Classification of the fibronectin variants with curvelets

International audienceThe role of the extracellular matrix (ECM) in the evolution of certain diseases (e.g. fibrosis, cancer) is generally accepted but yet to be completely understood. A numerical model that captures the physical properties of the ECM, could convey certain connections between the topology of its constituents and their associated biological features. This study addresses the analysis and modeling of fibrillar networks containing Fibronectin (FN) networks, a major ECM molecule, from 2D confocal microscopy images. We leveraged the advantages of the fast discrete curvelet transform (FDCT), in order to obtain a multiscale and multidirectional representation of the FN fibrillar networks. This step was validated by performing a classification among the different variants of FN upregulated in disease states with a multi-class classification algorithm, DAG-SVM. Subsequently, we designed a method to ensure the invariance to rotation of the curvelet features. Our results indicate that the curvelets offer an appropriate discriminative model for the FN networks, that is able to characterize the local fiber geometry

Fibronectin Extra Domains tune cellular responses and confer topographically distinct features to fibril networks

International audienceCellular fibronectin (FN; also known as FN1) variants harboring one or two alternatively spliced so-called extra domains (EDB and EDA) play a central bioregulatory role during development, repair processes and fibrosis. Yet, how the extra domains impact fibrillar assembly and function of the molecule remains unclear. Leveraging a unique biological toolset and image analysis pipeline for direct comparison of the variants, we demonstrate that the presence of one or both extra domains impacts FN assembly, function and physical properties of the matrix. When presented to FN-null fibroblasts, extra domain-containing variants differentially regulate pH homeostasis, survival, and TGF- β by tuning the magnitude of cellular responses, rather than triggering independent molecular switches. Numerical analyses of fiber topologies highlight significant differences in variant-specific structural features and provide a first step for the development of a generative model of FN networks to unravel assembly mechanisms and investigate the physical and functional versatility of extracellular matrix landscapes

Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

Endothelial cell interactions with their extracellular matrix are essential for vascular homeostasis and expansion. Large-scale proteomic analyses aimed at identifying components of integrin adhesion complexes have revealed the presence of several RNA binding proteins (RBPs) of which the functions at these sites remain poorly understood. Here, we explored the role of the RBP SAM68 (Src associated in mitosis, of 68 kDa) in endothelial cells. We found that SAM68 is transiently localized at the edge of spreading cells where it participates in membrane protrusive activity and the conversion of nascent adhesions to mechanically loaded focal adhesions by modulation of integrin signaling and local delivery of β-actin mRNA. Furthermore, SAM68 depletion impacts cell-matrix interactions and motility through induction of key matrix genes involved in vascular matrix assembly. In a 3D environment SAM68-dependent functions in both tip and stalk cells contribute to the process of sprouting angiogenesis. Altogether, our results identify the RBP SAM68 as a novel actor in the dynamic regulation of blood vessel networks

Nanometric axial resolution of fibronectin assembly units achieved with an efficient reconstruction approach for multi-angle-TIRF microscopy

International audienceHigh resolution imaging of molecules at the cell-substrate interface is required for understanding key biological processes. Here we propose a complete pipeline for multi-angle total internal reflection fluorescence microscopy (MA-TIRF) going from instrument design and calibration procedures to numerical reconstruction. Our custom setup is endowed with a homogeneous field illumination and precise excitation beam angle. Given a set of MA-TIRF acquisitions, we deploy an efficient joint deconvolution/reconstruction algorithm based on a variational formulation of the inverse problem. This algorithm offers the possibility of using various regularizations and can run on graphics processing unit (GPU) for rapid reconstruction. Moreover, it can be easily used with other MA-TIRF devices and we provide it as an open-source software. This ensemble has enabled us to visualize and measure with unprecedented nanometric resolution, the depth of molecular components of the fibronectin assembly machinery at the basal surface of endothelial cells

A Framework for Multi-angle Tirf Microscope Calibration

International audienceThis communication presents a pipeline for Multi-Angle TIRF calibration from the measurement of the incident angle to the model validation. This problem is of major importance when dealing with 3D reconstruction methods from a set of MA-TIRF acquisitions since the reconstruction accuracy highly depends on the agreement between the theoretical model and the physical system. One main issue is then to build phantom samples with known geometry, or known properties, in order to adjust and/or validate the model. This paper describes such a calibration procedure using a lens as phantom sample and proposes a new model validation experiment based on a dual-color co-localization

Counterbalancing anti-adhesive effects of Tenascin-C through fibronectin expression in endothelial cells

Abstract Cellular fibronectin (FN) and tenascin-C (TNC) are prominent development- and disease-associated matrix components with pro- and anti-adhesive activity, respectively. Whereas both are present in the tumour vasculature, their functional interplay on vascular endothelial cells remains unclear. We have previously shown that basally-oriented deposition of a FN matrix restricts motility and promotes junctional stability in cultured endothelial cells and that this effect is tightly coupled to expression of FN. Here we report that TNC induces FN expression in endothelial cells. This effect counteracts the potent anti-adhesive activity of TNC and leads to the assembly of a dense highly-branched subendothelial matrix that enhances tubulogenic activity. These findings suggest that pro-angiogenic remodelling of the perivascular matrix may involve TNC-induced upregulation of FN in endothelial cells

Fibronectin-guided migration of carcinoma collectives

International audienceFunctional interplay between tumour cells and their neoplastic extracellular matrix plays a decisive role in malignant progression of carcinomas. Here we provide a comprehensive data set of the human HNSCC-associated fibroblast matrisome. Although much attention has been paid to the deposit of collagen, we identify oncofetal fibronectin (FN) as a major and obligate component of the matrix assembled by stromal fibroblasts from head and neck squamous cell carcinomas (HNSCC). FN overexpression in tumours from 435 patients corresponds to an independent unfavourable prognostic indicator. We show that migration of carcinoma collectives on fibrillar FN-rich matrices is achieved through αvβ6 and α9β1 engagement, rather than α5β1. Moreover, αvβ6-driven migration occurs independently of latent TGF-β activation and Smad-dependent signalling in tumour epithelial cells. These results provide insights into the adhesion-dependent events at the tumour-stroma interface that govern the collective mode of migration adopted by carcinoma cells to invade surrounding stroma in HNSCC