Biology subjects

Legant, W. R.

Publications and source records attributed to Legant, W. R..

16 recordsLinked to original sources

bioRxiv2026

A custom two-in-one HIST and line-scanning confocal excitation module

Fluorescence microscopy applications often require specialized instruments that are optimized for different experimental goals. Here, we present a reconfigurable microscopy module that integrates highly inclined swept tile (HIST) illumination for high-sensitivity single-molecule imaging and line-scanning confocal microscopy for rapid and optically sectioned volumetric acquisition. The system shares major hardware components, including lasers, scanning optics, and detection hardware, while employing unique beam shaping pathways to enable rapid switching between modalities without realignment. We characterize the module performance by measuring the excitation beam profiles, the point spread functions (PSF), and the optical transfer functions (OTF) across 40x, 60x, and 100x magnifications and demonstrate imaging applications including diffraction-limited fixed and live-cell volumetric imaging, fluorescence recovery after photobleaching, and super-resolution DNA-PAINT and single particle tracking (SPT). We also demonstrate the capability to execute multimodal imaging workflows by performing confocal imaging for chromatin density classification correlated with SPT data of nuclear proteins with diverse functions. Together, these results demonstrate a versatile imaging platform capable of supporting complementary fluorescence imaging modalities within a single instrument.

bioengineering↗

bioRxiv2026

Nuclear SUN2 coordinates endothelial cell-matrix interactions to regulate blood vessel homeostasis and barrier function

Vascular endothelial cells respond to environmental forces to remodel vessels during development and to achieve homeostasis, and mis-regulated responses lead to vascular dysfunction and disease. The nucleus participates in force transduction to cell-matrix junctions via the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex that resides in the nuclear envelope, but how these forces are regulated and relayed is incompletely understood. We found that the LINC complex protein SUN2 is required for proper endothelial cell-matrix interactions that occur far from the nucleus and affect angiogenic expansion, vascular responses to flow, and barrier integrity. Endothelial cells lacking SUN2 had inappropriate flow responses and reduced expression of flow-mediated transcription factors in vitro and in vivo. Expression of several matrix and adhesion genes was reduced in SUN2-depleted cells, leading to defective extracellular matrix, dysmorphic focal adhesions resistant to dynamic turnover, and disturbed cell-matrix force distribution. Mechanistically, nuclear SUN2 affected dynamic regulation of the microtubule cytoskeleton that correlated with matrix metalloprotease-dependent barrier dysfunction. These findings indicate that nuclear SUN2 establishes and maintains blood vessel homeostasis by controlling microtubule-mediated effects on focal adhesion turnover and extracellular matrix properties, with implications for cardiovascular aging and diseases such as Marfan syndrome that affect vessel wall integrity.

cell biology↗

bioRxiv2026

Actin-membrane interface stress regulates Arp2/3-branched actin density during lamellipodial protrusion

Motile cells can sense and exert forces on the extracellular environment through dynamic actin networks. Increased stress against the polymerizing barbed ends of branched actin networks has been shown to lead to an increase in the density of these networks through a force feedback mechanism, though this phenomenon has not been explored through the examination of real-time responses of endogenous branched actin in cells. Here, we utilize mouse CRISPR knock-in fibroblasts with labeled Arp2/3 complex to identify cellular and extracellular conditions that regulate branched actin density and enrichment at the leading edge of lamellipodial protrusions. A common theme shared among all branched actin density-increasing conditions is higher levels of predicted interface stress between the plasma membrane and the barbed ends of the lamellipodial actin network. Among these conditions, we find that Arp2/3 is specifically required for robust spreading and protrusion in response to increased extracellular viscosity. Interestingly, time-lapse traction force microscopy of Arp2/3-dependent viscosity responses show significantly reduced changes in strain energy applied to the substrate when compared to spreading and motility utilizing cell-matrix adhesion. In addition, we find that increased extracellular viscosity can bypass the need for extracellular matrix proteins to support lamellipodial protrusion driven by optogenetic Rac activation. Our studies provide strong support for in vitro models of branched actin force feedback responses and further characterize an essential role for branched actin in mediating dramatic cell shape changes in response to increased extracellular viscosity.

cell biology↗

bioRxiv2026

Hydrodynamic dispersion drives viral-cellular contact for gene delivery in porous media

