Deciphering the molecular events controlling integrin activation

Integrins in adhesion sites (AS) mediate adhesion and force transmission to extracellular matrices essential for cell motility, proliferation and differentiation. Different fibronectin-binding integrins, simultaneously present in AS, perform distinct functions. Yet, how integrin dynamics control biochemical and biomechanical processes in AS was still elusive. In a precept study we unravel the key spatiotemporal molecular events leading to integrins activation by their main activator talin in mature AS (Rossier et al., Nat. Cell Biol., 2012). This study was performed in close synergistic collaboration with the group of Brahim Lounis (LP2N, Institut d’Optique, Bordeaux, France) and Jean-Baptiste Sibarita (IINS) and allowed the development of new strategies for super-resolution imaging and single protein tracking adapted to the study of adhesive structures. We performed single protein tracking (SPT) combined with PALM (sptPALM) and super-resolution microscopy to study integrins and talin displacements and distributions outside versus inside mature AS. We demonstrated that AS are specialized platforms priming integrins immobilization. Integrins are recruited to AS through a membrane diffusion/trapping mechanism, while talin is recruited to AS directly from the cytosol. Integrins reside in AS through free-diffusion and immobilization cycles, during which activation promotes immobilization. This is one of the pioneering studies using SPT/super-resolution to tackle in-depth the molecular activation of integrin in living cells. Therefore, in this study we started to build the methodology and the pedestal towards a molecular understanding of integrin activation during more integrated biological processes and pathologies.

Integrins β1 and β3 exhibit distinct dynamic nanoscale organizations inside focal adhesions.
Rossier O, Octeau V, Sibarita J-B, Leduc C, Tessier B, Nair D, Gatterdam V, Destaing O, Albigès-Rizo C, Tampé R, Cognet L, Choquet D, Lounis B, Giannone G.
Nature Cell Biology. 2012. 14,1057-67.

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The cancer cell glycocalyx mechanically primes integrin-dependent growth and survival

In collaboration with the group of Valerie Weaver (Cancer cell biologists, UCSF, USA), we studied how bulky membrane glycoproteins mechanically regulate integrin activation (Paszek et al., Nature, 2014). Mechanical perturbations of cell and tissue structures are playing a causal role in tumor development and progression. Malignancy is also associated with altered expression of glycans and glycoproteins that contribute to the glycocalyx. The group of V. Weaver constructed a glycoprotein expression-signature, which revealed that tumors express a disproportionate number of bulky glycoproteins. The aim of this study was to test whether a bulky glycocalyx could promote a tumor phenotype by increasing integrin-mediated adhesion. Experiments demonstrated that a thick glycocalyx, induced by over-expression of Mucin1, increases the membrane distance from the substrate and promote the formation of larger adhesion sites. This thick glycocalyx applies tension to integrin bonds, independent of actomyosin contractility, leading to integrin-dependent cell growth and survival even under minimally-adhesive conditions such as soft substrates. By studying the diffusive behavior of Mucin1 we demonstrated that integrin-dependent adhesion constitute a diffusive barrier for Mucin1, demonstrating a reciprocal mechanical regulation between integrins and the glycocalyx. Using tracking experiments we also demonstrated that bulky glycoproteins spatially regulate immobilization of activated integrins, experimentally proving that the glycocalyx controls integrin activation. Indeed, by studying the diffusive behavior of integrins we showed that Muc1 impedes the immobilization of active integrins outside of adhesive contacts, thus favoring integrin immobilization at sites of preexisting adhesion. Thus, control of membrane nano-topology by the glycocalyx could mechanically enhance integrin activation and foster metastatic progression. This study now implicates the glycocalyx as another physical structure, in addition to the extracellular matrix and cytoskeleton, whose mechanical perturbation contributes to the tumor phenotype.

The cancer cell glycocalyx mechanically primes integrin-dependent growth and survival.
Paszek MJ, Dufort CC, Rossier O, Bainer R, Mouw JK, Godula K, Hudak JE, Lakins JN, Wijekoon AC, Cassereau L, Rubashkin MG, Magbanua MJ, Thorn KS, Davidson MW, Rugo HS, Park JW, Hammer DA, Giannone G, Bertozzi CR, Weaver VM.
Nature. 2014. 511, 319–325.