Benoit Ladoux

Cytoskeletal coherence requires myosin-IIA contractility

Maintaining a physical connection across cytoplasm is crucial for many biological processes such as matrix force generation, cell motility, cell shape and tissue development. However, in the absence of stress fibers, the coherent structure that transmits force across the cytoplasm is not understood. We find that nonmuscle myosin-II (NMII) contraction of cytoplasmic actin filaments establishes a coherent cytoskeletal network irrespective of the nature of adhesive contacts. When NMII activity is inhibited during cell spreading by Rho kinase inhibition, blebbistatin, caldesmon overexpression or NMIIA RNAi, the symmetric traction forces are lost and cell spreading persists, causing cytoplasm fragmentation by membrane tension that results in ‘C’ or dendritic shapes. Moreover, local inactivation of NMII by chromophore-assisted laser inactivation causes local loss of coherence. Actin filament polymerization is also required for cytoplasmic coherence, but microtubules and intermediate filaments are dispensable. Loss of cytoplasmic coherence is accompanied by loss of circumferential actin bundles. We suggest that NMIIA creates a coherent actin network through the formation of circumferential actin bundles that mechanically link elements of the peripheral actin cytoskeleton where much of the force is generated during spreading.

Cooperative Retraction of Bundled Type IV Pili Enables Nanonewton Force Generation

The causative agent of gonorrhea, Neisseria gonorrhoeae, bears retractable filamentous appendages called type IV pili (Tfp). Tfp are used by many pathogenic and nonpathogenic bacteria to carry out a number of vital functions, including DNA uptake, twitching motility (crawling over surfaces), and attachment to host cells. In N. gonorrhoeae, Tfp binding to epithelial cells and the mechanical forces associated with this binding stimulate signaling cascades and gene expression that enhance infection. Retraction of a single Tfp filament generates forces of 50–100 piconewtons, but nothing is known, thus far, on the retraction force ability of multiple Tfp filaments, even though each bacterium expresses multiple Tfp and multiple bacteria interact during infection. We designed a micropillar assay system to measure Tfp retraction forces. This system consists of an array of force sensors made of elastic pillars that allow quantification of retraction forces from adherent N. gonorrhoeae bacteria. Electron microscopy and fluorescence microscopy were used in combination with this novel assay to assess the structures of Tfp. We show that Tfp can form bundles, which contain up to 8–10 Tfp filaments, that act as coordinated retractable units with forces up to 10 times greater than single filament retraction forces. Furthermore, single filament retraction forces are transient, whereas bundled filaments produce retraction forces that can be sustained. Alterations of noncovalent protein–protein interactions between Tfp can inhibit both bundle formation and high-amplitude retraction forces. Retraction forces build over time through the recruitment and bundling of multiple Tfp that pull cooperatively to generate forces in the nanonewton range. We propose that Tfp retraction can be synchronized through bundling, that Tfp bundle retraction can generate forces in the nanonewton range in vivo, and that such high forces could affect infection.

α-Catenin and vinculin cooperate to promote high E-cadherin-based adhesion strength.

