Biology subjects

Salamero, J.

Publications and source records attributed to Salamero, J..

8 recordsLinked to original sources

bioRxiv2024

Quantifying Cell Traction Forces at the Individual Fiber Scale in 3D: A Novel Approach Based on Deformable Photopolymerized Fiber Arrays

The forces exerted by cells upon the fibers of the extracellular matrix play a decisive role in cell motility in development and disease. How the local physical properties of the matrix (such as density, stiffness, orientation) affect cellular forces remains poorly understood. Existing approaches to measure cell 3D traction forces within fibrous substrates lack control over the local properties and rely on continuum approaches, not suited for measuring forces at the scale of individual fibers. A novel approach is proposed here to fabricate multilayer arrays of deformable fibers with defined geometrical and mechanical properties using two-photon polymerization. The fibers are characterized using Atomic Force Microscopy and span a wide range of sizes and mechanical properties. This approach is combined with a new reference-free method for measuring traction forces in 3D, which relies on automated segmentation of the fibers coupled with finite element modeling. The force measurement pipeline is applied to study forces exerted by adherent cells, endothelial cells, fibroblasts or macrophages, and reveals how these forces are influenced by fiber density and stiffness. Additionally, coupling to fast volumetric imaging with lattice light-sheet microscopy enables the measurement of the low-intensity and short-lived tractions exerted by amoeboid cells, such as dendritic cells.

biophysics↗

bioRxiv2023

DeepCristae, a CNN for the restoration of mitochondria cristae in live microscopy images

Mitochondria play an essential role in the life cycle of eukaryotic cells. However, we still dont know how their ultrastructure, like the cristae of the inner membrane, dynamically evolves to regulate these fundamental functions, in response to external conditions or during interaction with other cell components. Although high-resolution fluorescent microscopy coupled with recently developed innovative probes can reveal this structural organization, their long-term, fast and live 3D imaging remains challenging. To address this problem, we have developed a convolutional neural network, called DeepCristae, to restore mitochondria cristae in low spatial resolution microscopy images. Our network is trained from 2D STED images using a novel loss specifically designed for cristae restoration. To efficiently increase the size of the training set, we also developed a random image patch sampling centered on mitochondrial areas. To evaluate DeepCristae, quantitative assessments were carried out using metrics we derived by focusing on the mitochondria and cristae pixels rather than on the whole image as usual. Depending on the conditions of use indicated, DeepCristae works well on broad microscopy modalities (Stimulated Emission Depletion (STED), Live-SR, AiryScan and Lattice Light Sheet Microscopy). It is ultimately applied in the context of mitochondrial network dynamics during interaction with endo/lysosome membranes.

bioinformatics↗

bioRxiv2023

The recycling endosome biogenesis machinery coordinates BACE1 endosomal sorting and amyloid-Beta production

Alzheimers disease (AD) is the most common form of dementia worldwide. One of ADs main pathological hallmarks is the cerebral plaque deposits of {beta}-amyloid (A{beta}). A{beta} is generated through sequential enzymatic cleavage of the amyloid precursor protein (APP). The {beta}-secretase or {beta}-site APP-cleaving enzyme 1 (BACE1) initiates this cleavage and is thus key to regulate A{beta} formation. Both APP and BACE1 transit through the endolysosomal system but the exact nature of the compartment(s) where APP cleavage occurs as well as the molecular mechanisms that govern their endosomal sorting remain poorly known. Here we show that RAB11 not only regulates BACE1 transport from early/sorting endosomes (EEs/SEs) and drives the exocytosis of BACE1-containing recycling carriers. Moreover, recycling endosome-associated KIF13A, as well as its closely related homolog KIF13B, which are known RAB11 effectors involved in the biogenesis of recycling endosome (RE) from EEs/SEs, also participate in BACE1 endosomal sorting. Importantly, depletion of KIF13A or KIF13B leads to an increase in A{beta} generation. Depletion of the BLOC-1 complex, previously described as an essential partner for KIF13A-dependent RE biogenesis, also induces increased amount of A{beta}. Altogether, our findings support a model where the EEs/SEs represent a major organelle for A{beta} formation and identify the recycling endosome biogenesis machinery as a master coordinator of BACE1 endosomal sorting and transport.

cell biology↗

bioRxiv2022

STracking: a free and open-source python library for particle tracking and analysis

SummaryAnalysis of intra and extra cellular dynamic like vesicles transport involves particle tracking algorithms. Designing a particle tracking pipeline is a routine but tedious task. Therefore, particle dynamics analysis is often performed by combining several pieces of software (filtering, detection, tracking...) requiring a lot of manual operations, and therefore leading to poorly reproducible results. A good synergy between particle detector with a tracker is of paramount importance. In addition, a user-friendly interface to control the quality of estimated trajectories is necessary. To address these issues, we developed STracking a python library that allows to combine algorithms into standardized particle tracking pipelines. Availability and ImplementationSTracking is available as a python library using "pip install" and the source code is publicly available on GitHub (https://github.com/sylvainprigent/stracking). A graphical interface is available using two napari plugins: napari-stracking and napari-tracks-reader. These napari plugins can be installed via the napari plugins menu or using "pip install". The napari plugin source codes are available on GitHub (https://github.com/sylvainprigent/napari-tracks-reader, https://github.com/sylvainprigent/napari-stracking). Contactsylvain.prigent@inria.fr, cesar-augusto.valades-cruz@curie.fr

bioinformatics↗

bioRxiv2022

SPITFIR(e): A supermaneuverable algorithm for restoring 2D-3D fluorescence images and videos,and background subtraction

