Biology subjects

Roudot, P.

Publications and source records attributed to Roudot, P..

12 recordsLinked to original sources

bioRxiv2024

A quantitative pipeline for whole-mount deep imaging and multiscale analysis of gastruloids

Whole-mount 3D imaging at the cellular scale is a powerful tool for exploring complex processes during morphogenesis. In organoids, it allows examining tissue architecture, cell types, and morphology simultaneously in 3D models. However, cell packing in multilayered organoid tissues hinders both deep imaging and quantification of cell-scale processes. To address these challenges, we developed an experimental and computational pipeline to extract properties at scales ranging from cell to tissue. The experimental module is based on two-photon imaging of immunostained organoids. The computational module corrects for optical artifacts, performs accurate 3D nuclei segmentation and reliably quantifies gene expression. We provide the computational module as a user-friendly Python package called Tapenade, along with napari plugins which enable joint data processing and exploration across scales. We demonstrate the pipeline by quantifying 3D spatial patterns of gene expression and nuclear morphology in gastruloids, revealing how local cell deformations and gene co-expression relate to tissue-scale organization. This quantitative pipeline improves our understanding of gastruloid development, and lays the groundwork for a wide range of multi-layered organoids and tumoroids systems

biophysics↗

bioRxiv2022

Resolution doubling in light-sheet microscopy via oblique plane structured illumination

Structured illumination microscopy (SIM) doubles the spatial resolution of a fluorescence microscope without requiring high laser powers or specialized fluorophores. However, the excitation of out-of-focus fluorescence can accelerate photobleaching and phototoxicity. In contrast, light-sheet fluorescence microscopy (LSFM) largely avoids exciting out-of-focus fluorescence, thereby enabling volumetric imaging with low photo-bleaching and intrinsic optical sectioning. Combining SIM with LSFM would enable gentle 3D imaging at doubled resolution. However, multiple orientations of the illumination pattern, which are needed for isotropic resolution doubling in SIM, are challenging to implement in a light-sheet format. Here we show that multidirectional structured illumination can be implemented in oblique plane microscopy, a LSFM technique that uses a single objective for excitation and detection, in a straightforward manner. We demonstrate isotropic lateral resolution below 150nm, combined with lower photo-toxicity compared to traditional SIM systems and volumetric acquisition speed exceeding 1Hz.

biophysics↗

bioRxiv2021

Profiling site-specific cell states of Ewing Sarcoma xenografts in zebrafish

Tissue microenvironments affect the functional states of cancer cells, but determining these influences in vivo has remained a significant challenge. We present a quantitative high-resolution imaging assay of cancer cell morphology in zebrafish xenografts to probe functional adaptation to variable cell extrinsic cues and molecular interventions. We focus on Ewing Sarcoma, a pediatric cancer driven by a single oncogenic fusion protein EWSR1-FLI1, and with little to no additional somatic mutations, making it a prototypical form of cancer whose adaptation to microenvironments is likely driven by acute, non-genomic mechanisms. Using computer vision analysis of 3D cell shapes, we find systematic shifts in the distribution of cell morphotypes between distinct sites in the fish embryo. We also find site-specific morphological responses to differential expression of EWSR1-FLI1. Combining these data we propose a model where Ewing Sarcoma cancer cell plasticity is sensitive both to expression fluctuation of EWSR1-FLI1 and signals from the surrounding tissue microenvironment, with either or both factors possibly contributing to the oncogenic potential of these cells.

cancer 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

Real-Time Multi-Angle Projection Imaging of Biological Dynamics

We introduce a cost-effective and easily implemented scan unit which enables any camera-based microscope to perform projection imaging from diverse viewing angles. We demonstrate this capability on Lattice Light-Sheet and Oblique Plane Microscopy by rapidly delivering projection images with an uncompromised lateral resolution and high optical contrast. By imaging the sample from one or multiple perspectives, our method enables visualization of rapid biological processes, real time stereoscopic imaging as well as three-dimensional particle localization throughout a cellular volume from just two images. Furthermore, because our projection imaging technique provides intuitive three-dimensional renderings in real-time, it improves microscope usability, allows users to more-readily optimize instrument performance and identify biological phenomena of interest on-the-fly, while also reducing data overhead by a factor of >100. We leverage our rapid projection method to image cancer cell morpho-dynamics and calcium signaling in cultured neurons, to perform three-dimensional localization of genetically encoded nanoparticles, as well as to image orthogonal views of an embryonic Zebrafish heart simultaneously.

biophysics↗

bioRxiv2019

Community Standards for Open Cell Migration Data

Cell migration research has become a high-content field. However, the quantitative information encapsulated in these complex and high-dimensional datasets is not fully exploited due to the diversity of experimental protocols and non-standardised output formats. In addition, typically the datasets are not open for reuse. Making the data open and Findable, Accessible, Interoperable, and Reusable (FAIR) will enable meta-analysis, data integration, and data mining. Standardised data formats and controlled vocabularies are essential for building a suitable infrastructure for that purpose but are not available in the cell migration domain. We here present standardisation efforts by the Cell Migration Standardisation Organization, CMSO, an open community-driven organisation to facilitate the development of standards for cell migration data. This work will foster the development of improved algorithms and tools, and enable secondary analysis of public datasets, ultimately unlocking new knowledge of the complex biological process of cell migration.