Reactive biological processes often hinge on rare collisions between particles that occupy vastly different physical regimes, yet the transport physics that govern these encounters remain poorly understood. Biological cell-virus encounters offer a uniquely quantifiable instance of this general problem: collisions between particles whose transport is governed by entirely different physical mechanisms, yet whose interactions determine system-level function. In stagnant liquids, nanoscale viral vectors explore space only through slow Brownian diffusion, while microscale cells rapidly sediment, producing species separation that suppresses the virus-cell interaction interface. Here we show that liquid absorption into a dry, macroporous sponge enhances viral-cellular interactions by shifting the system into an advection-dispersion regime that circumvents this sedimentation-diffusion limit. By integrating experimental results with a multiscale simulation model, we demonstrate that the tortuous sponge porosity converts capillary-driven flow into convective mixing, driving orders-of-magnitude increases in viral-cellular collision rates. Coupling these dispersive transport dynamics with a probabilistic capture model reveals that hydrodynamic dispersion accounts for the multifold enhancement in viral-cellular transduction efficiency observed in porous sponges. These results provide a quantitative framework for emergent collision dynamics in complex porous media and establish a generalizable strategy to optimize active transport in spatiotemporally heterogeneous biological systems.

bioengineering↗

bioRxiv2024

Mapping the nuclear landscape with multiplexed super-resolution fluorescence microscopy

The nucleus coordinates many different processes. Visualizing how these are spatially organized requires imaging protein complexes, epigenetic marks, and DNA across scales from single molecules to the whole nucleus. To accomplish this, we developed a multiplexed imaging protocol to localize 13 different nuclear targets with nanometer precision in single cells. We show that nuclear specification into active and repressive states exists along a spectrum of length scales, emerging below one micron and becoming strengthened at the nanoscale with unique organizational principles in both heterochromatin and euchromatin. HP1- was positively correlated with DNA at the microscale but uncorrelated at the nanoscale. RNA Polymerase II, p300, and CDK9 were positively correlated at the microscale but became partitioned below 300 nm. Perturbing histone acetylation or transcription disrupted nanoscale organization but had less effect at the microscale. We envision that our imaging and analysis pipeline will be useful to reveal the organizational principles not only of the cell nucleus but also other cellular compartments.

cell biology↗

bioRxiv2024

A posterior approach to correct for focal plane offsets in lattice light sheet structured illumination microscopy

SignificanceLattice light sheet structured illumination microscopy (latticeSIM) has proven highly effective in producing 3D images with super resolution rapidly and with minimal photobleaching. However, due to the use of two separate objectives, sample-induced aberrations can result in an offset between the planes of excitation and detection, causing artifacts in the reconstructed images. AimWe introduce a posterior approach to detect and correct for the axial offset between the excitation and detection focal planes in latticeSIM and provide a method to minimize artifacts in the reconstructed images. ApproachWe utilized the residual phase information within the overlap regions of the laterally shifted structured illumination microscopy (SIM) information components in frequency space to retrieve the axial offset between the excitation and the detection focal planes in latticeSIM. ResultsWe validated our technique through simulations and experiments, encompassing a range of samples from fluorescent beads to subcellular structures of adherent cells. We also show utilizing transfer functions with the same axial offset as that which was present during the data acquisition results in reconstructed images with minimal artifacts and salvages otherwise unusable data. ConclusionWe envision that our method will be a valuable addition to restore image quality in latticeSIM datasets even for those acquired under non-ideal experimental conditions.

biophysics↗

bioRxiv2024

A series of spontaneously blinking dyes for super-resolution microscopy

Spontaneously blinking fluorophores permit the detection and localization of individual molecules without reducing buffers or caging groups, thus simplifying single-molecule localization microscopy (SMLM). The intrinsic blinking properties of such dyes are dictated by molecular structure and modulated by environment, which can limit utility. We report a series of tuned spontaneously blinking dyes with duty cycles that span two orders of magnitude, allowing facile SMLM in cells and dense biomolecular structures.

cell biology↗

bioRxiv2023

Correlative single molecule lattice light sheet imaging reveals the dynamic relationship between nucleosomes and the local chromatin environment

In the nucleus, biological processes are driven by proteins that diffuse through and bind to a meshwork of nucleic acid polymers. To better understand this interplay, we developed an imaging platform to simultaneously visualize single protein dynamics together with the local chromatin environment in live cells. Together with super-resolution imaging, new fluorescent probes, and biophysical modeling, we demonstrated that nucleosomes display differential diffusion and packing arrangements as chromatin density increases whereas the viscoelastic properties and accessibility of the interchromatin space remain constant. Perturbing nuclear functions impacted nucleosome diffusive properties in a manner that was dependent on local chromatin density and supportive of a model wherein transcription locally stabilizes nucleosomes while simultaneously allowing for the free exchange of nuclear proteins. Our results reveal that nuclear heterogeneity arises from both active and passive process and highlights the need to account for different organizational principals when modeling different chromatin environments.

