Biology subjects

Godin, M.

Publications and source records attributed to Godin, M..

10 recordsLinked to original sources

bioRxiv2026

ATAC-seq and MNase-seq Detect Distinct Modes of Chromatin Accessibility

Chromatin accessibility shapes the ability of transcription factors (TFs) and the transcriptional machinery to engage genomic DNA and therefore plays a central role in gene regulation. Two widely used approaches for profiling chromatin accessibility are micrococcal nuclease (MNase)-seq and assay for transposase-accessible chromatin (ATAC)-seq. ATAC-seq peaks are often thought to be equivalent to nucleosome-depleted regions (NDRs) that are defined by MNase-seq; however, these two measurements have not been systematically compared. Here, we perform a side-by-side comparison of ATAC-seq and MNase-seq in budding yeast and find a large discrepancy between ATAC-seq peaks and MNase-defined NDRs. We show that this discrepancy is not primarily due to the intrinsic differences between MNase and Tn5 enzymatic activity. Instead, ATAC-seq peaks and NDRs capture distinct chromatin states. Specifically, ATAC-seq peaks are enriched at dynamic nucleosomes associated with transcriptional co-regulators, including SAGA and SWI/SNF, whereas ATAC- NDRs mark more static nucleosome-free regions at promoters. Depletion of SWI/SNF reduces ATAC-seq signals without affecting most NDRs. Generation of NDRs and ATAC-seq peaks requires distinct TF properties, and native TFs differ in their ability to produce these two types of open chromatin. Finally, we find that the functional distinctions between ATAC-seq peaks and NDRs are widespread across eukaryotic species, including human cells. Together, our results provide new insights into the biological meaning of chromatin accessibility measured by these two assays.

molecular biology↗

bioRxiv2026

Catch-and-Display Immunoassay as an Accessible Platform for Digital Biomarker Detection

Digital immunoassays provide exceptional analytical sensitivity for detecting low-abundance biomarkers, but their broad adoption is limited by practical barriers. Commercial platforms are prohibitively expensive for routine use by individual laboratories, and laboratory-scale concepts typically describe specialized biosensors and sophisticated workflows. Here, we introduce a nanomembrane-based Catch-and-Display Immunoassay (CAD-IA) as a cost-effective and laboratory-friendly digital immunoassay for common research settings. In CAD-IA, fluorescent nanoparticles are "captured" by the nanoscale pores of ultrathin silicon nitride membranes through a pipette powered filtration. The captured nanoparticles serve as optically isolated hotspots for fluorescent immunocomplex formation when target antigen is present. Co-localization of the fluorescent particles and fluorescent immunocomplexes are then "displayed" and quantified by standard confocal microscopy to generate digital signals. CAD-IA is implemented using the {micro}SiM-DX (microfluidic device featuring an ultrathin silicon membrane for diagnostics) platform, which is manually assembled from mass produced, cost-effective components. Using the traumatic brain injury (TBI) biomarker S100B as a model, we demonstrate that CAD-IA provides consistent digital outputs and linear quantification with a dynamic range of at least two orders of magnitude when digital and analog analysis are combined on the same image sets. We further demonstrate that the assay maintains linearity in serum matrices and achieves suitable sensitivity (LoD = 0.02 g/mL) for clinically relevant diagnostic with the addition of tyramide signal amplification (TSA). While further optimization of CAD-IA is possible, these results constitute a proof-of-concept demonstration of a novel digital immunoassay deployable in most research laboratories.

bioengineering↗

bioRxiv2022

Targeting extracellular vesicle delivery to the lungs by microgel encapsulation

Extracellular vesicles (EVs) secreted by stem and progenitor cells have significant potential as cell-free cellular therapeutics. Yet, small EVs (<200 nm) are rapidly cleared after systemic administration, mainly by the liver, presenting challenges targeting EVs to a specific organ or tissue. Microencapsulation using natural nano-porous hydrogels (microgels) has been shown to enhance engraftment and increase the survival of transplanted cells. We sought to encapsulate EVs within microgels to target their delivery to the lung by virtue of their size-based retention within the pulmonary microcirculation. Mesenchymal stromal cell (MSC) derived EVs were labelled with the lipophilic dye (DiR) and encapsulated within agarose-gelatin microgels. Endothelial cells and bone marrow derived macrophages were able to take up EVs encapsulated in microgels in vitro, but less efficiently than the uptake of free EVs. Following intrajugular administration, microgel encapsulated EVs were selectively retained within the lungs for 72 hours, while free EVs were rapidly cleared by the liver. Furthermore, microgel loaded EVs demonstrated greater uptake by lung cells, in particular CD45+ immune cells, as assessed by flow cytometry compared to free EVs. Microencapsulation of EVs may be a novel tool for enhancing targeted delivery of EVs for future therapeutic applications.

bioengineering↗

bioRxiv2020

Time dependencies in the dynamic mechanical behavior of 3D microtissue cell cultures

