Focal adhesions (FAs) initiate chemical and mechanical signals involved in cell polarity, migration, proliferation and differentiation. Super-resolution microscopy revealed that FAs are organized at the nanoscale into functional layers from the lower plasma membrane to the upper actin cytoskeleton. Yet, how FAs proteins are guided into specific nano-layers to promote interaction with given targets is unknown. Using single protein tracking, super-resolution microscopy and functional assays, we link the molecular behavior and 3D nanoscale localization of kindlin with its function in integrin activation inside FAs. We show that immobilization of integrins in FAs depends on interaction with kindlin. Unlike talin, kindlin displays free diffusion along the plasma membrane outside and inside FAs. We demonstrate that the kindlin Pleckstrin Homology domain promotes membrane diffusion and localization to the membrane-proximal integrin nano-layer, necessary for kindlin enrichment and function in FAs. Using kindlin-deficient cells, we show that kindlin membrane localization and diffusion are crucial for integrin activation, cell spreading and FAs formation. Thus, kindlin uses a different route than talin to reach and activate integrins, providing a possible molecular basis for their complementarity during integrin activation.

The spatial organization of vascular endothelial growth factor (VEGF) signaling is a key determinant of vascular patterning during development and tissue repair. How VEGF signaling becomes spatially restricted and the role of VEGF secreting astrocytes in this process remains poorly understood. Using a VEGF-GFP fusion protein and confocal time-lapse microscopy, we observed the intracellular routing, secretion and immobilization of VEGF in scratch-activated living astrocytes. We found VEGF to be directly transported to cell-extracellular matrix attachments where it is incorporated into fibronectin fibrils. VEGF accumulated at β1 integrin containing fibrillar adhesions and was translocated along the cell surface prior to internalization and degradation. We also found that only the astrocyte-derived, matrix-bound, and not soluble VEGF decreases β1 integrin turnover in fibrillar adhesions. We suggest that polarized VEGF release and ECM remodeling by VEGF secreting cells is key to control the local concentration and signaling of VEGF. Our findings highlight the importance of astrocytes in directing VEGF functions and identify these mechanisms as promising target for angiogenic approaches.

Heterodimeric integrin receptors control cell adhesion, migration and extracellular matrix assembly. While the α integrin subunit determines extracellular ligand specificity, the β integrin chain binds to an acidic residue of the ligand, and cytoplasmic adapter protein families such as talins, kindlins and paxillin, to form mechanosensing cell matrix adhesions. Alternative splicing of the β1 integrin cytoplasmic tail creates ubiquitously expressed β1A, and the heart and skeletal musclespecific β1D form. To study the physiological difference between these forms, we developed fluorescent β1 integrins and analyzed their dynamics, localization, and cytoplasmic adapter recruitment and effects on cell proliferation. On fibronectin, GFP-tagged β1A integrin showed dynamic exchange in peripheral focal adhesions, and long, central fibrillar adhesions. In contrast, GFP-β1D integrins exchanged slowly, forming immobile and short central adhesions. While adhesion recruitment of GFP-β1A integrin was sensitive to C-terminal tail mutagenesis, GFP-β1D integrin was recruited independently of the distal NPXY motif. In addition, a P786A mutation in the proximal, talin-binding NPXY 783 motif switched β1D to a highly dynamic integrin. In contrast, the inverse A786P mutation in β1A integrin interfered with paxillin recruitment and proliferation. Thus, differential β1 integrin splicing controls integrin-dependent adhesion signaling, to adapt to the specific physiological needs of differentiated muscle cells.

Diabetic nephropathy, a devastating consequence of diabetes mellitus, is characterized by the accumulation of extracellular matrix (ECM) that disrupts the kidney’s filtration apparatus. Elevated glucose levels increase the deposition of a fibronectin (FN) matrix by mesangial cells, the primary matrix-producing cells of the kidney, and also increase acetyl-CoA leading to higher levels of lysine acetylation. Here, we investigated the connection between acetylation and the ECM and show that treatment of mesangial cells with deacetylase inhibitors increases both acetylation and FN matrix assembly compared to untreated cells. The matrix effects were linked to lysine 794 (K794) in the β1 integrin cytoplasmic domain based on studies of cells expressing acetylated (K794Q) and non-acetylated (K794R) mimetics. β1(K794Q) cells assembled significantly more FN matrix than wildtype β1 cells, while the non-acetylated β1(K794R) form was inactive. We show that mutation of K794 affects FN assembly by stimulating integrin-FN binding activity and cell contractility. Wildtype and β1(K794Q) cells but not β1(K794R) cells further increased their FN matrix when stimulated with deacetylase inhibitors indicating that increased acetylation on other proteins is required for maximum FN assembly. Thus, lysine acetylation provides a mechanism for glucose-induced fibrosis by up-regulation of FN matrix assembly.

Focal adhesions (FAs) initiate chemical and mechanical signals involved in cell polarity, migration, proliferation and differentiation. Super-resolution microscopy revealed that FAs are organized at the nanoscale into functional layers from the lower plasma membrane to the upper actin cytoskeleton. Yet, how FAs proteins are guided into specific nano-layers to promote interaction with given targets is unknown. Using single protein tracking, super-resolution microscopy and functional assays, we linked the molecular behavior and tridimensional nanoscale localization of kindlin with its function in integrin activation inside FAs. We show that immobilization of integrins in FAs depends on interaction with kindlin. Unlike talin, kindlin displayed free diffusion along the plasma membrane outside and inside FAs. We demonstrate that the kindlin Pleckstrin Homology domain promotes membrane diffusion and localization to the membrane-proximal integrin nano-layer, necessary for kindlin enrichment and function in FAs. Using kindlin-deficient cells, we show that kindlin membrane localization and diffusion are crucial for integrin activation during cell adhesion and spreading. Thus, kindlin uses a different route than talin to reach and activate integrins, providing a possible molecular basis for their complementarity during integrin activation.