biophysics↗

bioRxiv2023

Smart Lattice Light Sheet Microscopy for imaging rare and complex cellular events

Light sheet microscopes enable rapid, high-resolution imaging of biological specimens; however, biological processes span a variety of spatiotemporal scales. Moreover, long-term phenotypes are often instigated by rare or fleeting biological events that are difficult to capture with a single imaging modality and constant imaging parameters. To overcome this limitation, we present smartLLSM, a microscope that incorporates AI-based instrument control to autonomously switch between epifluorescent inverted imaging and lattice light sheet microscopy. We apply this technology to two major scenarios. First, we demonstrate that the instrument provides population-level statistics of cell cycle states across thousands of cells on a coverslip. Second, we show that by using real-time image feedback to switch between imaging modes, the instrument autonomously captures multicolor 3D datasets or 4D time-lapse movies of dividing cells at rates that dramatically exceed human capabilities. Quantitative image analysis on high-content + high-throughput datasets reveal kinetochore and chromosome dynamics in dividing cells and determine the effects of drug perturbation on cells in specific mitotic stages. This new methodology enables efficient detection of rare events within a heterogeneous cell population and records these processes with high spatiotemporal 4D imaging over statistically significant replicates.

biophysics↗

bioRxiv2022

Patient-derived extracellular matrix demonstrates role of COL3A1 in blood vessel mechanics

Vascular Ehlers-Danlos Syndrome (vEDS) is a rare autosomal dominant disease caused by mutations in the COL3A1 gene, which renders patients susceptible to aneurysm and arterial dissection and rupture. To determine the role of COL3A1 variants in the biochemical and biophysical properties of human arterial ECM, we developed a method for synthesizing ECM directly from vEDS donor fibroblasts. We found that the protein content of the ECM generated from vEDS donor fibroblasts differed significantly from ECM from healthy donors, including upregulation of collagen subtypes and other proteins related to ECM structural integrity. We further found that ECM generated from a donor with a glycine substitution mutation was characterized by increased glycosaminoglycan content and unique viscoelastic mechanical properties, including increased time constant for stress relaxation, resulting in a decrease in migratory speed of human aortic endothelial cells when seeded on the ECM. Collectively, these results demonstrate that causal COL3A1 mutations lead to the synthesis of ECM that differs in composition, structure, and mechanical properties from healthy donors. These results further suggest that ECM mechanical properties could serve as a prognostic indicator for patients with vEDS, and the insights provided by the approach demonstrate the broader utility of cell-derived ECM in disease modeling.

bioengineering↗

bioRxiv2022

Zyxin and non-muscle myosin are required for single fibroblast durotaxis, but Rho-kinase activity and the Arp2/3 complex are dispensable

Durotaxis, migration of cells directed by stiffness gradient, is critical in development and disease. To study the molecular determinants of single cell durotaxis, we developed an all-in-one photopolymerized hydrogel system containing areas of stiffness gradients with different slopes, along with uniform stiffness (soft and stiff) regions. We find that fibroblasts rely on non-muscle myosin II (NMII) activity and the LIM-domain protein zyxin for durotaxis on both steep and shallow stiffness gradients. Importantly, unlike haptotaxis, the Arp2/3 complex is dispensable for durotaxis on both stiffness gradients. Lack of Arp2/3 results in a filopodia-based durotactic migration that is equally efficient as that of lamellipodia-based durotactic migration. Finally, we reveal an essential role for the actin-bundler fascin in the formation and asymmetric distribution of filopodia during filopodia-based durotaxis in shallow, but not steep, stiffness gradient. Together, our all-in-one hydrogel system can serve as a platform to identify, discriminate, and characterize stiffness gradient specific molecular mechanisms that cells employ to efficiently durotax.

cell biology↗

bioRxiv2020

u-track 3D: measuring and interrogating intracellular dynamics in three dimensions.

