Biology subjects

Lee, R. M.

Publications and source records attributed to Lee, R. M..

10 recordsLinked to original sources

bioRxiv2026

Losing the split ends: unraveling the mechanisms of fiber branching during blood clot formation

The coagulation cascade generates a three-dimensional fibrin gel that seals wounds and prevents the loss of blood. Extensive research has defined the structural and mechanical characteristics of fibrin gels and the various biochemical, physiological, and pathological factors that alter those properties. However, there has been no direct visualization of fibrin polymerization with the spatial and temporal resolution to characterize the fibrin branching process. Thus, a mechanistic explanation for the origins of fibrin structures remains unresolved, with several hypothesized models for how fibrin branching occurs. Using SimView light-sheet microscopy, we captured videos of fibrin fibers forming junctions and gelling. Results: We observed four distinct pathways for fibrin fibers to interact and bind, all predicated on the observation that tumbling and diffusing fibers collide and stick together. Branching occurred due to the binding of mature fibers, not through the splitting of fibers or through the lateral association of small fibrin oligomers (protofibrils), as had been previously hypothesized. Diffusion simulations of rod-like fibers confirmed the branching mechanisms, the relative frequency of the various binding pathways, and the distribution of fiber angles after binding. Scanning Electron Microscopy (SEM) imaging also corroborated the relative frequency of various types of fibrin junctions. Moreover, SEM enabled the visualization of junctions at different timepoints, demonstrating that additional fibrin strands adhere to the initial binding site, strengthening the interaction. These results establish a mechanistic framework for fibrin branching and gel formation and define an innovative experimental approach for future interrogations of fibrin polymerization.

biophysics↗

bioRxiv2026

Myo1e/f regulate phagocytic podosomes to promote efficient cup closure in macrophages

Phagocytosis requires coordinated remodeling of the actin cytoskeleton to generate protrusive and contractile forces that drive target engulfment. Class I myosins Myo1e and Myo1f (Myo1e/f) have been implicated in linking the plasma membrane to the actin network, but their specific roles during Fc-receptor-mediated phagocytosis remain unclear. Using CRISPR-edited RAW 264.7 macrophages lacking Myo1e and Myo1f, we show that double knockout (dKO) cells exhibit markedly reduced uptake of IgG-coated beads, a phenotype that is partially rescued by re-expression of either myosin. Lattice-light-sheet and confocal imaging revealed distinct F-actin architectures corresponding to the various stages of cup progression, including basal podosome-like adhesions, individual phagocytic podosomes (actin teeth) along the rim of the cup, and a contractile phagocytic ring formed by the reorganization of podosomes into a higher-order network. In Myo1e/f- deficient cells, podosome formation was diminished, actin teeth were largely absent, and the phagocytic ring formed prematurely, which was often accompanied by stalled cup progression and repeated engulfment attempts. Myo1e/f localized both to podosomes and to the inner surface of the phagocytic ring, non-muscle myosin II (NM2) localized to the outer surface, and the absence of Myo1e/f correlated with the diffuse distribution of NM2. In addition, Myo1e/f-deficient macrophages exhibited increased trogocytosis of antibody-opsonized HL-60 cells, indicating a shift from whole-target engulfment toward partial target ingestion. These results suggest that Myo1e/f coordinate spatial and temporal transitions between protrusive and contractile actin networks, thereby ensuring efficient phagocytic cup progression. Our findings highlight a dual role for Myo1e/f in adhesion regulation and force balance during macrophage phagocytosis.

cell biology↗

bioRxiv2026

Adapting Upright Light Sheet Fluorescence Microscopy for Imaging at Air-Liquid Interface

