Paxillin phase separation promotes focal adhesion assembly and integrin signaling.

Focal adhesions (FAs) are transmembrane protein assemblies mediating cell-matrix connection. Although protein liquid-liquid phase separation (LLPS) has been tied to the organization and dynamics of FAs, the underlying mechanisms remain unclear. Here, we experimentally tune the LLPS of PXN/Paxillin, an essential scaffold protein of FAs, by utilizing a light-inducible Cry2 system in different cell types. In addition to nucleating FA components, light-triggered PXN LLPS potently activates integrin signaling and subsequently accelerates cell spreading. In contrast to the homotypic interaction-driven LLPS of PXN in vitro, PXN condensates in cells are associated with the plasma membrane and modulated by actomyosin contraction and client proteins of FAs. Interestingly, non-specific weak intermolecular interactions synergize with specific molecular interactions to mediate the multicomponent condensation of PXN and are efficient in promoting FA assembly and integrin signaling. Thus, our data establish an active role of the PXN phase transition into a condensed membrane-associated compartment in promoting the assembly/maturation of FAs.

Development of a High-Throughput Fluorescence Polarization Assay to Detect Inhibitors of the FAK-Paxillin Interaction.

Focal adhesion kinase (FAK) is a promising cancer drug target due to its massive overexpression in multiple solid tumors and its critical role in the integration of signals that control proliferation, invasion, apoptosis, and metastasis. Previous FAK drug discovery and high-throughput screening have exclusively focused on the identification of inhibitors that target the kinase domain of FAK. Because FAK is both a kinase and scaffolding protein, the development of novel screening assays that detect inhibitors of FAK protein-protein interactions remains a critical need. In this report, we describe the development of a high-throughput fluorescence polarization (FP) screening assay that measures the interactions between FAK and paxillin, a focal adhesion-associated protein. We designed a tetramethylrhodamine (TAMRA)-tagged paxillin peptide based on the paxillin LD2 motif that binds to the focal adhesion targeting (FAT) domain with significant dynamic range, specificity, variability, stability, and a Z'-factor suitable for high-throughput screening. In addition, we performed a pilot screen of 1593 compounds using this FP assay, showing its feasibility for high-throughput drug screening. Finally, we identified three compounds that show dose-dependent competition of FAT-paxillin binding. This assay represents the first described high-throughput screening assay for FAK scaffold inhibitors and can accelerate drug discovery efforts for this promising drug target.

Spatial arrangement of LD motif-interacting residues on focal adhesion targeting domain of Focal Adhesion Kinase determine domain-motif interaction affinity and specificity.

BACKGROUND: Leucine rich Aspartate motifs (LD motifs) are molecular recognition motifs on Paxillin that recognize LD-motif binding domains (LDBD) of a number of focal adhesion proteins in order to carry out downstream signaling and actin cytoskeleton remodeling. In this study, we identified structural features within LDBDs that influence their binding affinity with Paxillin LD motifs. METHODS: Various point mutants of focal adhesion targeting (FAT) domain of Focal Adhesion Kinase (FAK) were created by moving a key Lysine residue two and three helical turns in order to match the unique conformations as observed in LDBDs of two other focal adhesion proteins, Vinculin and CCM3. RESULTS: This led to identify a mutant of FAT domain of FAK, named as FAT(NV) (Asn992 of FAT domain was replaced by Val), with remarkable high affinity for LD1 (Kd = 1.5 µM vs no-binding with wild type) and LD2 peptides (Kd = 7.2 µM vs 63 µM with wild type). Consistently, the focal adhesions of MCF7 cells expressing FAK(NV) were highly stable (turnover rate = 1.25 × 10-5 µm2/s) as compared to wild type FAK transfected cells (turnover rate = 1.5 × 10-3 µm2/s). CONCLUSIONS: We observed that the relative disposition of key LD binding amino-acids at LDBD surface, hydrophobic burial of long Leucine side chains of LD-motifs and complementarity of charged surfaces are the key factors determining the binding affinities of LD motifs with LDBDs. GENERAL SIGNIFICANCE: Our study will help in protein engineering of FAT domain of FAK by modulating FAK-LD motif interactions which have implications in cellular focal adhesions and cell migration.

