Deciphering the molecular links between actomyosin force, integrin regulation and ECM properties through an in vitro reconstitution – RECAMECA

We reconstitute a minimal adhesion complex made of an integrin-containing lipid bilayer that couples actomyosin cables, via regulatory proteins on one side, and a micropatterned ECM on the other side. Single particle tracking microscopy of FA components in our assay, where the concentration of the components, actomyosin force and the physical properties of the ECM are modulatable, unambiguously identify mechanosensitive elementary reactions. The physiological relevance of our in vitro findings is tested in cells systematically.

The central point of this project is the study of the mechanosensitivity of an artificial focal adhesion. We started by purifying the transmembrane integrin receptor to establish our system. We have successfully purified the alphaIIb beta3 integrin from platelets which we will first use as a model. We have developed the incorporation of this integrin into a supported lipid bilayer. To mimic the formation of focal adhesions, we micropattered this membranes into islands covered with extracellular matrix. In this biochemical assay, the effect of actomysoin force on the diffusion of fluorescent proteins such as integrin and its activators talin-1 and kindlin-2 is cuurently assessed by FRAP (Fluorescence Recovery After Photobleaching) at I2BC. In parrallel, we develop a strategy to track single proteins movements to decipher changes in the nanoscale organisation of integrin and its regulators in response to force. A first series of experiments has been carried out at IINS to track the movement of photo-switchable fluorescent kindlin-2 in this assay. The above strategies should bring new insights into the force-dependent inside-out regulation of integrin. At CINAM, the observation of the binding of integrin-containing giant unilamellar vesicles (GUVs) to ECM-coated surfaces has been assessed by RICM. In this experimental configuration, GUVs spread and open-up, offering access for regulators such as talin. It is therefore possible to study the “outside-in” effect of the integrin binding to ECM on the recruitment of the cytoplasmic regulators of integrin.

We will continue to describe the regulation of integrins by talin and kindlin and start the study of tensin which is supposed to regulate integrins. We will also test the effect of the elasticity of the extracellular matrix by mimicking these natural polymers with synthetic gels with well-controlled mechanical properties. Finally, to facilitate the experiments, we will develop a minimal version of the integrin.

Talin dissociates from RIAM and associates to vinculin sequentially in response to the actomyosin force.
Vigouroux C, Henriot V, Le Clainche C.
Nat Commun. 2020 Jun 19;11(1):3116. doi: 10.1038/s41467-020-16922-1.

Actin polymerization downstream of integrins:
signaling pathways and mechanotransduction
Romero S, Le Clainche C, Gautreau AM.
Biochem J. 2020 Jan 17;477(1):1-21. doi: 10.1042/BCJ20170719.

Cells adapt to the properties of their environment. Focal adhesions (FAs) are important mechanosensors that adapt their integrin-mediated adhesion to the internal force of the actomyosin cytoskeleton and to the physical properties of the extracellular matrix (ECM). The objective of our project is to decipher the elementary links between actomyosin force, integrin regulation and ECM properties, underlying FA mechanosensitivity. To this end, we will reconstitute a minimal adhesion complex made of an integrin-containing lipid bilayer that couples actomyosin cables, via regulatory proteins on one side, and a micropatterned ECM on the other side. Single particle tracking microscopy of FA components in our assay, where the concentration of the components, actomyosin force and the physical properties of the ECM are modulatable, will unambiguously identify mechanosensitive elementary reactions. The physiological relevance of our in vitro findings will be tested in cells systematically.