Biology subjects

Boujemaa-Paterski, R.

Publications and source records attributed to Boujemaa-Paterski, R..

6 recordsLinked to original sources

bioRxiv2025

VASP regulates the polar organization of adhesion-associated actin filaments

Focal adhesions (FAs) are dynamic macromolecular assemblies that anchor the actin cytoskeleton to the extracellular matrix via integrin receptors, thereby regulating cell morphology and migration. Although FA maturation and organization have been extensively studied, it remains unclear how regulatory proteins influence the 3D architecture of FAs. Here, we show that loss of the vasodilator-stimulated phosphoprotein (VASP) impairs adhesion dynamics. We employed CRISPR/Cas9-mediated knockout of VASP and/or the mechanosensitive adaptor protein zyxin to investigate their respective roles in actin-adhesion coupling. Loss of VASP and zyxin correlates with altered FA morphology and impaired dynamics. Using cryo-electron tomography (cryo-ET), we resolved the polarity of individual actin filaments associated with FAs and identified a contractility-related actin layer enriched with tropomyosin. VASP and zyxin are required for the assembly of dense and aligned actin bundles with uniform polarity, oriented with their barbed ends towards the cell edge. In contrast, the tropomyosin-decorated dorsal actin layer remains unaffected by these perturbations. Our findings reveal distinct, layered architectures within FAs and underscore the cooperative role of VASP and zyxin in stabilizing the organization of actin filaments at functional adhesion sites.

cell biology↗

bioRxiv2024

The molecular basis of lamin-specific chromatin interactions

In the cell nucleus, chromatin is anchored to the nuclear lamina, a network of lamin filaments and binding proteins that underly the inner nuclear membrane. The nuclear lamina is involved in chromatin organisation through the interaction of lamina-associated domains (LADs) within the densely packed heterochromatin regions. Employing cryo-focused ion beam (cryo-FIB) milling in conjunction with cryo-electron tomography (cryo-ET), we analysed the distribution of nucleosomes at the lamin-chromatin interface. Depletion of lamin A/C reduced the concentration of nucleosomes at the nuclear periphery, suggesting that lamins are directly involved in the interaction with chromatin. Using cryo-electron microscopy (cryo-EM), we then identified the specific binding motif of the lamin A tail domain that interacts with nucleosomes, distinguishing it from the other lamin isoforms. Furthermore, we examined chromatin structure dynamics using a genome-wide analysis that revealed lamin-dependent macroscopic-scale alterations in gene expression and chromatin remodelling. Our findings provide detailed insights into the dynamic and structural interplay between lamin isoforms and chromatin, molecular interactions which are shaping chromatin architecture and epigenetic regulation.

cell biology↗

bioRxiv2023

Vimentin filaments integrate low complexity domains in a highly complex helical structure

Intermediate filaments (IFs) are integral components of the cytoskeleton. They provide cells with tissue-specific mechanical properties and are involved in numerous cellular processes. Due to their intricate architecture, a 3D structure of IFs has remained elusive. Here we use cryo-focused ion beam milling, cryo-electron microscopy and tomography, to obtain a 3D structure of vimentin IFs (VIFs). VIFs assemble into a modular, densely-packed and highly-ordered helical symmetric structure of 40 -helices in cross-section, organized into 5 protofibrils. Surprisingly, the intrinsically disordered head domains form an amyloid-like fiber in the center of VIFs, while the intrinsically disordered tails form lateral connections between the protofibrils. Our findings demonstrate how protein domains of low sequence complexity can complement well-folded protein domains to construct a biopolymer with striking strength and stretchability.

biochemistry↗

bioRxiv2022

A network of mixed actin polarity in the leading edge of spreading cells

Physical interactions of cells with the underlying extracellular matrix (ECM) play key roles in multiple cellular processes, such as tissue morphogenesis, cell motility, wound healing, cancer invasion and metastasis. The actin cytoskeletal network is a central driver and regulator of cellular dynamics, that produces membrane protrusions such as lamellipodia and filopodia. In this study, we examined actin organization in the expanding lamellipodia during the early stages of cell spreading. To gain insight into the 3D actin organization, at a molecular resolution, we plated cultured fibroblasts on galectin-8 coated EM grids, an ECM protein presents in disease states. We then combined cryo-electron tomography (cryo-ET) with advanced image processing tools for reconstructing the structure of F-actin in the lamellipodia. This approach enabled us to resolve the polarity and orientation of the filaments, and the structure of the Arp2/3 complexes associated with F-actin branches. We show here that F-actin in lamellipodial protrusions forms a dense network with three distinct sub-domains. One consists primarily of radial filaments, with their barbed ends pointing towards the membrane, the other is enriched with parallel filaments that run between the radial fibers, in addition to an intermediate sub-domain. Surprisingly, a minor, yet significant ([~]10%) population of actin filaments, are oriented with their barbed ends towards the cell center. Our results provide novel structural insights into F-actin assembly and dynamic reorganization in the leading edge of spreading cells.

cell biology↗

bioRxiv2022

How talin allosterically activates vinculin

The talin-vinculin axis is a key mechanosensing component of cellular focal adhesions. How talin and vinculin respond to forces and regulate one another remains unclear. By combining single molecule magnetic tweezer experiments, Molecular Dynamics simulations, actin bundling assays, and adhesion assembly experiments in live cells, we here discover a two-ways allosteric network within vinculin as a regulator of the talin-vinculin interaction. We directly observe a maturation process of vinculin upon talin binding which reinforces the binding to talin at a rate of 0.03 s-1. This allosteric transition can compete with force-induced dissociation of vinculin from talin only at 7-10 pN. Mimicking the allosteric activation by mutation yields a vinculin molecule that bundles actin and localizes to focal adhesions in a force-independent manner. Hence, the allosteric switch confines talin-vinculin interactions and focal adhesion build-up to intermediate force levels. The allosteric vinculin mutant is a valuable molecular tool to further dissect the mechanical and biochemical signalling circuits at focal adhesions and elsewhere.

biophysics↗

bioRxiv2018

Reconstitution of the equilibrium state of dynamic actin networks

Principles of regulation of actin network dimensions, fundamentally important for cell functions, remain unclear. We studied in vitro and in silico the effect of key parameters, actin density, ADF/Cofilin concentration and network width on the network length. In the presence of ADF/Cofilin, networks reached equilibrium and became globally treadmilling. At the trailing edge, the network disintegrated into large fragments. A mathematical model predicts the network length as a function of width, actin and ADF/Cofilin concentrations. Local depletion of ADF/Cofilin by binding to actin is significant, leading to wider networks growing longer. A single rate of breaking network nodes, proportional to ADF/Cofilin density and inversely proportional to the square of the actin density, can account for the disassembly dynamics. Selective disassembly of heterogeneous networks by ADF/Cofilin controls steering during motility. Our results establish general principles on how the dynamic equilibrium state of actin network emerges from biochemical and structural feedbacks.

cell biology↗