While fluorescent microscopy imaging has become the spearhead of modern biology as it is able to generate long-term videos depicting 4D nanoscale cell behaviors, it is still limited by the optical aberrations and the photon budget available in the specimen and to some extend to photo-toxicity. A direct consequence is the necessity to develop flexible and "off-road" algorithms in order to recover structural details and improve spatial resolution, which is critical when pushing the illumination to the low levels in order to limit photo-damages. Moreover, as the processing of very large temporal series of images considerably slows down the analysis, special attention must be paid to the feasibility and scalability of the developed restoration algorithms. To address these specifications, we present a very flexible method designed to restore 2D-3D+Time fluorescent images and subtract undesirable out-of-focus background. We assume that the images are sparse and piece-wise smooth, and are corrupted by mixed Poisson-Gaussian noise. To recover the unknown image, we consider a novel convex and non-quadratic regularizer Sparse Hessian Variation) defined as the mixed norms which gathers image intensity and spatial second-order derivatives. This resulting restoration algorithm named SPITFIR(e) (SParse fIT for Fluorescence Image Restoration) utilizes the primal-dual optimization principle for energy minimization and can be used to process large images acquired with varied fluorescence microscopy modalities. It is nearly parameter-free as the practitioner needs only to specify the amount of desired sparsity (weak, moderate, high). Experimental results in lattice light sheet, stimulated emission depletion, multifocus microscopy, spinning disk confocal, and wide-field microscopy demonstrate the generic ability of the SPITFIR(e) algorithm to efficiently reduce noise and blur, and to subtract undesirable fluorescent background, while avoiding the emergence of deconvolution artifacts.

bioinformatics↗

bioRxiv2021

BioImageIT: Open-source framework for integration of image data-management with analysis

Open science and FAIR principles have become major topics in the field of bioimaging. This is due to both new data acquisition technologies that generate large datasets, and new analysis approaches that automate data mining with high accuracy. Nevertheless, data are rarely shared and rigorously annotated because it requires a lot of manual and tedious management tasks and software packaging. We present BioImageIT, an open-source framework for integrating data management according to FAIR principles with data processing.

bioinformatics↗

bioRxiv2020

HPM Live μ for full CLEM workflow

With the development of advanced imaging methods that took place in the last decade, the spatial correlation of microscopic and spectroscopic information - known as multimodal imaging or correlative microscopy (CM) - has become a broadly applied technique to explore biological and biomedical materials at different length scales. Among the many different combinations of techniques, Correlative Light and Electron Microscopy (CLEM) has become the flagship of this revolution. Where light (mainly fluorescence) microscopy can be used directly for the live imaging of cells and tissues, for almost all applications, electron microscopy (EM) requires fixation of the biological materials. Although sample preparation for EM is traditionally done by chemical fixation and embedding in a resin, rapid cryogenic fixation (vitrification) has become a popular way to avoid the formation of artefacts related to the chemical fixation/embedding procedures. During vitrification, the water in the sample transforms into an amorphous ice, keeping the ultrastructure of the biological sample as close as possible to the native state. One immediate benefit of this cryo-arrest is the preservation of protein fluorescence, allowing multi-step multi-modal imaging techniques for CLEM. To further explore the potential of cryo-fixation, we developed a high-pressure freezing (HPF) system that allows vitrification under different environmental parameters and applied it in different CLEM workflows. In this chapter, we introduce our novel HPF live instrument with a focus on its coupling to a light microscope. We elaborate on the optimization of sample preservation and the time needed to capture a biological event, going from live imaging to cryo-arrest using HPF. We will address the adaptation of HPF to novel correlation workflows related to the forthcoming transition from imaging 2D (cell monolayers) to imaging 3D samples (tissue) and the associated importance of homogeneous deep vitrification. Lastly, we will discuss the potential of our HPM within CLEM protocols especially for correlating live imaging using the Zeiss LSM900 with electron microscopy.

biophysics↗

bioRxiv2020

Kinetics of Plasmodium midgut invasion in Anopheles mosquitoes

Malaria-causing Plasmodium parasites traverse the mosquito midgut cells to establish infection at the basal side of the midgut. This dynamic process is a determinant of mosquito vector competence, yet the kinetics of the parasite migration is not well understood. Here we used transgenic mosquitoes of two Anopheles species and a Plasmodium berghei fluorescence reporter line to track parasite passage through the mosquito tissues at high spatial resolution. We provide new quantitative insight into malaria parasite invasion in African and Indian Anopheles species and demonstrate that species-specific kinetics of Plasmodium invasion is shaped by the mosquito complement-like system. Author SummaryThe traversal of the mosquito midgut cells is one of the critical stages in the life cycle of malaria parasites. Motile parasite forms, called ookinetes, traverse the midgut epithelium in a dynamic process which is not fully understood. Here, we harnessed transgenic reporters to track invasion of Plasmodium parasites in African and Indian mosquito species. We found important differences in parasite dynamics between the two anopheline species and demonstrated an unexpected role of mosquito complement-like system in regulation of parasite invasion.

immunology↗