Structural Basis of Paxillin Recruitment by Kindlin-2 in Regulating Cell Adhesion.

Activation of cell surface receptor integrin has been extensively studied as the first key step to trigger cell adhesion, but the subsequent events, widely regarded as integrin "outside-in" signaling to form supramolecular complexes (focal adhesions [FAs]) to promote dynamic cell adhesion, remain poorly elucidated. Integrin activator kindlin-2 was recently found to associate with paxillin in nascent FAs, implicating an early yet undefined integrin outside-in signaling event. Here we show structurally that kindlin-2 recognizes paxillin via a distinct interface involving the ubiquitin-like kindlin-2 F0 domain and the paxillin LIM4 domain. The interface is adjacent to the membrane binding site of kindlin-2 F0, suggesting a mechanism for kindlin-2 to recruit paxillin to the membrane-proximal site where FA assembly is initiated. Disruption of the interface impaired the localization of paxillin, causing strong defects in FA assembly and cell migration. These data unveil a structural basis of the kindlin-2/paxillin interaction in controlling dynamic cell adhesion.

Development of a Fragment-Based Screening Assay for the Focal Adhesion Targeting Domain Using SPR and NMR.

The Focal Adhesion Targeting (FAT) domain of Focal Adhesion Kinase (FAK) is a promising drug target since FAK is overexpressed in many malignancies and promotes cancer cell metastasis. The FAT domain serves as a scaffolding protein, and its interaction with the protein paxillin localizes FAK to focal adhesions. Various studies have highlighted the importance of FAT-paxillin binding in tumor growth, cell invasion, and metastasis. Targeting this interaction through high-throughput screening (HTS) provides a challenge due to the large and complex binding interface. In this report, we describe a novel approach to targeting FAT through fragment-based drug discovery (FBDD). We developed two fragment-based screening assays-a primary SPR assay and a secondary heteronuclear single quantum coherence nuclear magnetic resonance (HSQC-NMR) assay. For SPR, we designed an AviTag construct, optimized SPR buffer conditions, and created mutant controls. For NMR, resonance backbone assignments of the human FAT domain were obtained for the HSQC assay. A 189-compound fragment library from Enamine was screened through our primary SPR assay to demonstrate the feasibility of a FAT-FBDD pipeline, with 19 initial hit compounds. A final total of 11 validated hits were identified after secondary screening on NMR. This screening pipeline is the first FBDD screen of the FAT domain reported and represents a valid method for further drug discovery efforts on this difficult target.

Structural basis of the target-binding mode of the G protein-coupled receptor kinase-interacting protein in the regulation of focal adhesion dynamics.

Focal adhesions (FAs) are specialized sites where intracellular cytoskeleton elements connect to the extracellular matrix and thereby control cell motility. FA assembly depends on various scaffold proteins, including the G protein-coupled receptor kinase-interacting protein 1 (GIT1), paxillin, and liprin-α. Although liprin-α and paxillin are known to competitively interact with GIT1, the molecular basis governing these interactions remains elusive. To uncover the underlying mechanisms of how GIT1 is involved in FA assembly by alternatively binding to liprin-α and paxillin, here we solved the crystal structures of GIT1 in complex with liprin-α and paxillin at 1.8 and 2.6 Å resolutions, respectively. These structures revealed that the paxillin-binding domain (PBD) of GIT1 employs distinct binding modes to recognize a single α-helix of liprin-α and the LD4 motif of paxillin. Structure-based design of protein variants produced two binding-deficient GIT1 variants; specifically, these variants lost the ability to interact with liprin-α only or with both liprin-α and paxillin. Expressing the GIT1 variants in COS7 cells, we discovered that the two PBD-meditated interactions play different roles in either recruiting GIT1 to FA or facilitating FA assembly. Additionally, we demonstrate that, unlike for the known binding mode of the FAT domain to LD motifs, the PBD of GIT1 uses different surface patches to achieve high selectivity in LD motif recognition. In summary, our results have uncovered the mechanisms by which GIT1's PBD recognizes cognate paxillin and liprin-α structures, information we anticipate will be useful for future investigations of GIT1-protein interactions in cells.

