Increased ligand binding to cellular integrins (“activation”) plays important roles in processes such as development, cell migration, extracellular matrix assembly, tumor metastasis and hemostasis and thrombosis[1-5]. Integrin activation encompasses both increased integrin monomer affinity and increased receptor clustering[6] and depends on integrin-talin interactions[5]. Loss of kindlins results in reduced activation of integrins[7-13]. Kindlins might promote talin binding to integrins through a cooperative mechanism[5, 14-16]; however, kindlins do not increase talin association with integrins[17]. Here we report that, unlike talin head domain (THD), kindlin-3 caused little effect on the affinity of purified monomeric αIIbβ3, and it didn’t enhance activation by THD. Furthermore, studies with ligands of varying valency showed that kindlins primarily increased cellular αIIbβ3 avidity rather than monomer affinity. In platelets or nucleated cells, loss of kindlins markedly reduced αIIbβ3 binding to multivalent but not monovalent ligands. Finally, silencing of kindlins reduced the clustering of ligand-occupied αIIbβ3 as revealed by total internal reflection fluorescence (TIRF) and electron microscopy. Thus, in contrast to talins, kindlins have little primary effect on integrin αIIbβ3 affinity for monovalent ligands and increase multivalent ligand binding by promoting the clustering of talin-activated integrins.

The leading edge of migrating cells contains rapidly translocating activated integrins associated with growing actin filaments that form "sticky fingers" that sense extracellular matrix and guide cell migration. Here we utilized indirect bimolecular fluorescence complementation (BiFC) to visualize a molecular complex containing an MRL protein (RIAM or lamellipodin), talin, and activated integrins in living cells. This complex localizes at the tips of growing actin filaments in lamellipodial and filopodial protrusions, thus corresponding to the tips of the "sticky fingers." Formation of the complex requires talin to form a bridge between the MRL protein and the integrins. Moreover, disruption of the MRL protein-integrin-talin (MIT) complex markedly impairs cell protrusion. These data reveal the molecular basis of the formation of "sticky fingers" at the leading edge of migrating cells and show that an MIT complex drives these protrusions.

No abstract

PAK4 is a metazoan-specific kinase acting downstream of Cdc42. Here we describe the structure of human PAK4 in complex with Inka1, a potent endogenous kinase inhibitor. Using single mammalian cells containing crystals 50 μm in length, we have determined the in cellulo crystal structure at 2.95 Å resolution, which reveals the details of how the PAK4 catalytic domain binds cellular ATP and the Inka1 inhibitor. The crystal lattice consists only of PAK4–PAK4 contacts, which form a hexagonal array with channels of 80 Å in diameter that run the length of the crystal. The crystal accommodates a variety of other proteins when fused to the kinase inhibitor. Inka1–GFP was used to monitor the process crystal formation in living cells. Similar derivatives of Inka1 will allow us to study the effects of PAK4 inhibition in cells and model organisms, to allow better validation of therapeutic agents targeting PAK4.

Talin-mediated integrin activation drives integrin-based adhesions. A simple binary switch—vinculin competitively displacing RIAM from talin—is found to play a central role in the maturation and evolving functions of integrin-based adhesions.

No abstract

Background: ArgBP2 is a cytoskeletal adaptor protein down-regulated in tumors. Results: ArgBP2 binds to ␣-actinin through a conserved protein domain. ArgBP2 expression is associated with cross-linked F-actin stress fibers. Conclusion: ArgBP2 inhibits cell migration via its interaction with ␣-actinin, which links it to the actomyosin network; this is negatively regulated by PKA. Significance: We propose a reason for reduced ArgBP2 expression in cancer metastasis.

Olfactory dysfunction is an early and prevalent symptom of Alzheimer’s disease (AD) and the olfactory bulb is a nexus of beta-amyloid plaque and tau neurofibrillary tangle (NFT) pathology during early AD progression. To mitigate the accumulation of misfolded proteins, an endoplasmic reticulum stress response called the unfolded protein response (UPR) occurs in the AD hippocampus. However, chronic UPR activation can lead to apoptosis and the upregulation of beta-amyloid and tau production. Therefore, UPR activation in the olfactory system could be one of the first changes in AD. In this study, we investigated whether two proteins that signal UPR activation are expressed in the olfactory system of AD cases with low or high amounts of aggregate pathology. We used immunohistochemistry to label two markers of UPR activation (p-PERK and p-eIF2α) concomitantly with neuronal markers (NeuN and PGP9.5) and pathology markers (beta-amyloid and tau) in the olfactory bulb, piriform cortex, entorhinal cortex and the CA1 region of the hippocampus in AD and normal cases. We show that UPR activation, as indicated by p-PERK and p-eIF2α expression, is significantly increased throughout the olfactory system in AD cases with low (Braak stage III-IV) and high-level (Braak stage V-VI) pathology. We further show that UPR activation occurs in the mitral cells and in the anterior olfactory nucleus of the olfactory bulb where tau and amyloid pathology is abundant. However, UPR activation is not present in neurons when they contain NFTs and only rarely occurs in neurons containing diffuse tau aggregates. We conclude that UPR activation is prevalent in all regions of the olfactory system and support previous findings suggesting that UPR activation likely precedes NFT formation. Our data indicate that chronic UPR activation in the olfactory system might contribute to the olfactory dysfunction that occurs early in the pathogenesis of AD.