Cell spreading requires the coupling of actin-driven membrane protrusion and integrin-mediated adhesion to the extracellular matrix. The integrin-activating adaptor protein kindlin-2 plays a central role for cell adhesion and membrane protrusion by directly binding and recruiting paxillin to nascent adhesions. Here, we report that kindlin-2 has a dual role during initial cell spreading: it binds paxillin via the pleckstrin homology and F0 domains to activate Rac1, and it directly associates with the Arp2/3 complex to induce Rac1-mediated membrane protrusions. Consistently, abrogation of kindlin-2 binding to Arp2/3 impairs lamellipodia formation and cell spreading. Our findings identify kindlin-2 as a key protein that couples cell adhesion by activating integrins and the induction of membrane protrusions by activating Rac1 and supplying Rac1 with the Arp2/3 complex.

Integrins require an activation step prior to ligand binding and signaling. How talin and kindlin contribute to these events in non-hematopoietic cells is poorly understood. Here we report that fibroblasts lacking either talin or kindlin failed to activate β1 integrins, adhere to fibronectin (FN) or maintain their integrins in a high affinity conformation induced by Mn(2+). Despite compromised integrin activation and adhesion, Mn(2+) enabled talin- but not kindlin-deficient cells to initiate spreading on FN. This isotropic spreading was induced by the ability of kindlin to directly bind paxillin, which in turn bound focal adhesion kinase (FAK) resulting in FAK activation and the formation of lamellipodia. Our findings show that talin and kindlin cooperatively activate integrins leading to FN binding and adhesion, and that kindlin subsequently assembles an essential signaling node at newly formed adhesion sites in a talin-independent manner.

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.

Integrins require an activation step before ligand binding and signaling that is mediated by talin and kindlin binding to the β integrin cytosolic domain (β-tail). Conflicting reports exist about the contribution of phosphorylation of a conserved threonine motif in the β1-tail (β1-pT788/pT789) to integrin activation. We show that widely used and commercially available antibodies against β1-pT788/pT789 integrin do not detect specific β1-pT788/pT789 integrin signals in immunoblots of several human and mouse cell lysates but bind bi-phosphorylated threonine residues in numerous proteins, which were identified by mass spectrometry experiments. Furthermore, we found that fibroblasts and epithelial cells expressing the phospho-mimicking β1-TT788/789DD integrin failed to activate β1 integrins and displayed reduced integrin ligand binding, adhesion initiation and cell spreading. These cellular defects are specifically caused by the inability of kindlin to bind β1-tail polypeptides carrying a phosphorylated threonine motif or phospho-mimicking TT788/789DD substitutions. Our findings indicate that the double-threonine motif in β1-class integrins is not a major phosphorylation site but if phosphorylated would curb integrin function.

Cell migration is mediated by the dynamic remodeling of focal adhesions (FAs). Recently, an important role of endosomal signaling in regulation of cell migration was recognized. Here, we show an essential function for late endosomes carrying the p14-MP1 (LAMTOR2/3) complex in FA dynamics. p14-MP1-positive endosomes move to the cell periphery along microtubules (MTs) in a kinesin1- and Arl8b-dependent manner. There they specifically target FAs to regulate FA turnover, which is required for cell migration. Using genetically modified fibroblasts from p14-deficient mice and Arl8b-depleted cells, we demonstrate that MT plus end-directed traffic of p14-MP1-positive endosomes triggered IQGAP1 disassociation from FAs. The release of IQGAP was required for FA dynamics. Taken together, our results suggest that late endosomes contribute to the regulation of cell migration by transporting the p14-MP1 scaffold complex to the vicinity of FAs.

The mammalian target of rapamycin complex 1 (mTORC1) integrates mitogenic and stress signals to control growth and metabolism. Activation of mTORC1 by amino acids and growth factors involves recruitment of the complex to the lysosomal membrane and is further supported by lysosome distribution to the cell periphery. Here, we show that translocation of lysosomes toward the cell periphery brings mTORC1 into proximity with focal adhesions (FAs). We demonstrate that FAs constitute discrete plasma membrane hubs mediating growth factor signaling and amino acid input into the cell. FAs, as well as the translocation of lysosome-bound mTORC1 to their vicinity, contribute to both peripheral and intracellular mTORC1 activity. Conversely, lysosomal distribution to the cell periphery is dispensable for the activation of mTORC1 constitutively targeted to FAs. This study advances our understanding of spatial mTORC1 regulation by demonstrating that the localization of mTORC1 to FAs is both necessary and sufficient for its activation by growth-promoting stimuli.

Kindlin-1 is an integrin tail binding protein that controls integrin activation. Mutations in the FERMT-1 gene, which encodes for Kindlin-1, lead to Kindler syndrome in man, which is characterized by skin blistering, premature skin aging and skin cancer of unknown etiology. Here we show that loss of Kindlin-1 in mouse keratinocytes recapitulates Kindler syndrome and also produces enlarged and hyperactive stem cell compartments, which lead to hyperthickened epidermis, ectopic hair follicle development and increased skin tumor susceptibility. Mechanistically, Kindlin-1 controls keratinocyte adhesion through β1-class integrins and proliferation and differentiation of cutaneous epithelial stem cells by promoting α(v)β(6) integrin-mediated transforming growth factor-β (TGF-β) activation and inhibiting Wnt-β-catenin signaling through integrin-independent regulation of Wnt ligand expression. Our findings assign Kindlin-1 the previously unknown and essential task of controlling cutaneous epithelial stem cell homeostasis by balancing TGF-β-mediated growth-inhibitory signals and Wnt-β-catenin-mediated growth-promoting signals.

The disassembly of integrin-containing focal adhesions (FAs) at mitotic entry is essential for cell rounding, mitotic retraction fibre formation, bipolar spindle positioning and chromosome segregation. The mechanism that drives FA disassembly at mitotic entry is unknown. Here, we show that the CDK1-cyclin B1 complex phosphorylates the integrin activator kindlin, which results in the recruitment of the cullin 9-FBXL10 ubiquitin ligase complex that mediates kindlin ubiquitination and degradation. This molecular pathway is essential for FA disassembly and cell rounding, as phospho-inhibitory mutations of the CDK1 motif prevent kindlin degradation, FA disassembly and mitotic cell rounding. Conversely, phospho-mimetic mutations promote kindlin degradation in interphase, accelerate mitotic cell rounding and impair mitotic retraction fibre formation. Despite the opposing effects on kindlin stability, both types of mutations cause severe mitotic spindle defects, apoptosis and aneuploidy. Thus, the exquisite regulation of kindlin levels at mitotic entry is essential for cells to progress accurately through mitosis.

To enter mitosis, most adherent animal cells reduce adhesion, which is followed by cell rounding. How mitotic cells regulate adhesion to neighboring cells and extracellular matrix (ECM) proteins is poorly understood. Here we report that, similar to interphase, mitotic cells can employ integrins to initiate adhesion to the ECM in a kindlin- and talin-dependent manner. However, unlike interphase cells, we find that mitotic cells cannot engage newly bound integrins to actomyosin via talin or vinculin to reinforce adhesion. We show that the missing actin connection of newly bound integrins leads to transient ECM-binding and prevents cell spreading during mitosis. Furthermore, β1 integrins strengthen the adhesion of mitotic cells to adjacent cells, which is supported by vinculin, kindlin, and talin1. We conclude that this dual role of integrins in mitosis weakens the cell-ECM adhesion and strengthens the cell-cell adhesion to prevent delamination of the rounding and dividing cell.