Roles of paxillin phosphorylation in IL-3 withdrawal-induced Ba/F3 cell apoptosis.

BACKGROUND: Ba/F3, a mouse pro-B cell line, is dependent on IL-3 for its survival and proliferation. IL-3 withdrawal causes cells to round, stop in G1 phase, then undergo apoptosis. Additionally, IL-3 is known to induce tyrosine phosphorylation of paxillin, a scaffold and signaling protein. We previously determined that overexpression of paxillin prohibited Ba/F3 cell apoptosis induced by IL-3 withdrawal. OBJECTIVE: Address whether phosphorylation is essential for the anti-apoptotic effect of overexpressed paxillin. METHODS: Mutations were introduced into paxillin cDNA at five phosphorylation sites-Y31F, Y40F, Y118F, Y181F, S273A, or S273D. After overexpression of paxillin mutants in Ba/F3 cells, the apoptotic proportions of cell populations were measured by an annexin V conjugation assay while cells were undergoing IL-3 withdrawal. RESULTS: The anti-apoptotic effect of paxillin overexpression was abolished by site-directed mutagenesis replacing Y31, Y40, Y118, and Y181 with phenylalanine, and S273 with aspartic acid. In contrast, the mutation replacing S273 with alanine had no effect on the anti-apoptotic effect. CONCLUSION: The above results suggest that paxillin-mediated phosphorylation at Y31, Y40, Y118, and Y181 is essential for the anti-apoptotic effect of paxillin overexpression in Ba/F3 cells and contributes to the cell survival signaling pathway triggered by IL-3. Conversely, phosphorylation at S273 is involved in the negative regulation of the anti-apoptotic action of overexpressed paxillin.

Paxillin phosphorylation at serine 273 and its effects on Rac, Rho and adhesion dynamics.

Focal adhesions are protein complexes that anchor cells to the extracellular matrix. During migration, the growth and disassembly of these structures are spatiotemporally regulated, with new adhesions forming at the leading edge of the cell and mature adhesions disassembling at the rear. Signalling proteins and structural cytoskeletal components tightly regulate adhesion dynamics. Paxillin, an adaptor protein within adhesions, is one of these proteins. Its phosphorylation at serine 273 (S273) is crucial for maintaining fast adhesion assembly and disassembly. Paxillin is known to bind to a GIT1-ßPIX-PAK1 complex, which increases the local activation of the small GTPase Rac. To understand quantitatively the behaviour of this system and how it relates to adhesion assembly/disassembly, we developed a mathematical model describing the dynamics of the small GTPases Rac and Rho as determined by paxillin S273 phosphorylation. Our model revealed that the system possesses bistability, where switching between uninduced (active Rho) and induced (active Rac) states can occur through a change in rate of paxillin phosphorylation or PAK1 activation. The bistable switch is characterized by the presence of memory, minimal change in the levels of active Rac and Rho within the induced and uninduced states, respectively, and the limited regime of monostability associated with the uninduced state. These results were validated experimentally by showing the presence of bimodality in adhesion assembly and disassembly rates, and demonstrating that Rac activity increases after treating Chinese Hamster Ovary cells with okadaic acid (a paxillin phosphatase inhibitor), followed by a modest recovery after 20 min washout. Spatial gradients of phosphorylated paxillin in a reaction-diffusion model gave rise to distinct regions of Rac and Rho activities, resembling polarization of a cell into front and rear. Perturbing several parameters of the model also revealed important insights into how signalling components upstream and downstream of paxillin phosphorylation affect dynamics.

