Focal adhesions are controlled by microtubules through local contractility regulation.

Microtubules regulate cell polarity and migration via local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with talin, the major component of focal adhesions. Here, local optogenetic activation of KANK1-mediated microtubule/talin linkage promoted microtubule targeting to an individual focal adhesion and subsequent withdrawal, resulting in focal adhesion centripetal sliding and rapid disassembly. This sliding is preceded by a local increase of traction force due to accumulation of myosin-II and actin in the proximity of the focal adhesion. Knockdown of the Rho activator GEF-H1 prevented development of traction force and abolished sliding and disassembly of focal adhesions upon KANK1 activation. Other players participating in microtubule-driven, KANK-dependent focal adhesion disassembly include kinases ROCK, PAK, and FAK, as well as microtubules/focal adhesion-associated proteins kinesin-1, APC, and αTAT. Based on these data, we develop a mathematical model for a microtubule-driven focal adhesion disruption involving local GEF-H1/RhoA/ROCK-dependent activation of contractility, which is consistent with experimental data.

Genome-Wide CRISPR Screen Identifies an NF2-Adherens Junction Mechanistic Dependency for Cardiac Lineage.

BACKGROUND: Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic protein)/Wnt-signaling pathways. However, the mechanisms and how regulatory roadblocks are removed with specific developmental signaling pathways remain unclear. METHODS: We conducted a genome-wide CRISPR screen to uncover essential regulators of cardiomyocyte specification in human embryonic stem cells using a myosin heavy chain 6 (MYH6)-GFP (green fluorescence protein) reporter system. After an independent secondary single guide ribonucleic acid validation of 25 candidates, we identified NF2 (neurofibromin 2), a moesin-ezrin-radixin like (MERLIN) tumor suppressor, as an upstream driver of early cardiomyocyte lineage specification. Independent monoclonal NF2 knockouts were generated using CRISPR-Cas9, and cell states were inferred through bulk RNA sequencing and protein expression analysis across differentiation time points. Terminal lineage differentiation was assessed by using an in vitro 2-dimensional-micropatterned gastruloid model, trilineage differentiation, and cardiomyocyte differentiation. Protein interaction and post-translation modification of NF2 with its interacting partners were assessed using site-directed mutagenesis, coimmunoprecipitation, and proximity ligation assays. RESULTS: Transcriptional regulation and trajectory inference from NF2-null cells reveal the loss of cardiomyocyte identity and the acquisition of nonmesodermal identity. Sustained elevation of early mesoderm lineage repressor SOX2 and upregulation of late anticardiac regulators CDX2 and MSX1 in NF2 knockout cells reflect a necessary role for NF2 in removing regulatory roadblocks. Furthermore, we found that NF2 and AMOT (angiomotin) cooperatively bind to YAP (yes-associated protein) during mesendoderm formation, thereby preventing YAP activation, independent of canonical MST (mammalian sterile 20-like serine-threonine protein kinase)-LATS (large tumor suppressor serine-threonine protein kinase) signaling. Mechanistically, cardiomyocyte lineage identity was rescued by wild-type and NF2 serine-518 phosphomutants, but not NF2 FERM (ezrin-radixin-meosin homology protein) domain blue-box mutants, demonstrating that the critical FERM domain-dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for early cardiomyocyte lineage differentiation. CONCLUSIONS: These results provide mechanistic insight into the essential role of NF2 during early epithelial-mesenchymal transition by sequestering the repressive effect of YAP and relieving regulatory roadblocks en route to cardiomyocytes.

Structural basis for the distinct roles of non-conserved Pro116 and conserved Tyr124 of BCH domain of yeast p50RhoGAP.

p50RhoGAP is a key protein that interacts with and downregulates the small GTPase RhoA. p50RhoGAP is a multifunctional protein containing the BNIP-2 and Cdc42GAP Homology (BCH) domain that facilitates protein-protein interactions and lipid binding and the GAP domain that regulates active RhoA population. We recently solved the structure of the BCH domain from yeast p50RhoGAP (YBCH) and showed that it maintains the adjacent GAP domain in an auto-inhibited state through the ß5 strand. Our previous WT YBCH structure shows that a unique kink at position 116 thought to be made by a proline residue between alpha helices α6 and α7 is essential for the formation of intertwined dimer from asymmetric monomers. Here we sought to establish the role and impact of this Pro116. However, the kink persists in the structure of P116A mutant YBCH domain, suggesting that the scaffold is not dictated by the proline residue at this position. We further identified Tyr124 (or Tyr188 in HBCH) as a conserved residue in the crucial ß5 strand. Extending to the human ortholog, when substituted to acidic residues, Tyr188D or Tyr188E, we observed an increase in RhoA binding and self-dimerization, indicative of a loss of inhibition of the GAP domain by the BCH domain. These results point to distinct roles and impact of the non-conserved and conserved amino acid positions in regulating the structural and functional complexity of the BCH domain.

