β1A Integrin Is a Master Regulator of Invadosome Organization and Function

Use of patterned surfaces, reverse genetics, and time-controlled photoinactivation showed that β1 but not β3 integrins are required for invadosome formation, self-assembly, and stabilization into a ring structure. The activation state of β1 as well as its phosphorylation by protein kinase C on Ser785 control these process and link to the degradative function

Paxillin and Hic-5 Interaction with Vinculin Is Differentially Regulated by Rac1 and RhoA

Cell migration is of paramount importance to organism development and maintenance as well as multiple pathological processes, including cancer metastasis. The RhoGTPases Rac1 and RhoA are indispensable for cell migration as they regulate cell protrusion, cell-extracellular matrix (ECM) interactions and force transduction. However, the consequences of their activity at a molecular level within the cell remain undetermined. Using a combination of FRET, FRAP and biochemical analyses we show that the interactions between the focal adhesion proteins vinculin and paxillin, as well as the closely related family member Hic-5 are spatially and reciprocally regulated by the activity of Rac1 and RhoA. Vinculin in its active conformation interacts with either paxillin or Hic-5 in adhesions in response to Rac1 and RhoA activation respectively, while inactive vinculin interacts with paxillin in the membrane following Rac1 inhibition. Additionally, Rac1 specifically regulates the dynamics of paxillin as well as its binding partner and F-actin interacting protein actopaxin (α-parvin) in adhesions. Furthermore, FRET analysis of protein:protein interactions within cell adhesions formed in 3D matrices revealed that, in contrast to 2D systems vinculin interacts preferentially with Hic-5. This study provides new insight into the complexity of cell-ECM adhesions in both 2D and 3D matrices by providing the first description of RhoGTPase-coordinated protein:protein interactions in a cellular microenvironment. These data identify discrete roles for paxillin and Hic-5 in Rac1 and RhoA-dependent cell adhesion formation and maturation; processes essential for productive cell migration

FAK phosphorylation at Tyr-925 regulates cross-talk between focal adhesion turnover and cell protrusion

FAK plays a key role in the regulation of cell migration. The authors show that the phosphorylation status of FAK at Tyr-925 is involved in FA turnover, formation of FAs, and increase in cell edge protrusion, together with activation of the p130CAS/Rac1 signaling pathway

SHARPIN is an endogenous inhibitor of beta 1-integrin activation

Regulated activation of integrins is critical for cell adhesion, motility and tissue homeostasis. Talin and Kindlins activate β1-integrins, but the counteracting inhibiting mechanisms are poorly defined. Here we identified SHARPIN as an important inactivator of β1-integrins in an RNAi-screen. SHARPIN inhibited β1-integrin functions in human cancer cells and primary leukocytes. Fibroblasts, leukocytes and keratinocytes from SHARPIN-deficient mice exhibited increased β1-integrin activity which was fully rescued by re-expression of SHARPIN. SHARPIN directly bound to a conserved cytoplasmic region of integrin α-subunits and inhibited recruitment of Talin and Kindlin to the integrin. Therefore, SHARPIN inhibits the critical switching of β1-integrins from inactive to active conformations

Integrins β1 and β3 exhibit distinct dynamic nanoscale organizations inside focal adhesions

Integrins in focal adhesions (FAs) mediate adhesion and force transmission to extracellular matrices crucial for cell motility, proliferation and differentiation1,2. Different αβ-integrins binding to fibronectin (FN) perform distinct functions3-5 and are simultaneously present in FAs. Although the static nanoscale organization of FAs was described6, explaining how individual dynamics of specific integrins control biochemical and biomechanical events in FAs is still elusive. Combining single protein tracking and super-resolution imaging we show that β3- and β1-integrins act as distinct adhesion units displaying specific dynamics and nano-organizations within FAs. Integrins reside in FAs through free-diffusion and immobilization cycles, thus integrins are not constantly active inside FAs. Integrin activation promotes immobilization, stabilized in FAs by simultaneous FN and actin binding proteins (ABPs) connections. The integrin activator talin7, is recruited in FAs from the cytosol without membrane free-diffusion, spatially restricting integrin immobilization to FAs. Talin immobilization zones are concentrated and mainly stationary in FA, consistent with the formation of stable β-integrin/talin connections. β3-integrin immobilizations are concentrated and stationary within FAs, whereas β1-integrins are sparse displaying eventually rearward movements. Thus, differential transmission of F-actin motion to FN occurs through specific integrins within FAs. This dynamic nano-partitioning of β-integrins within FAs could control local forces and signaling necessary for distinct cellular functions such as migration and extracellular matrices remodeling