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Focal adhesions (FAs) link the extracellular matrix to the actin cytoskeleton to mediate cell adhesion, migration, mechanosensing and signalling. FAs have conserved nanoscale protein organization, suggesting that the position of proteins within FAs regulates their activity and function. Vinculin binds different FA proteins to mediate distinct cellular functions, but how vinculin’s interactions are spatiotemporally organized within FAs is unknown. Using interferometric photoactivation localization super-resolution microscopy to assay vinculin nanoscale localization and a FRET biosensor to assay vinculin conformation, we found that upward repositioning within the FA during FA maturation facilitates vinculin activation and mechanical reinforcement of FAs. Inactive vinculin localizes to the lower integrin signalling layer in FAs by binding to phospho-paxillin. Talin binding activates vinculin and targets active vinculin higher in FAs where vinculin can engage retrograde actin flow. Thus, specific protein interactions are spatially segregated within FAs at the nanoscale to regulate vinculin activation and function. Waterman and colleagues use super-resolution microscopy and biosensor technology to characterize the spatiotemporal regulation of the protein interactions within focal adhesions that control vinculin activation and function during focal adhesion maturation.

The focal adhesion kinase (FAK) scaffold provides FAK-targeted cancer therapeutics with greater efficacy and specificity than traditional kinase inhibitors. The FAK scaffold function largely involves the interaction between FAK’s focal adhesion targeting (FAT) domain and paxillin, ultimately regulating many hallmarks of cancer. We report the design of paxillin LD-motif mimetics that successfully inhibit the FAT-paxillin interaction. Chemical and biochemical screening identifies stapled peptide 1907, a high affinity binder of the FAT four-helix bundle with ~100-fold greater binding affinity than the native LD2-sequence. The X-ray co-crystal structure of the FAT-1907 complex is solved. Myristoylated 1907-analog, peptide 2012, delocalizes FAK from focal adhesions, induces cancer cell apoptosis, reduces in vitro viability and invasion, and decreases tumor burden in B16F10 melanoma female mice. Enzymatic FAK inhibition produces no comparable effects. Herein, we describe a biologically potent therapeutic strategy to target the FAK-paxillin complex, a previously deemed undruggable protein-protein interaction. Here, the authors develop hydrocarbon-stapled paxillin-mimetic peptides that act as selective and potent focal adhesion kinase (FAK) scaffold inhibitors to displace FAK from focal adhesions. Treatment shows reduced cancer cell survival in vitro and reduced tumor burden in vivo.

Paxillin (PXN), a key component of the focal adhesion complex, has been associated with cancer progression, but the underlying mechanisms are poorly understood. The purpose of this study was to elucidate mechanisms by which PXN affects cancer growth and progression, which we addressed using cancer patient data, cell lines, and orthotopic mouse models. We demonstrated a previously unrecognized mechanism whereby nuclear PXN enhances angiogenesis by transcriptionally regulating SRC expression. SRC, in turn, increases PLAT expression through NF-ĸB activation; PLAT promotes angiogenesis via LRP1 in endothelial cells. PXN silencing in ovarian cancer mouse models reduced angiogenesis, tumor growth, and metastasis. These findings provide a new understanding of the role of PXN in regulating tumor angiogenesis and growth.

Solid cancers like pancreatic ductal adenocarcinoma (PDAC), a type of pancreatic cancer, frequently exploit nerves for rapid dissemination. This neural invasion (NI) is an independent prognostic factor in PDAC, but insufficiently modeled in genetically engineered mouse models (GEMM) of PDAC. Here, we systematically screened for human-like NI in Europe's largest repository of GEMM of PDAC, comprising 295 different genotypes. This phenotype screen uncovered 2 GEMMs of PDAC with human-like NI, which are both characterized by pancreas-specific overexpression of transforming growth factor α (TGF-α) and conditional depletion of p53. Mechanistically, cancer-cell-derived TGF-α upregulated CCL2 secretion from sensory neurons, which induced hyperphosphorylation of the cytoskeletal protein paxillin via CCR4 on cancer cells. This activated the cancer migration machinery and filopodia formation toward neurons. Disrupting CCR4 or paxillin activity limited NI and dampened tumor size and tumor innervation. In human PDAC, phospho-paxillin and TGF-α-expression constituted strong prognostic factors. Therefore, we believe that the TGF-α-CCL2-CCR4-p-paxillin axis is a clinically actionable target for constraining NI and tumor progression in PDAC.

Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min −1 . The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics. Live cell super-resolution imaging requires a high temporal resolution, which remains a challenge. Here the authors combine photo-activated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI) to achieve high spatiotemporal resolution and quantitative imaging of focal adhesion dynamics.

