throughput smFRET analysis of freely diffusing nucleic acid molecules and associated proteins

Single-molecule Forster resonance energy transfer (smFRET) is a powerful technique for nanometer-scale studies of single molecules. Solution-based smFRET, in particular, can be used to study equilibrium intra- and intermolecular conformations, binding/unbinding events and conformational changes under biologically relevant conditions without ensemble averaging. However, single-spot smFRET measurements in solution are slow. Here, we detail a high-throughput smFRET approach that extends the traditional single-spot confocal geometry to a multispot one. The excitation spots are optically conjugated to two custom silicon single photon avalanche diode (SPAD) arrays. Two-color excitation is implemented using a periodic acceptor excitation (PAX), allowing distinguishing between singly- and doubly-labeled molecules. We demonstrate the ability of this setup to rapidly and accurately determine FRET efficiencies and population stoichiometries by pooling the data collected independently from the multiple spots. We also show how the high throughput of this approach can be used to increase the temporal resolution of single-molecule FRET population characterization from minutes to seconds. Combined with microfluidics, this high-throughput approach will enable simple real-time kinetic studies as well as powerful molecular screening applications.

biophysics↗

bioRxiv2018

Monte-Carlo Diffusion-Enhanced Photon Inference: Distance Distributions and Conformational Dynamics in single-molecule FRET

Single-molecule Forster Resonance Energy Transfer (smFRET) is utilized to study the structure and dynamics of many bio-molecules, such as proteins, DNA and their various complexes. The structural assessment is based on the well-known Forster relationship between the measured efficiency of energy transfer between a donor (D) and an acceptor (A) dye and the distance between them. Classical smFRET analysis methods called photon distribution analysis (PDA) take into account photon shot-noise, D-A distance distribution and, more recently, interconversion between states in order to extract accurate distance information. It is known that rapid D-A distance fluctuations on the order of the D lifetime (or shorter) can increase the measured mean FRET efficiency and thus decrease the estimated D-A distance. Nonetheless, this effect has been so far neglected in smFRET experiments, potentially leading to biases in estimated distances.\n\nHere we introduce a PDA approach dubbed Monte-Carlo-diffusion-enhanced photon inference (MC-DEPI). MC-DEPI recolor detected photons of smFRET experiments taking into account dynamics of D-A distance fluctuations, multiple interconverting states and photo-blinking. Using this approach, we show how different underlying conditions may yield identical FRET histograms and how the additional information from fluorescence decays helps distinguishing between the different conditions. We also introduce a machine learning fitting approach for retrieving the D-A distance distribution, decoupled from the above-mentioned effects. We show that distance interpretation of smFRET experiments of even the simplest dsDNA is nontrivial and requires decoupling the effects of rapid D-A distance fluctuations on FRET in order to avoid systematic biases in the estimation of the D-A distance distribution.

biophysics↗