Characterizing the time-dependent mechanical properties of cells is not only necessary to determine how they deform, but also to fully understand how external forces trigger biochemical-signaling cascades to govern their behavior. Presently mechanical properties are largely assessed by applying local shear or compressive forces on single cells in isolation grown on non-physiological 2D surfaces. In comparison, we developed the microfabricated vacuum actuated stretcher to measure tensile loading of 3D multicellular microtissue cultures. With this approach, we assessed here the time-dependent stress relaxation and recovery responses of microtissues, and quantified the spatial remodeling that follows step length changes. Unlike previous results, stress relaxation and recovery in microtissues measured over a range of step amplitudes and pharmacological treatments followed a stretched exponential behavior describing a broad distribution of inter-related timescales. Furthermore, despite a performed compendium of experiments, all responses led to a single linear relationship between the residual elasticity and degree of stress relaxation, suggesting that these mechanical properties are coupled through interactions between structural elements and the association of cells with their matrix. Lastly, although stress relaxation could be quantitatively and spatially linked to recovery, they differed greatly in their dynamics; while stress recovery behaved as a linear process, relaxation time constants changed with an inverse power law with step size. This assessment of microtissues offers insights into how the collective behavior of cells in a 3D collagen matrix generate the dynamic mechanical properties of tissues, which is necessary to understanding how cells deform and sense mechanical forces in the body.

biophysics↗

bioRxiv2020

Mechanical stretch sustains myofibroblast phenotype and function in microtissues through latent TGF-β1 activation

Fibrosis is a leading cause of death in developed countries that is characterized by a progressive deterioration of tissue mechanical behavior. Developing methods to study tissue mechanics and myofibroblast activation may lead to new targets for therapeutic treatments that are urgently needed. Microtissue arrays are a promising approach to conduct relatively high throughput research into fibrosis as they recapitulate key biomechanical aspects of the disease through a relevant 3D extracellular environment. In early work, our group developed a device called the MVAS-force to stretch microtissues while enabling simultaneous assessment of their dynamic mechanical behavior. Here we investigated TGF-{beta}1 induced fibroblast to myofibroblast differentiation in microtissue cultures using our MVAS-force device through assessing -SMA expression, contractility and stiffness. By doing so, we linked cell-level phenotypic changes to functional changes that characterize the clinical manifestation of fibrotic disease. As expected, TGF-{beta}1 treatment promoted a myofibroblastic phenotype and microtissues became stiffer and possessed increased contractility. Furthermore, these changes were partially reversible upon TGF-{beta}1 withdrawal. In contrast, however, long-term cyclic stretching maintained myofibroblast activation. Furthermore stretching had no effect compared static cultures when TGF-{beta}1 receptors were inhibited and stretching promoted myofibroblast differentiation when given latent TGF-{beta}1. Together these results suggest that external mechanical stretch may activate latent TGF-{beta}1 and might be a powerful stimulus for continued myofibroblast activation to progress fibrosis. Further exploration of this pathway with our approach may yield new insights into myofibroblast activation and more effective therapeutic treatments for fibrosis. Insight boxUsing a novel high-throughput approach, we quantified the effects of dynamic mechanical stretching on the phenotype and function of cells in 3D microtissue cultures during myofibroblast activation with TGF-{beta}1 treatment and subsequent withdrawal. Our findings show that mechanical stretch may activate endogenously produced latent TGF-{beta}1 to maintain the presence and activity of myofibroblasts after tissue injury. Importantly, through this feed forward mechanism, mechanical stretch might be a powerful stimulus that directs tissues away from recovery and towards the development of fibrosis.

cell biology↗

bioRxiv2019

Structural and mechanical remodeling of the cytoskeleton studied in 3D microtissues under acute dynamic stretch

When stretched, cells cultured on 2D substrates share a universal softening and fluidization response that arises from poorly understood remodeling of well-conserved cytoskeletal elements. It is known, however, that the structure and distribution of the cytoskeleton is profoundly influenced by the dimensionality of a cells environment. Therefore, in this study we aimed to determine whether cells cultured in a 3D matrix share this softening behavior and to link it to cytoskeletal remodeling. To achieve this, we developed a high-throughput approach to measure the dynamic mechanical properties of cells and allow for sub-cellular imaging within physiologically relevant 3D microtissues. We found that fibroblast, smooth muscle and skeletal muscle microtissues strain softened but did not fluidize, and upon loading cessation, they regained their initial mechanical properties. Furthermore, microtissue prestress decreased with the strain amplitude to maintain a constant mean tension. This adaptation under an auxotonic condition resulted in lengthening. A filamentous actin cytoskeleton was required, and responses were mirrored by changes to actin remodeling rates and visual evidence of stretch-induced actin depolymerization. Our new approach for assessing cell mechanics has linked behaviors seen in 2D cultures to a 3D matrix, and connected remodeling of the cytoskeleton to homeostatic mechanical regulation of tissues.

biophysics↗

bioRxiv2019

Mechanotransduction of strain regulates an invasive phenotype in newly transformed epithelial cells