bioinformatics↗

bioRxiv2019

Direct Arp2/3-vinculin binding is essential for cell spreading, but only on compliant substrates and in 3D

Cells modify their shape in response to the extracellular environment through dynamic remodeling of the actin cytoskeleton by actin-binding proteins (ABPs) 1-4. The relation between actin dynamics and spreading is well-understood for cells on flat glass coverslips; much less is known about cell morphogenesis in compliant three-dimensional environments, and, in particular, how ABPs contribute to this process 5. Here, we knocked-out a diverse set of ABPs, and evaluated the effect of each on cell spreading on planar glass surfaces (2D) and in reconstituted collagen gels (3D). Our morphometric analyses identify the Arp2/3 complex and its associated regulatory genes among the ABPs that contribute most strongly to cell spreading in 3D, but marginally in 2D. Cells lacking Arp3 have reduced spreading specifically in 3D, and display stiffness-dependent cell-matrix adhesion defects. Through manipulation of vinculin activity, we determine that the Arp3 knock-out phenotype largely arises from the lack of direct interaction between vinculin and Arp2/3 complex. This interaction is dispensable for cell spreading in 2D. These data highlight that actin architectural features necessary for adhesion formation and cell spreading in 3D are efficiently compensated on flat and stiff surfaces.

cell biology↗

bioRxiv2019

Light-sheet microscopy with isotropic, sub-micron resolution and solvent-independent large-scale imaging

We present cleared tissue Axially Swept Light-Sheet Microscopy (ctASLM), which achieves sub-micron isotropic resolution, high optical sectioning capability, and large field of view imaging (870x870 m2) over a broad range of immersion media. ctASLM can image live, expanded, and both aqueous and organic chemically cleared tissue preparations and provides 2- to 5-fold better axial resolution than confocal or other reported cleared tissue light-sheet microscopes. We image millimeter-sized tissues with sub-micron 3D resolution, which enabled us to perform automated detection of cells and subcellular features such as dendritic spines.

bioengineering↗

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↗

bioRxiv2018

Universal Light-Sheet Generation with Field Synthesis

We introduce Field Synthesis, a theorem that can be used to synthesize any scanned or dithered light-sheet, including those used in lattice light-sheet microscopy (LLSM), from an incoherent superposition of one-dimensional intensity distributions. This user-friendly and modular approach offers a drastically simplified optical design, higher light-throughput, simultaneous multicolor illumination, and a 100% spatial duty cycle, thereby providing uncompromised biological imaging with decreased rates of photobleaching.

bioengineering↗

bioRxiv2018

Mutant p53 triggers a dynamin-1/APPL1 endosome feedback loop that regulates β1 integrin recycling and migration

Multiple mechanisms contribute to cancer cell progression and metastatic activity, including changes in endocytic trafficking and signaling of cell surface receptors. We report that gain-of-function (GOF) mutant p53 expression enhances {beta} integrin and EGF receptor recycling and increases cell migration by triggering a positive feedback loop involving the activation of dynamin-1 (Dyn1) and accumulation of a spatially-restricted subpopulation of APPL1-positive perimeter endosomes. DNM1 is upregulated at both the mRNA and protein levels in a manner dependent on expression of GOF mutant p53. Perimeter APPL1 endosomes are required for rapid recycling of EGFR and {beta}1 integrins and modulate Akt signaling and Dyn1 activation to create the positive feedback loop that culminates in increased focal adhesion turnover and cell migration. Thus, Dyn1- and Akt-dependent perimeter APPL1 endosomes function as a nexus, integrating signaling and receptor trafficking, that can be co-opted by cancer cells for mutant p53-driven migration and invasion.

cell biology↗

bioRxiv2018

Hyperactive Rac1 drives MAPK-independent proliferation in melanoma by assembly of a mechanosensitive dendritic actin network

AbstractCancer cells use a variety of mechanisms to subvert growth regulation and overcome environmental challenges. Often, these same mechanisms enable cancer cells to also develop resistance to targeted therapies. Here, we describe how a hyperactivating mutation of the Rac1 GTPase (Rac1P29S) harnesses Rac1s role as a regulator of actin polymer assembly to sustain cell cycle progression in growth limiting conditions. This proliferative advantage supports metastatic colonization of melanoma cells and confers insensitivity to inhibitors of the mitogen-activated protein kinase (MAPK) pathway, a frequent target for melanoma treatment. Rac1P29S bypasses the MAPK axis through a mechanism that necessitates cell-matrix attachment, however, does not depend on integrin-mediated focal adhesion assembly and focal adhesion kinase signaling. Even without involvement of canonical adhesion signaling, cells carrying the Rac1P29S mutation show elevated traction upon drug treatment and require mechanical resistance from their surrounding matrix to gain a proliferative advantage. We describe an alternative arm for cell mechanosensing, whereby actin polymerization against a matrix of minimal rigidity organizes biochemical cues to drive proliferative signals. Hyperactivation of Rac1 by the P29S mutation channels this pathway in melanoma through Arp 2/3-dependent formation of a constrained actin brush network that results in the inactivation of tumor suppressor NF2/Merlin. These data suggest an alternative mechanism for mechanosensitive growth regulation that can be hijacked by cancer cells to circumvent the adverse conditions of foreign microenvironments or drug treatment.

cell biology↗