Dissecting single-cell molecular spatiotemporal mobility and clustering at focal adhesions in polarised cells by fluorescence fluctuation spectroscopy methods.

Quantitative fluorescence fluctuation spectroscopy from optical microscopy datasets is a very powerful tool to resolve multiple spatiotemporal cellular and subcellular processes at the molecular level. In particular, raster image correlation spectroscopy (RICS) and number and brightness analyses (N&B) yield molecular mobility and clustering dynamic information extracted from real-time cellular processes. This quantitative information can be inferred in a highly flexible and detailed manner, i.e. 1) at the localisation level: from full-frame datasets and multiple regions of interest within; and 2) at the temporal level: not only from full-frame and multiple regions, but also intermediate temporal events. Here we build on previous research in deciphering the molecular dynamics of paxillin, a main component of focal adhesions. Cells use focal adhesions to attach to the extracellular matrix and interact with their local environment. Through focal adhesions and other adhesion structures, cells sense their local environment and respond accordingly; due to this continuous communication, these structures can be highly dynamic depending on the extracellular characteristics. By using a previously well-characterised model like paxillin, we examine the powerful sensitivity and some limitations of RICS and N&B analyses. We show that cells upon contact to different surfaces show differential self-assembly dynamics in terms of molecular diffusion and oligomerisation. In addition, single-cell studies show that these dynamics change gradually following an antero-posterior gradient.

Paxillin actions in the nucleus.

Paxillin is a group III LIM domain protein that is best characterized as a cytoplasmic scaffold/adaptor protein that functions primarily as a mediator of focal adhesion. However, emerging studies indicate that paxillin's functions are far broader. Not only does paxillin appear to regulate cytoplasmic kinase signaling, but it also cycles between the cytoplasm and nucleus, and may serve as an important regulator of mRNA trafficking and subsequent translation. Herein, we provide some insights suggesting that paxillin, like its relative Hic-5, has nuclear binding partners and mediates critical processes within the nucleus, at least in part functioning as coregulator of nuclear receptors and nuclear kinases to mediate genomic signaling.

Marker-free method for accurate alignment between correlated light, cryo-light, and electron cryo-microscopy data using sample support features.

Combining fluorescence microscopy with electron cryo-tomography allows, in principle, spatial localization of tagged macromolecular assemblies and structural features within the cellular environment. To allow precise localization and scale integration between the two disparate imaging modalities, accurate alignment procedures are needed. Here, we describe a marker-free method for aligning images from light or cryo-light fluorescence microscopy and from electron cryo-microscopy that takes advantage of sample support features, namely the holes in the carbon film. We find that the accuracy of this method, as judged by prediction errors of the hole center coordinates, is better than 100 nm.

Structural and functional insights into the interaction between the Cas family scaffolding protein p130Cas and the focal adhesion-associated protein paxillin.

The Cas family scaffolding protein p130Cas is a Src substrate localized in focal adhesions (FAs) and functions in integrin signaling to promote cell motility, invasion, proliferation, and survival. p130Cas targeting to FAs is essential for its tyrosine phosphorylation and downstream signaling. Although the N-terminal SH3 domain is important for p130Cas localization, it has also been reported that the C-terminal region is involved in p130Cas FA targeting. The C-terminal region of p130Cas or Cas family homology domain (CCHD) has been reported to adopt a structure similar to that of the focal adhesion kinase C-terminal focal adhesion-targeting domain. The mechanism by which the CCHD promotes FA targeting of p130Cas, however, remains unclear. In this study, using a calorimetry approach, we identified the first LD motif (LD1) of the FA-associated protein paxillin as the binding partner of the p130Cas CCHD (in a 1:1 stoichiometry with a Kd ∼4.2 µm) and elucidated the structure of the p130Cas CCHD in complex with the paxillin LD1 motif by X-ray crystallography. Of note, a comparison of the CCHD/LD1 complex with a previously solved structure of CCHD in complex with the SH2-containing protein NSP3 revealed that LD1 had almost identical positioning of key hydrophobic and acidic residues relative to NSP3. Because paxillin is one of the key scaffold molecules in FAs, we propose that the interaction between the p130Cas CCHD and the LD1 motif of paxillin plays an important role in p130Cas FA targeting.