Mutant VPS35-D620N induces motor dysfunction and impairs DAT-mediated dopamine recycling pathway.

The D620N mutation in vacuolar protein sorting protein 35 (VPS35) gene has been identified to be linked to late onset familial Parkinson disease (PD). However, the pathophysiological roles of VPS35-D620N in PD remain unclear. Here, we generated the transgenic Caenorhabditis elegans overexpressing either human wild type or PD-linked mutant VPS35-D620N in neurons. C. elegans expressing VPS35-D620N, compared with non-transgenic controls, showed movement disorders and dopaminergic neuron loss. VPS35-D620N worms displayed more swimming induced paralysis but showed no defects in BSR assays, thus indicating the disruption of dopamine (DA) recycling back inside neurons. Moreover, VPS35 formed a protein interaction complex with DA transporter (DAT), RAB5, RAB11 and FAM21. In contrast, the VPS35-D620N mutant destabilized these interactions, thus disrupting DAT transport from early endosomes to recycling endosomes, and decreasing DAT at the cell surface. These effects together increased DA in synaptic clefts, and led to dopaminergic neuron degeneration and motor dysfunction. Treatment with reserpine significantly decreased the swimming induced paralysis in VPS35-D620N worms, as compared with vehicle treated VPS35-D620N worms. Our studies not only provide novel insights into the mechanisms of VPS35-D620N-induced dopaminergic neuron degeneration and motor dysfunction via disruption of DAT function and the DA signaling pathway but also indicate a potential strategy to treat VPS35-D620N-related PD and other disorders.

Structural basis for p50RhoGAP BCH domain-mediated regulation of Rho inactivation.

Spatiotemporal regulation of signaling cascades is crucial for various biological pathways, under the control of a range of scaffolding proteins. The BNIP-2 and Cdc42GAP Homology (BCH) domain is a highly conserved module that targets small GTPases and their regulators. Proteins bearing BCH domains are key for driving cell elongation, retraction, membrane protrusion, and other aspects of active morphogenesis during cell migration, myoblast differentiation, and neuritogenesis. We previously showed that the BCH domain of p50RhoGAP (ARHGAP1) sequesters RhoA from inactivation by its adjacent GAP domain; however, the underlying molecular mechanism for RhoA inactivation by p50RhoGAP remains unknown. Here, we report the crystal structure of the BCH domain of p50RhoGAP Schizosaccharomyces pombe and model the human p50RhoGAP BCH domain to understand its regulatory function using in vitro and cell line studies. We show that the BCH domain adopts an intertwined dimeric structure with asymmetric monomers and harbors a unique RhoA-binding loop and a lipid-binding pocket that anchors prenylated RhoA. Interestingly, the ß5-strand of the BCH domain is involved in an intermolecular ß-sheet, which is crucial for inhibition of the adjacent GAP domain. A destabilizing mutation in the ß5-strand triggers the release of the GAP domain from autoinhibition. This renders p50RhoGAP active, thereby leading to RhoA inactivation and increased self-association of p50RhoGAP molecules via their BCH domains. Our results offer key insight into the concerted spatiotemporal regulation of Rho activity by BCH domain-containing proteins.

Mechanobiology of Tumor Growth.

Over the past decade, researchers have highlighted the importance of mechanical cues of the metastatic niche such as matrix stiffness, topography, mechanical stresses, and deformation on cells in influencing tumor growth and proliferation. Understanding the cellular and molecular basis and fine-tuning the mechano-response of cancer cells to this niche could lead to new and novel therapeutic interventions. In this review, we discuss the importance of mechanical cues surrounding tumor microenvironment that govern the growth and progression of cancer. We also highlight some emergent principles underlying the mechanosensing and mechanotransduction mechanisms that link cellular responses such as gene expression to phenotypic changes arising from such external cues. Recent technological advancements to visualize, quantify, model, and test these crucial steps with great precision will further advance our understanding of this phenomenon. We will conclude by showcasing potential applications of mechanobiology in controlling cancer growth as alternative cancer treatment regimes.

Activation of AKT-mTOR Signaling Directs Tenogenesis of Mesenchymal Stem Cells.