Our organs and tissues are in constant motion, exposing epithelial cells to mechanical stretch. However, how these external forces impact cellular morphology, organization and dynamics in healthy and diseased tissues is still being elucidated. In several studies, we and others have shown how mechanical stresses and strains in the epithelium can modulate the dynamics and invasiveness of transformed cells. Carcinoma, the most common type of cancer, develops in the sheets of cells forming the epithelium and lining our organs and cavities. It usually begins with the transformation of a single cell via the activation of oncogenes such as Ras. Here, we show in a model system how mechanical stretch in epithelial sheets results in a more invasive phenotype in transformed cells. Cyclic strain prevents the formation of strong circumferential belts of actin in RasV12 cells and greatly promoting the formation of RasV12 protrusions, typical of a more invasive phenotype. We also show that RasV12 and wild type MDCK cells possess distinct sensitivity to strain. External forces remodel their actin cytoskeletons and adhesion complexes differently, resulting in a more invasive system dynamic. Our work demonstrates that the Rho-ROCK mechanotransduction pathway is involved in regulating a mechanically-induced switch to a more invasive phenotype. In a mechanically dynamic microenvironment, transformed cells exhibit drastically different cellular dynamics and movements when compared to static conditions. They grow larger invasive protrusions, potentially making them harder to be eliminated from healthy tissues. The insights gained in this study reveal the complex dynamics at play in healthy and transformed epithelial cells which are found in a mechanically active microenvironment.

biophysics↗

bioRxiv2018

Time dependence of cellular responses to dynamic and complex strain fields

Exposing cells to an unconventional sequence of physical cues can reveal subtleties of cellular sensing and response mechanisms. We investigated the mechanoresponse of cyclically-stretched fibroblasts under a spatially non-uniform strain field which was subjected to repeated changes in stretching directions over 55 hours. A polydimethylsiloxane microfluidic stretcher array optimized for complex staining procedures and imaging was developed to generate biologically relevant strain and strain gradient amplitudes. We demonstrated that cells can successfully reorient themselves repeatedly, as the main cyclical stretching direction is consecutively switched between two perpendicular directions every 11 hours. Importantly, from one reorientation to the next, the extent to which cells reorient themselves perpendicularly to the local strain direction progressively decreases, while their tendency to align perpendicularly to the strain gradient direction tends to increase. We demonstrate that these results are consistent with our finding that cellular responses to strains and strain gradients occur on two distinct time scales, the latter being slower. Overall, our results reveal the absence of major irreversible cellular changes that compromise the ability to sense and reorient to changing strain directions under the conditions of this experiment. On the other hand, we show how the history of strain field dynamics can influence the cellular realignment behavior, due to the interplay of complex time-dependent responses.

cell biology↗

bioRxiv2017

A vacuum-actuated microtissue stretcher for long-term exposure to oscillatory strain within a 3D matrix

Although our understanding of cellular behavior in response to extracellular biological and mechanical stimuli has greatly advanced using conventional 2D cell culture methods, these techniques lack physiological relevance. We developed the microtissue vacuum-actuated stretcher (MVAS) to probe cellular behavior within a 3D multicellular environment composed of innate matrix protein, and in response to continuous uniaxial stretch. The MVAS consists of an array of fifty self-assembled microtissues bordered by vacuum chambers. When a vacuum is applied, the microtissues stretch in plane allowing live imaging. The MVAS is highly suitable for biomedical research and pharmaceutical discovery due to a high-throughput array format and scalable fabrication steps outlined in this paper. We validated our approach by characterizing the bulk microtissue strain, the microtissue strain field and single cell strain, and by assessing F-actin expression in response to chronic cyclic strain of 10%. The MVAS was shown to be capable of delivering reproducible dynamic bulk strain amplitudes up to 13% and the strain field had local maxima around each of the cantilevers. The strain at the single cell level was found to be 10.4% less than the microtissue axial strain due to cellular rotation. Chronic cyclic strain produced a 35% increase in F-actin expression consistent with previously observed cytoskeletal reinforcement in 2D cell culture. The MVAS may further our understanding of the reciprocity shared between cells and their environment, which is critical to meaningful biomedical research and successful therapeutic approaches.

bioengineering↗

bioRxiv2016

Cell Mechanosensing and Avoidance of Strain Gradients

Cyclically stretched cells are known to exhibit a strain-induced reorientation response. In this study, we comprehensively analyse this behaviour for human fibroblasts subjected to a highly non-uniform strain field within a polymethylsiloxane microdevice. We demonstrate a strong correlation between the strain amplitude and the degree of cell alignment perpendicular to the principal strain direction (stretching avoidance). Analogously, our results indicate that the strain gradient amplitude and direction also regulate this reorientation through a coordinated gradient avoidance response. We stipulate that strain gradients are thus biologically relevant mechanical cues sensed by cells. To gain insight into the underlying mechanosensing processes, we also studied focal adhesion reorganization and the effect of modulating myosin-II contractility. The extracted focal adhesion orientation distributions are similar to those obtained for the cell bodies, and their density is increased by the presence of stretching forces. Moreover, it was found that the myosin-II activity promoter calyculin-A has little effect on the cellular response, while the inhibitor blebbistatin suppresses cell and focal adhesion alignment and reduces focal adhesion density. These results confirm that similar internal structures involved in sensing and responding to strain direction and amplitude are also key players in strain gradient mechanosensing and avoidance.

biophysics↗