Light sheet fluorescence microscopy (LSFM) is increasingly appreciated as the gold standard for gentle, volumetric imaging with fast acquisition speeds and/or long imaging durations. However, the often-constrained sample space of these microscopes has precluded a specific class of biological specimens from being studied with these tools: those requiring an air-liquid interface (ALI). Here, we present a device for robust imaging at ALI on an upright light sheet microscope with dipping objectives. We demonstrate the system using three relevant use-cases: ex vivo embryonic mouse salivary glands, human epidermal equivalent cultures, and in vivo adult Drosophila melanogaster brains. While the device presented is engineered for one specific light sheet microscope design, it provides a blueprint for easy adaptation to other systems. In doing so, it can potentially spur the use of LSFM for model systems that have so far been unable to take advantage of this powerful technology.

bioengineering↗

bioRxiv2026

Measuring Amorphous Motion: Application of Optical Flow to Three-Dimensional Fluorescence Microscopy Images

Motion is an essential component of any living system. It is rich with information, but it is often challenging to quantitatively extract biologically informative results from the motion apparent in microscopy images. This challenge is exacerbated by the wide variety in biological movement, which often takes the form of difficult-to-segment amorphous structures undergoing complex motion. An image processing technique known as optical flow can capture motion at each pixel in an image, thus bypassing the need for object segmentation or a priori definition of motion types. This makes it a powerful tool for quantitative assessment of biological systems from the protein to organism scale. However, despite its flexibility and strengths for analyzing fluorescence microscopy images, its adoption in the bioimaging community has been limited by the availability of easy-to-use tools and guidance in results interpretation. Here we describe an optical flow tool, OpticalFlow3D, that can be run in Python or MATLAB and is compatible with three-dimensional microscopy images. Using biological examples across length scales, we illustrate how OpticalFlow3D can enable new biological insight.

bioinformatics↗

bioRxiv2026

Mechanically-induced Septin Networks Protect Nuclear Integrity

The cytoskeleton is a key mediator of mechanical interactions in cells, but specific contributions of septins remains unclear. Septins preferentially localize with a subset of actin stress fibers positioned under the nucleus, where they are situated between the membrane and stress fibers. Removing the nucleus from the cell results in the loss of these subnuclear septin-decorated stress fibers. Surprisingly, however, their formation can be rescued using a large glass bead in place of the nucleus. Similarly, applying a compressive force to the cell via confinement, whether externally or through internally generated actomyosin forces, results in increased septin accumulation in regions where the nucleus engages the cell cortex. Finally, loss of septin filaments via knockdown of SEPT7 increases the likelihood of nuclear membrane rupture during confinement. Together these data suggest that septins act as a dynamic mechanosensitive protective mechanism to buffer mechanical forces on the nucleus.

cell biology↗

bioRxiv2023

Metastatic breast cancer cells have reduced calcium and actin response after ATP-P2Y2 signaling

The tumor microenvironment and wound healing after injury both contain extremely high concentrations of the extracellular signaling molecule, adenosine triphosphate (ATP) compared to normal tissue. P2Y2 receptor, an ATP-activated purinergic receptor, is typically associated with pulmonary, endothelial, and neurological cell signaling. Here we report its role and importance in breast epithelial cell signaling and how it is altered in metastatic breast cancer. In response to ATP activation, P2Y2 receptor signaling causes an increase of intracellular Ca2+ in non-tumorigenic breast epithelial cells, while their tumorigenic and metastatic counterparts have significantly reduced Ca2+ responses. The non-tumorigenic cells respond to increased Ca2+ with actin polymerization and localization to cell edges, while the metastatic cells remained unaffected. The increase in intracellular Ca2+ after ATP stimulation was blunted using a P2Y2 antagonist, which also prevented actin mobilization and caused cell dissemination from spheroids in non-tumorigenic breast epithelial cells. Furthermore, the lack of Ca2+ concentration changes and actin mobilization in the metastatic breast cancer cells could be due to reduced P2Y2 expression, which correlates with poorer overall survival in breast cancer patients. This study elucidates rapid changes that occur after elevated intracellular Ca2+ in breast epithelial cells and how metastatic cancer cells have adapted to evade this cellular response.

