Abstract
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.
Highlights
The transition from unicellular organisms to multicellular animals coincides with the emergence of dedicated cell adhesion molecules such as cadherins and integrins[1]
Interpretation of focal adhesion composition and dynamics in a cellular context is complicated by the fact that multiple integrins as well as diverse extracellular matrix ligands are present for any given cell type[21,22]
These complications can be circumvented by employing chimeric integrins, which have been used before to dissect the role of the integrin cytoplasmic domain in receptor localization in non-adherent cells[25]
Summary
The transition from unicellular organisms to multicellular animals (metazoans) coincides with the emergence of dedicated cell adhesion molecules such as cadherins and integrins[1]. FA analysis has been further hampered by the spatial and temporal heterogeneity of these structures within a single cell and the current lack of an in vitro reconstituted system with recombinant purified components These shortcomings prompted us to establish a minimal system with chimeric integrin β subunits, which can be clustered by multivalent ligands to stimulate initial protein recruitment. We observe differential recruitment of additional focal adhesion proteins including kindlin and paxillin but not vinculin, indicating direct versus indirect association of these cytoplasmic proteins with different integrin β subunits This system allows us to test the evolutionary conservation of talin binding to integrin cytoplasmic domains encoded in the genome of unicellular protists such as Capsaspora owczarzaki. Our findings demonstrate the usefulness of chimeric integrins to resolve the initial molecular events at integrin cytoplasmic tails in a cellular context, and provide first evidence that integrin activity regulation by talin evolved before the emergence of metazoans
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