Focal adhesions (FAs) are multiprotein structures that link the intracellular cytoskeleton to the extracellular matrix. They mediate cell adhesion and migration, crucial to many (patho-) physiological processes. We examined in two cell types from different species the binding dynamics of functionally related FA protein pairs: paxillin and vinculin versus zyxin and VASP. In photobleaching experiments ~40% of paxillin and vinculin remained stably associated with a FA for over half an hour. Zyxin and VASP predominantly displayed more transient interactions. We show protein binding dynamics are influenced by FA location and orientation. In FAs located close to the edge of the adherent membrane paxillin, zyxin and VASP were more dynamic and had larger bound fractions. Zyxin and VASP were also more dynamic and had larger bound fractions at FAs perpendicular compared to parallel to this edge. Finally, we developed a photoconversion assay to specifically visualise stably bound proteins within subcellular structures and organelles. This revealed that while paxillin and vinculin are distributed evenly throughout FAs, their stably bound fractions form small clusters within the FA-complex. These clusters are more concentrated for paxillin than for vinculin and are mostly found at the proximal half of the FA where actin also enters.

Cell migration requires the fine spatiotemporal integration of many proteins that regulate the fundamental processes that drive cell movement. Focal adhesion (FA) dynamics is a continuous process involving coordination between FA and actin cytoskeleton, which is essential for cell migration. We studied the spatiotemporal relationship between the dynamics of focal adhesion kinase (FAK) and paxillin at FAs in the protrusion of living endothelial cells. Concurrent dual-color imaging showed that FAK was assembled at FA first, which was followed by paxillin recruitment to the FA. By tracking and quantifying FAK and paxillin in migrating cells, the normalized FAK/Paxillin fluorescence intensity (FI) ratio is > 1 (≈4 fold) at cell front, ≈1 at cell center and < 1 at cell rear. The significantly higher FAK FI than paxillin FI at cell front indicates that the assembly of FAK-FAs occurs ahead of paxillin at cell front. To determine the time difference between the assemblies of FAK and paxillin at nascent FAs, FAs containing both FAK and paxillin were quantified by image analysis and time correlation. The results show that FAK assembles at the nascent FAs earlier than paxillin in the protrusions at cell front.

Colorectal cancer (CRC) is the third most common cancer worldwide, and metastasis is strongly associated with poor prognosis in patients with CRC. We have previously found that the expression and phosphorylation of paxillin (PXN) play an important role in the metastatic potential of breast cancer. This study examined the potential role of PXN in CRC metastasis. Resected tumor specimens from 92 patients with CRC were subjected to immunohistochemical analysis of PXN levels. Three human CRC cell lines, HCT116, LoVo, and SW480 were used for scratch and transwell invasion assays to examine the effects of PXN over-expression. RNA sequencing was performed to obtain the expression profiles under PXN over-expression. High levels of PXN were significantly correlated with advanced stage, higher carcinoembryonic antigen and carbohydrate antigen 19-9 levels, and poorer overall survival. The migration ability of CRC cells was enhanced by exogenous PXN over-expression, but this enhancement was not observed in cells harboring exogenously mutated PXN at Tyr31 or Tyr88 phosphorylation sites. In PXN-over-expressing cells, TNF-α signaling via NF-[Formula: see text]B was positively enriched. PXN expression and phosphorylation at Tyr31 or Tyr88 may influence the migration and invasion of CRC cells. PXN expression and phosphorylation at Tyr31 or Tyr88 are promising targets for evaluating prognosis and treating CRC.

Migration of human glioma cells (hGCs) within the brain parenchyma makes glioblastoma one of the most aggressive and lethal tumors. Studies of the cellular and molecular mechanisms underlying hGC migration are hindered by the limitations of existing glioma models. Here we developed a dorsal root ganglion axon-oligodendrocyte-hGC co-culture to study in real time the migration and interaction of hGCs with their microenvironment. hGCs interact with myelinated and non-myelinated axons through the formation of pseudopodia. Isolation of pseudopodia-localized polysome-bound RNA reveals transcripts of Lck, Paxillin, Crk-II , and Rac1 that undergo local translation. Inhibition of Lck phosphorylation using a small-molecule inhibitor (Lck-I), blocks the phosphorylation of Paxillin and Crk-II, the formation of pseudopodia and the migration of hGCs. In vivo intraventricular administration of the Lck-I using an orthotopic xenograft glioma model, results in statistically significant inhibition of tumor size and significant down-regulation of Nanog-targeted genes, which are associated with glioblastoma patient survival. Moreover, treatment of human glioma stem cells (hGSCs) with Lck-I, results in significant inhibition of self-renewal and tumor-sphere formation. The involvement of Lck in different levels of glioma malignant progression, such as migration, tumor growth, and regulation of cancer stemness, makes Lck a potentially important therapeutic target for human glioblastomas.

Paxillin and kindlin are essential regulatory proteins involved in cell adhesion, migration, and signal transduction. Paxillin influences cytoskeletal dynamics by interacting with multiple signaling proteins, while kindlin regulates integrin activation, affecting adhesion and motility. This review examines the structures and functions of these proteins, focusing on their roles in cancer progression, immune response, and therapeutic potential. The cooperation between paxillin and kindlin in integrin activation and focal adhesion dynamics offers valuable insights into tumor metastasis, immune function, and tissue repair.