Particle tracking is a ubiquitous task in the study of dynamic molecular and cellular processes through microscopy. Light-sheet microscopy has opened a path to acquiring complete cell volumes for investigation in 3-dimensions (3D). However, hypothesis formulation and quantitative analysis have remained difficult due to fundamental challenges in the visualization and the verification of large and dense sets of 3D particle trajectories. Here we describe u-track 3D, a software package that addresses these two challenges. Building on the established framework of particle association in space and time implemented for 2D time-lapse sequences, we first report a complete and versatile pipeline for particle tracking in 3D. We then present the concept of dynamic region of interest (dynROI), which allows an experimenter to interact with dynamic 3D processes in 2D views amenable to visual inspection. Third, we present an estimator of trackability which defines a score for every trajectory, thereby overcoming the challenges of trajectory validation by visual inspection. With these combined strategies, u-track 3D provides a framework for the unbiased study of molecular processes in complex volumetric sequences.

cell biology↗

bioRxiv2020

Deep learning enables fast and dense single-molecule localization with high accuracy

Single-molecule localization microscopy (SMLM) has had remarkable success in imaging cellular structures with nanometer resolution, but the need for activating only single isolated emitters limits imaging speed and labeling density. Here, we overcome this major limitation using deep learning. We developed DECODE, a computational tool that can localize single emitters at high density in 3D with highest accuracy for a large range of imaging modalities and conditions. In a public software benchmark competition, it outperformed all other fitters on 12 out of 12 data-sets when comparing both detection accuracy and localization error, often by a substantial margin. DECODE allowed us to take live-cell SMLM data with reduced light exposure in just 3 seconds and to image microtubules at ultra-high labeling density. Packaged for simple installation and use, DECODE will enable many labs to reduce imaging times and increase localization density in SMLM.

biophysics↗

bioRxiv2020

APC mutant cells exploit compensatory chromosome alterations to restore tumour cell fitness

Cancer cells tolerate copy number alterations (CNAs) of genomic regions that are lethal to non-cancer cells. Certain CNAs are preferentially associated with specific cancer types and lineages, but the mechanisms underlying the emergence and selection of specific CNAs remain unclear. Adenomatous polyposis coli (APC) mutations induce mitotic errors, but their impact on tumor evolution remains elusive. We investigated APC function in cultured cells and tumors and found that its loss led to {beta}-catenin accumulation at centrosomes, suppressing its maturation through inhibition of key centrosome regulators, including Aurora kinase A (AURKA) that promotes tumor growth. These defects collectively reduced cellular fitness, leading to impaired mitotic fidelity and delayed cell cycle progression. However, in APC-mutant tumors, AURKA activity was maintained, at least in part, through the amplification of chromosomes harboring AURKA and its activator genes, yet this alone was insufficient to fully restore proliferation: aberrant chromosomal reorganization also emerged and contributed to the adaptive fitness of APC-mutant cells. Such a process of adaptive CNA selection provides a framework for understanding how specific CNAs are selected to counteract disadvantages imposed by genetic alterations during tumor progression, providing one key insight into how specific CNAs are selected in this context.

cell biology↗

bioRxiv2019

Super-resolution Imaging Reveals 3D Structure and Organizing Mechanism of Accessible Chromatin

Access to cis-regulatory elements packaged in chromatin is essential for directing gene expression and cell viability. Here, we report a super-resolution imaging strategy, 3D ATAC-PALM, that enables direct visualization of the entire accessible genome. We found that active chromosomal segments are organized into spatially-segregated accessible chromatin domains (ACDs). Rapid depletion of CTCF or Cohesin (RAD21 subunit) induced enhanced ACD clustering, reduced physical separation between intrachromosomal ACDs, and differentially regulated ACD compaction. Experimental perturbations and polymer modeling suggest that dynamic protein-protein and protein-DNA interactions within ACDs couple with loop extrusion to organize ACD topology. Dysorganization of ACDs upon CTCF or Cohesin loss alters transcription factor binding and target search dynamics in living cells. These results uncover fundamental mechanisms underpinning the formation of 3D genome architecture and its pivotal function in transcriptional regulation.

molecular biology↗

bioRxiv2018

Augmin-mediated amplification of long-lived spindle microtubules directs plus-ends to kinetochores

Dividing cells reorganize their microtubule cytoskeleton into a bipolar spindle, which moves one set of sister chromatids to each nascent daughter cell. Early spindle assembly models postulated that spindle-pole-derived microtubules search the cytoplasmic space until they randomly encounter a kinetochore to form a stable attachment. More recent work uncovered several additional, centrosome-independent microtubule generation pathways, but the contributions of each pathway to spindle assembly have remained unclear. Here, we combined live microscopy and mathematical modeling to show that most microtubules nucleate at non-centrosomal regions in dividing human cells. Using a live-cell probe that selectively labels aged microtubule lattices, we demonstrate that the distribution of growing microtubule plus-ends can be almost entirely explained by Augmin-dependent amplification of long-lived microtubules. By ultra-fast 3D lattice light-sheet microscopy, we observed that this mechanism results in a strong directional bias of microtubule growth towards individual kinetochores. Our systematic quantification of spindle dynamics reveals highly coordinated microtubule growth during kinetochore-fiber assembly.

cell biology↗