Biology subjects

Sumrall, E. R.

Publications and source records attributed to Sumrall, E. R..

4 recordsLinked to original sources

bioRxiv2025

Surface-Tethering Enhances Precision in Measuring Diffusion Within 3D Protein Condensates

Biomolecular condensates, or membraneless organelles, play pivotal roles in cellular organization by compartmentalizing biochemical reactions and regulating diverse processes such as RNA metabolism, signal transduction, and stress response. Super-resolved imaging and single-molecule tracking are essential for probing the internal dynamics of these condensates, yet intrinsic Brownian (thermal capillary wave) motion of the entire condensate in vitro could introduce artifacts into diffusion measurements, confounding the interpretation of molecular mobility. Here, we systematically assess and address this question using both experiments and simulations. We deploy three surface-tethering strategies--using biotinylated DNA, protein, or antibody tethers--to immobilize FUS protein condensates on passivated glass surfaces. We show that tethering effectively suppresses the global translational and rotational Brownian motion of the entire condensate, eliminating inherent measurement artifacts while preserving their spherical appearance and native liquid-like properties. Quantitative analysis reveals that untethered condensates systematically overestimate or underestimate molecular diffusion coefficients and step sizes, particularly for slowly diffusing structured mRNAs, while rapidly diffusing unstructured RNAs are unaffected due to temporal scale separation. Comparative evaluation of tethering strategies demonstrates tunable control over condensate stability and internal dynamics, with implications for optimizing experimental design. Finally, simulations spanning the full physiological parameter space enable us to provide practical guidelines for assessing whether, and to what extent, tethering is beneficial, based on condensate size and the diffusion properties of the biomolecule of interest. Our findings establish surface tethering as a necessary and robust approach for accurate quantification of intra-condensate molecular dynamics, providing a methodological framework for future studies of membraneless organelles. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/659185v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1f0ae11org.highwire.dtl.DTLVardef@341574org.highwire.dtl.DTLVardef@16c75daorg.highwire.dtl.DTLVardef@1ad5ed1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

ER tethering and active transport govern condensate diffusion during hyperosmotic stress

BackgroundHyperosmotic shock and the resulting cell volume compression are commonly experienced by organs such as the kidneys, causing rapid formation of hyperosmotic phase separation (HOPS) condensates in the cytoplasm and nucleoplasm. Although the tight relationship between hyperosmotic shock and condensation has been characterized, the dynamics of biomolecular condensates in hyperosmotically compressed cells and their regulatory mechanisms remain largely unknown. ResultsWe used live-cell single-particle tracking (SPT) across different time scales to systematically characterize the dynamics of HOPS condensates formed by model protein mRNA decapping enzyme 1A (DCP1A). We found that HOPS condensates predominantly exhibited sub-diffusion rather than free diffusion, whereas some ([~]2%) exhibited short super-diffusion. Using tools measuring spatial accessibility inside cells and fluorescence labels for specific cellular organelles, we further revealed the origins of sub-diffusion and super-diffusion as endoplasmic reticulum (ER) attachment and coupling to microtubule-dependent active transport, respectively. Further, we reconstructed an accessibility map of the hyperosmotically compressed cell from trajectories of genetically encoded multimeric nanoparticles (GEMs), revealing that the cytoplasm of a compressed cell remains highly accessible without significant local corrals. ConclusionsIn contrast to prior portrayals of the cytosolic space as static and constrained, our data suggest that the cytosol of a hyperosmotically compressed cell remains dynamic and accessible. Meanwhile, hyperosmotic and potentially other condensates can be spatially organized through docking to membrane structures, with intermittent episodes of long-range transport. These insights broaden our understanding of the physical environment within cells under hyperosmotic shock and provide a model for spatiotemporal organization of condensates via docking or coupling to existing cellular structures and processes.

biophysics↗

bioRxiv2024

Single molecule tracking reveals nanodomains in biomolecular condensates

Biomolecular condensates regulate cellular physiology by sequestering and processing RNAs and proteins, yet how these processes are locally tuned within condensates remains unclear. Moreover, in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), condensates undergo liquid-to-solid phase transitions, but capturing early intermediates in this process has been challenging. Here, we present a surface multi-tethering approach to achieve intra-condensate single-molecule tracking of fluorescently labeled RNA and protein molecules within liquid-like condensates. Using RNA-binding protein Fused in Sarcoma (FUS) as a model for condensates implicated in ALS, we discover that RNA and protein diffusion is confined within distinct nanometer-scale domains, or nanodomains, which exhibit unique connectivity and chemical environments. During condensate aging, these nanodomains reposition, facilitating FUS fibrilization at the condensate surface, a transition enhanced by FDA-approved ALS drugs. Our findings demonstrate that nanodomain formation governs condensate function by modulating biomolecule sequestration and percolation, offering insights into condensate aging and disease-related transitions.

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↗