cancer biology↗

bioRxiv2022

Propagation dynamics of electrotactic motility in large epithelial cell sheets

Directional migration initiated at the wound edge leads epithelial sheets to migrate in wound healing. How such coherent migration is achieved is not well understood. Here we used electric fields to induce robust migration of sheets of human keratinocytes and developed an in silico model to characterize initiation and propagation of epithelial collective migration. Electric fields initiate increase in migrations directionality and speed at the leading edge. The increases propagate across the epithelial sheets, resulting in directional migration of cell sheets as coherent units. Both the experimental and in silico models demonstrated vector-like integration of the electric and default directional cues at the free edge in space and time. The resultant collective migration is remarkably consistent in experiments and modeling, both qualitatively and quantitatively. The keratinocyte model thus faithfully reflects key features of epithelial migration as a coherent tissue in vivo, e.g. that leading cells lead, and that epithelium maintains cell- cell junction.

cell biology↗

bioRxiv2021

Label-free Cell Tracking Enables Collective Motion Phenotyping in Epithelial Monolayers

Collective cell migration is an umbrella term for a rich variety of cell behaviors, whose distinct character is essential for biological function, notably for cancer metastasis. One essential feature of collective behavior is the motion of cells relative to their immediate neighbors. We introduce an AI-based pipeline to segment and track cell nuclei from phase contrast images. Nuclei segmentation is based on a U-Net convolutional neural network trained on images with nucleus staining. Tracking, based on the Crocker-Grier algorithm, quantifies nuclei movement and allows for robust downstream analysis of collective motion. Since the AI algorithm required no new training data, our approach promises to be applicable to and yield new insights for vast libraries of existing collective motion images. In a systematic analysis of a cell line panel with oncogenic mutations, we find that the collective rearrangement metric, D2min, which reflects non-affine motion, shows promise as an indicator of metastatic potential. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/472148v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@82a35corg.highwire.dtl.DTLVardef@b31442org.highwire.dtl.DTLVardef@f703aborg.highwire.dtl.DTLVardef@1120d98_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIVersatile AI-based algorithm can robustly identify individual cells and track their motion from phase contrast images. C_LIO_LIAnalysis of motion of cells relative to nearby neighbors distinguishes weakly tumorigenic (KRas) and metastatic (KRas/PTEN-/-) cell lines. C_LI

biophysics↗

bioRxiv2020

Distinct Roles of Tumor-Associated Mutations in Collective Cell Migration

Recent evidence suggests that groups of cells are more likely to form clinically dangerous metastatic tumors, emphasizing the importance of understanding mechanisms underlying collective behavior. The emergent collective behavior of migrating cell sheets in vitro has been shown to be disrupted in tumorigenic cells but the connection between this behavior and in vivo tumorigenicity is unclear. Here we use particle image velocimetry to measure a multi-dimensional collective migration phenotype for genetically defined cell lines that range in their in vivo behavior from non-tumorigenic to aggressively metastatic. By using cells with controlled mutations, we show that PTEN deletion enhances collective migration, while Ras activation suppresses it, even when combined with PTEN deletion. These opposing effects on collective migration phenotype of two mutations that are frequently found in patient tumors could be exploited in clinical assessments of metastatic potential or in the development of novel treatments for metastatic disease.

biophysics↗

bioRxiv2019

Quantifying topography-guided actin dynamics across scales using optical flow

Periodic surface topographies with feature sizes comparable to those of in vivo collagen fibers are used to measure and compare actin dynamics for two representative cell types that have markedly different migratory modes and physiological purposes: slowly migrating epithelial MCF10A cells and polarizing, fast migrating, neutrophil-like HL60 cells. Both cell types exhibit reproducible guidance of actin waves (esotaxis) on these topographies, enabling quantitative comparisons of actin dynamics. We adapt a computer-vision algorithm, optical flow, to measure the directions of actin waves at the submicron scale. Clustering the optical flow into regions that move in similar directions enables micron-scale measurements of actin-wave speed and direction. Although the speed and morphology of actin waves differ between MCF10A and HL60 cells, the underlying actin guidance by nanotopography is similar in both cell types at the micron and sub-micron scales.

biophysics↗