Podosome Force Generation Machinery: A Local Balance between Protrusion at the Core and Traction at the Ring.

Determining how cells generate and transduce mechanical forces at the nanoscale is a major technical challenge for the understanding of numerous physiological and pathological processes. Podosomes are submicrometer cell structures with a columnar F-actin core surrounded by a ring of adhesion proteins, which possess the singular ability to protrude into and probe the extracellular matrix. Using protrusion force microscopy, we have previously shown that single podosomes produce local nanoscale protrusions on the extracellular environment. However, how cellular forces are distributed to allow this protruding mechanism is still unknown. To investigate the molecular machinery of protrusion force generation, we performed mechanical simulations and developed quantitative image analyses of nanoscale architectural and mechanical measurements. First, in silico modeling showed that the deformations of the substrate made by podosomes require protrusion forces to be balanced by local traction forces at the immediate core periphery where the adhesion ring is located. Second, we showed that three-ring proteins are required for actin polymerization and protrusion force generation. Third, using DONALD, a 3D nanoscopy technique that provides 20 nm isotropic localization precision, we related force generation to the molecular extension of talin within the podosome ring, which requires vinculin and paxillin, indicating that the ring sustains mechanical tension. Our work demonstrates that the ring is a site of tension, balancing protrusion at the core. This local coupling of opposing forces forms the basis of protrusion and reveals the podosome as a nanoscale autonomous force generator.

Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions.

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.

Roles of paxillin family members in adhesion and ECM degradation coupling at invadosomes.

Invadosomes are acto-adhesive structures able to both bind the extracellular matrix (ECM) and digest it. Paxillin family members-paxillin, Hic-5, and leupaxin-are implicated in mechanosensing and turnover of adhesion sites, but the contribution of each paxillin family protein to invadosome activities is unclear. We use genetic approaches to show that paxillin and Hic-5 have both redundant and distinctive functions in invadosome formation. The essential function of paxillin-like activity is based on the coordinated activity of LD motifs and LIM domains, which support invadosome assembly and morphology, respectively. However, paxillin preferentially regulates invadosome assembly, whereas Hic-5 regulates the coupling between ECM degradation and acto-adhesive functions. Mass spectrometry analysis revealed new partners that are important for paxillin and Hic-5 specificities: paxillin regulates the acto-adhesive machinery through janus kinase 1 (JAK1), whereas Hic-5 controls ECM degradation via IQGAP1. Integrating the redundancy and specificities of paxillin and Hic-5 in a functional complex provides insights into the coupling between the acto-adhesive and ECM-degradative machineries in invadosomes.

Autophagy Promotes Focal Adhesion Disassembly and Cell Motility of Metastatic Tumor Cells through the Direct Interaction of Paxillin with LC3.

Autophagy is a conserved catabolic process that plays a housekeeping role in eliminating protein aggregates and organelles and is activated during nutrient deprivation to generate metabolites and energy. Autophagy plays a significant role in tumorigenesis, although opposing context-dependent functions of autophagy in cancer have complicated efforts to target autophagy for therapeutic purposes. We demonstrate that autophagy inhibition reduces tumor cell migration and invasion in vitro and attenuates metastasis in vivo. Numerous abnormally large focal adhesions (FAs) accumulate in autophagy-deficient tumor cells, reflecting a role for autophagy in FA disassembly through targeted degradation of paxillin. We demonstrate that paxillin interacts with processed LC3 through a conserved LIR motif in the amino-terminal end of paxillin and that this interaction is regulated by oncogenic SRC activity. Together, these data establish a function for autophagy in FA turnover, tumor cell motility, and metastasis.

