Synergistic phase separation of two pathways promotes integrin clustering and nascent adhesion formation.
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ABSTRACT:
SUBMITTER: Case LB
PROVIDER: S-EPMC8791637 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Integrins and integrin-dependent cell-matrix adhesions are essential for a number of physiological processes. Integrin function is tightly regulated via binding of cytoplasmic proteins to integrin intracellular domains. Yet, the complexity of cell-matrix adhesions in mammals, with more than 150 core adhesome proteins, complicates the analysis of integrin-associated protein complexes. Interestingly, the evolutionary origin of integrins dates back before the transition from unicellular life to complex multicellular animals. Though unicellular relatives of metazoa have a less complex adhesome, nothing is known about the initial steps of integrin activation and adhesion complex assembly in protozoa. Therefore, we developed a minimal, microscope-based system using chimeric integrins to investigate receptor-proximal events during focal adhesion assembly. Clustering of the human integrin β1 tail led to recruitment of talin, kindlin, and paxillin and mutation of the known talin binding site abolished recruitment of this protein. Proteins indirectly linked to integrins, such as vinculin, migfilin, p130CAS, or zyxin were not enriched around the integrin β1 tail. With the exception of integrin β4 and integrin β8, the cytoplasmic domains of all human integrin β subunits supported talin binding. Likewise, the cytoplasmic domains of integrin β subunits expressed by the protozoan Capsaspora owczarzaki readily recruited talin and this interaction was based on an evolutionary conserved NPXY/F amino acid motif. The results we present here validate the use of our novel microscopic assay to uncover details of integrin-based protein-protein interactions in a cellular context and suggest that talin binding to integrin β cytoplasmic tails is an ancient feature of integrin regulation.
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Project description:Integrin adhesion complexes are essential membrane-associated cellular compartments for metazoan life. The formation of initial integrin adhesion complexes is a dynamic process involving focal adhesion proteins assembled at the integrin cytoplasmic tails and the inner leaflet of the plasma membrane. The weak multivalent protein interactions within the complex and with the plasma membrane suggest that liquid-liquid phase separation could play a role in the nascent adhesion assembly. Here, we report that solid-supported lipid membranes supplemented with phosphoinositides induce the phase separation of minimal integrin adhesion condensates composed of integrin β1 tails, kindlin, talin, paxillin, and FAK at physiological ionic strengths and protein concentrations. We show that the presence of phosphoinositides is key to enriching kindlin and talin on the lipid membrane, which is necessary to further induce the phase separation of paxillin and FAK at the membrane. Our data demonstrate that lipid membrane surfaces set the local solvent conditions for steering the membrane-localized phase separation even in a regime where no condensate formation of proteins occurs in bulk solution.
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