The family of integrin transmembrane receptors is essential for the normal function of multicellular organisms by facilitating cellextracellular matrix adhesion. The vitronectin-binding integrin αVβ5 localizes to focal adhesions (FAs) as well as poorly characterized flat clathrin lattices (FCLs). Here, we show that, in human keratinocytes, αVβ5 is predominantly found in FCLs, and formation of the αVβ5containing FCLs requires the presence of vitronectin as ligand, Ca 2+ , and the clathrin adaptor proteins ARH (also known as LDLRAP1), Numb and EPS15/EPS15L1. Integrin chimeras, containing the extracellular and transmembrane domains of β5 and the cytoplasmic domains of β1 or β3, almost exclusively localize in FAs. Interestingly, lowering actomyosin-mediated contractility promotes integrin redistribution to FLCs in an integrin tail-dependent manner, while increasing cellular tension favors αVβ5 clustering in FAs. Our findings strongly indicate that clustering of integrin αVβ5 in FCLs is dictated by the β5 subunit cytoplasmic domain, cellular tension and recruitment of specific adaptor proteins to the β5 subunit cytoplasmic domains.

Hemidesmosomes are specialized cell-matrix adhesion structures that are associated with the keratin cytoskeleton. Although the adhesion function of hemidesmosomes has been extensively studied, their role in mechanosignaling and transduction remains largely unexplored. Here, we show that keratinocytes lacking hemidesmosomal integrin α6β4 exhibit increased focal adhesion formation, cell spreading, and traction-force generation. Moreover, disruption of the interaction between α6β4 and intermediate filaments or laminin-332 results in similar phenotypical changes. We further demonstrate that integrin α6β4 regulates the activity of the mechanosensitive transcriptional regulator YAP through inhibition of Rho–ROCK–MLC– and FAK–PI3K–dependent signaling pathways. Additionally, increased tension caused by impaired hemidesmosome assembly leads to a redistribution of integrin αVβ5 from clathrin lattices to focal adhesions. Our results reveal a novel role for hemidesmosomes as regulators of cellular mechanical forces and establish the existence of a mechanical coupling between adhesion complexes.

Physical forces regulate numerous biological processes during development, physiology, and pathology. Forces between the external environment and intracellular actin cytoskeleton are primarily transmitted through integrin-containing focal adhesions and cadherin-containing adherens junctions. Crosstalk between these complexes is well established and modulates the mechanical landscape of the cell. However, integrins and cadherins constitute large families of adhesion receptors and form multiple complexes by interacting with different ligands, adaptor proteins, and cytoskeletal filaments. Recent findings indicate that integrin-containing hemidesmosomes oppose force transduction and traction force generation by focal adhesions. The cytolinker plectin mediates this crosstalk by coupling intermediate filaments to the actin cytoskeleton. Similarly, cadherins in desmosomes might modulate force generation by adherens junctions. Moreover, mechanotransduction can be influenced by podosomes, clathrin lattices, and tetraspanin-enriched microdomains. This review discusses mechanotransduction by multiple integrin-and cadherin-based cell adhesion complexes, which together with the associated cytoskeleton form an integrated network that allows cells to sense, process, and respond to their physical environment.

The vitronectin receptor integrin αVβ5 can reside in two distinct adhesion structures: focal adhesions (FAs) and flat clathrin lattices (FCLs). Here we investigated the mechanism that regulates the subcellular distribution of β5 in keratinocytes and show that β5 has approximately 7- and 5-fold higher affinity for the clathrin adaptors ARH and Numb, respectively, than for talin; all proteins that bind to the membrane-proximal NPxY motif of the β5 cytoplasmic domain. Using mass spectrometry, we identified β5 interactors including the Rho GEFs p115Rho-GEF and GEF-H1, and the serine protein kinase MARK2; depletion of which diminishes the clustering of β5 in FCLs. Substitution of two serines (S759/762) in the β5 cytoplasmic domain with phospho-mimetic glutamates causes a shift in the localization of β5 from FAs into FCLs without affecting the interactions with MARK2, p115Rho-GEF or GEF-H1. Instead, we demonstrate that changes in the actomyosin-based cellular contractility by ectopic expression of activated Rho or disruption of microtubules regulates β5 localization. Finally, we present evidence that β5 in either FAs or FCLs functions to promote adhesion to vitronectin, cell spreading, and proliferation.

Integrins mediate cell adhesion by connecting the extracellular matrix to the intracellular cytoskeleton and orchestrate signal transduction in response to chemical and mechanical stimuli by interacting with many cytoplasmic proteins. We used BioID to interrogate the interactomes of β1 and β3 integrins in epithelial cells and identified PEAK1 as an interactor of the RGD-binding integrins α5β1, αVβ3, and αVβ5 in focal adhesions. We demonstrate that the interaction between integrins and PEAK1 occurs indirectly through Tensin3, requiring both the membrane-proximal NPxY motif on the integrin β tail and binding of the SH2 domain of Tensin3 to phosphorylated Tyr-635 on PEAK1. Phosphorylation of Tyr-635 is mediated by Src and regulates cell migration. Additionally, we found that Shc1 localizes in focal adhesions in a PEAK1 phosphorylated Tyr-1188–dependent fashion. Besides binding Shc1, PEAK1 also associates with a protein cluster that mediates late EGFR/Shc1 signaling. We propose a model in which PEAK1 binds Tensin3 and Shc1 to converge integrin and growth factor receptor signal transduction.