Deciphering Mode of Action of Functionally Important Regions in the Intrinsically Disordered Paxillin (Residues 1-313) Using Its Interaction with FAT (Focal Adhesion Targeting Domain of Focal Adhesion Kinase).

Intrinsically disordered proteins (IDPs) play a major role in various cellular functions ranging from transcription to cell migration. Mutations/modifications in such IDPs are shown to be associated with various diseases. Current strategies to study the mode of action and regulatory mechanisms of disordered proteins at the structural level are time consuming and challenging. Therefore, using simple and swift strategies for identifying functionally important regions in unstructured segments and understanding their underlying mechanisms is critical for many applications. Here we propose a simple strategy that employs dissection of human paxillin (residues 1-313) that comprises intrinsically disordered regions, followed by its interaction study using FAT (Focal adhesion targeting domain of focal adhesion kinase) as its binding partner to retrace structural behavior. Our findings show that the paxillin interaction with FAT exhibits a masking and unmasking effect by a putative intra-molecular regulatory region. This phenomenon suggests how cancer associated mutations in paxillin affect its interactions with Focal Adhesion Kinase (FAK). The strategy could be used to decipher the mode of regulations and identify functionally relevant constructs for other studies.

Engineering Synthetic Antibody Inhibitors Specific for LD2 or LD4 Motifs of Paxillin.

Focal adhesion protein paxillin links integrin and growth factor signaling to actin cytoskeleton. Most of paxillin signaling activity is regulated via leucine-rich LD motifs (LD1-LD5) located at the N-terminus. Here, we demonstrate a method to engineer highly selective synthetic antibodies (sABs) against LD2 and LD4 that are binding sites for focal adhesion kinase (FAK) and other proteins. Phage display selections against peptides were used to generate sABs recognizing each LD motif. In the obtained X-ray crystal structures of the LD-sAB complexes, the LD motifs are helical and bind sABs through a hydrophobic side, similarly as in the structures with natural paxillin partners. The sABs are capable of pulling down endogenous paxillin in complex with FAK and can visualize paxillin in focal adhesions in cells. They were also used as selective inhibitors to effectively compete with focal adhesion targeting domain of FAK for the binding to LD2 and LD4. The sABs are tools for investigation of paxillin LD binding "platforms" and are capable of inhibiting paxillin interactions, thereby useful as potential therapeutics in the future.

Molecular mechanism of vinculin activation and nanoscale spatial organization in focal adhesions.

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.

Gastrin-stimulated Gα13 Activation of Rgnef Protein (ArhGEF28) in DLD-1 Colon Carcinoma Cells.

The guanine nucleotide exchange factor Rgnef (also known as ArhGEF28 or p190RhoGEF) promotes colon carcinoma cell motility and tumor progression via interaction with focal adhesion kinase (FAK). Mechanisms of Rgnef activation downstream of integrin or G protein-coupled receptors remain undefined. In the absence of a recognized G protein signaling homology domain in Rgnef, no proximal linkage to G proteins was known. Utilizing multiple methods, we have identified Rgnef as a new effector for Gα13 downstream of gastrin and the type 2 cholecystokinin receptor. In DLD-1 colon carcinoma cells depleted of Gα13, gastrin-induced FAK Tyr(P)-397 and paxillin Tyr(P)-31 phosphorylation were reduced. RhoA GTP binding and promoter activity were increased by Rgnef in combination with active Gα13. Rgnef co-immunoprecipitated with activated Gα13Q226L but not Gα12Q229L. The Rgnef C-terminal (CT, 1279-1582) region was sufficient for co-immunoprecipitation, and Rgnef-CT exogenous expression prevented Gα13-stimulated SRE activity. A domain at the C terminus of the protein close to the FAK binding domain is necessary to bind to Gα13. Point mutations of Rgnef-CT residues disrupt association with active Gα13 but not Gαq. These results show that Rgnef functions as an effector of Gα13 signaling and that this linkage may mediate FAK activation in DLD-1 colon carcinoma cells.