Background: Cadherin interactions with catenins are crucial for intercellular adhesion. Results: αE-catenin and vinculin cooperate to promote the time-dependent reinforcement of cadherin-mediated adhesions. Conclusion: αE-catenin and vinculin form a mechanoresponsive link between cadherin and the underlying actin cytoskeleton. Significance: The force-dependent modulation of α-catenin and vinculin recruitment contributes to the development of cadherin adhesion strength. Maintaining cell cohesiveness within tissues requires that intercellular adhesions develop sufficient strength to support traction forces applied by myosin motors and by neighboring cells. Cadherins are transmembrane receptors that mediate intercellular adhesion. The cadherin cytoplasmic domain recruits several partners, including catenins and vinculin, at sites of cell-cell adhesion. Our study used force measurements to address the role of αE-catenin and vinculin in the regulation of the strength of E-cadherin-based adhesion. αE-catenin-deficient cells display only weak aggregation and fail to strengthen intercellular adhesion over time, a process rescued by the expression of αE-catenin or chimeric E-cadherin·αE-catenins, including a chimera lacking the αE-catenin dimerization domain. Interestingly, an αE-catenin mutant lacking the modulation and actin-binding domains restores cadherin-dependent cell-cell contacts but cannot strengthen intercellular adhesion. The expression of αE-catenin mutated in its vinculin-binding site is defective in its ability to rescue cadherin-based adhesion strength in cells lacking αE-catenin. Vinculin depletion or the overexpression of the αE-catenin modulation domain strongly decreases E-cadherin-mediated adhesion strength. This supports the notion that both molecules are required for intercellular contact maturation. Furthermore, stretching of cell doublets increases vinculin recruitment and α18 anti-αE-catenin conformational epitope immunostaining at cell-cell contacts. Taken together, our results indicate that αE-catenin and vinculin cooperatively support intercellular adhesion strengthening, probably via a mechanoresponsive link between the E-cadherin·β-catenin complexes and the underlying actin cytoskeleton.

Adhesion on Microstructured Surfaces

Using a homemade setup, we investigated the adhesion between soft elastic substrates bearing surface microstructures (array of caps [resp., holes] of height [resp., depth] h) and a smooth surface of the same rubber. In the framework of the classical model developed by Johnson, Kendall, and Roberts, we show the following. (i) The existence of a critical height h c for the microstructures, resulting from a competition between the adhesion energy and the elastic deformation energy necessary to invade the pattern: for h < h c , the bead and the substrate are in intimate contact even when the applied force is zero, and for h > h c , an air film remains intercalated in the microstructure, and the contact is limited to the top of the caps or between the holes. The transition between these two states can be induced by increasing the squeezing force. (ii) The adhesion energy, W, of intimate contacts (h < h c ) decreases as the height increases. Suspended contacts correspond to a low adhesion and a nearly Hertzian behavior. Using simple scaling arguments and a two-level energetic description (single microstructure and whole contact) we propose a semiquantitative description of these observations.

Techniques to measure pilus retraction forces.

The importance of physical forces in biology is becoming more appreciated. Neisseria gonorrhoeaehas become a paradigm for the study of physical forces in the bacterial world. Cycles of elongations and retractions of Type IV pili enables N. gonorrhoeaebacteria to exert forces on its environment, forces that play major roles in the life cycle of this pathogen. In order to better understand the role of these forces, there is a need to fully characterize them. Here, we present two different techniques, optical tweezers and Polyacrylamide MicroPillars (PoMPs), for measuring pilus retraction forces. Initially designed for N. gonorrhoeae, these assays can be readily modified to study other pilus-bearing bacteria including Neisseria meningitidis.

High-resolution imaging of cellular processes across textured surfaces using an indexed-matched elastomer.

Understanding and controlling how cells interact with the microenvironment has emerged as a prominent field in bioengineering, stem cell research and in the development of the next generation of in vitro assays as well as organs on a chip. Changing the local rheology or the nanotextured surface of substrates has proved an efficient approach to improve cell lineage differentiation, to control cell migration properties and to understand environmental sensing processes. However, introducing substrate surface textures often alters the ability to image cells with high precision, compromising our understanding of molecular mechanisms at stake in environmental sensing. In this paper, we demonstrate how nano/microstructured surfaces can be molded from an elastomeric material with a refractive index matched to the cell culture medium. Once made biocompatible, contrast imaging (differential interference contrast, phase contrast) and high-resolution fluorescence imaging of subcellular structures can be implemented through the textured surface using an inverted microscope. Simultaneous traction force measurements by micropost deflection were also performed, demonstrating the potential of our approach to study cell-environment interactions, sensing processes and cellular force generation with unprecedented resolution.