Tendon repair is a clinical challenge because of the limited understanding on tenogenesis. The synthesis of type I collagen (Collagen I) and other extracellular matrix are essential for tendon differentiation and homeostasis. Current studies on tenogenesis focused mostly on the tenogenic transcriptional factors while the signaling controlling tenogenesis on translational level remains largely unknown. Here, we showed that mechanistic target of rapamycin (mTOR) signaling was activated by protenogenic growth factor, transforming growth factors beta1, and insulin-like growth factor-I. The expression of mTOR was upregulated during tenogenesis of mesenchymal stem cells (MSCs). Moreover, mTOR was downregulated in human tendinopathy tissues and was inactivated upon statin treatment. Both inhibition and depletion of AKT or mTOR significantly reduced type I collagen production and impaired tenogenesis of MSCs. Tendon specific-ablation of mTOR resulted in tendon defect and reduction of Collagen I. However, there is no evident downregulation of tendon associated collagens at the transcription level. Our study demonstrated that AKT-mTOR axis is a key mediator of tendon differentiation and provided a novel therapeutic target for tendinopathy and tendon injuries. Stem Cells 2018;36:527-539.

Actomyosin contractility drives bile regurgitation as an early response during obstructive cholestasis.

BACKGROUND & AIMS: A wide range of liver diseases manifest as biliary obstruction, or cholestasis. However, the sequence of molecular events triggered as part of the early hepatocellular homeostatic response in obstructive cholestasis is poorly elucidated. Pericanalicular actin is known to accumulate during obstructive cholestasis. Therefore, we hypothesized that the pericanalicular actin cortex undergoes significant remodeling as a regulatory response to obstructive cholestasis. METHODS: In vivo investigations were performed in a bile duct-ligated mouse model. Actomyosin contractility was assessed using sandwich-cultured rat hepatocytes transfected with various fluorescently labeled proteins and pharmacological inhibitors of actomyosin contractility. RESULTS: Actomyosin contractility induces transient deformations along the canalicular membrane, a process we have termed inward blebbing. We show that these membrane intrusions are initiated by local ruptures in the pericanalicular actin cortex; and they typically retract following repair by actin polymerization and actomyosin contraction. However, above a certain osmotic pressure threshold, these inward blebs pinch away from the canalicular membrane into the hepatocyte cytoplasm as large vesicles (2-8µm). Importantly, we show that these vesicles aid in the regurgitation of bile from the bile canaliculi. CONCLUSION: Actomyosin contractility induces the formation of bile-regurgitative vesicles, thus serving as an early homeostatic mechanism against increased biliary pressure during cholestasis. LAY SUMMARY: Bile canaliculi expand and contract in response to the amount of secreted bile, and resistance from the surrounding actin bundles. Further expansion due to bile duct blockade leads to the formation of inward blebs, which carry away excess bile to prevent bile build up in the canaliculi.

Epidermal growth factor activates the Rho GTPase-activating protein (GAP) Deleted in Liver Cancer 1 via focal adhesion kinase and protein phosphatase 2A.

Deleted in Liver Cancer 1 (DLC1) is a RHO GTPase-activating protein (GAP) that negatively regulates RHO. Through its GAP activity, it modulates the actin cytoskeleton network and focal adhesion dynamics, ultimately leading to suppression of cell invasion and metastasis. Despite its presence in various structural and signaling components, little is known about how the activity of DLC1 is regulated at focal adhesions. Here we show that EGF stimulation activates the GAP activity of DLC1 through a concerted mechanism involving DLC1 phosphorylation by MEK/ERK and its subsequent dephosphorylation by protein phosphatase 2A (PP2A) and inhibition of focal adhesion kinase by MEK/ERK to allow the binding between DLC1 and PP2A. Phosphoproteomics and mutation studies revealed that threonine 301 and serine 308 on DLC1, known previously to be mutated in certain cancers, are required for DLC1-PP2A interaction and the subsequent activation of DLC1 upon their dephosphorylation. The intricate interplay of this "MEK/ERK-focal adhesion kinase-DLC1-PP2A" quartet provides a novel checkpoint in the spatiotemporal control of cell spreading and cell motility.

Structural Basis for a Unique ATP Synthase Core Complex from Nanoarcheaum equitans.

ATP synthesis is a critical and universal life process carried out by ATP synthases. Whereas eukaryotic and prokaryotic ATP synthases are well characterized, archaeal ATP synthases are relatively poorly understood. The hyperthermophilic archaeal parasite, Nanoarcheaum equitans, lacks several subunits of the ATP synthase and is suspected to be energetically dependent on its host, Ignicoccus hospitalis. This suggests that this ATP synthase might be a rudimentary machine. Here, we report the crystal structures and biophysical studies of the regulatory subunit, NeqB, the apo-NeqAB, and NeqAB in complex with nucleotides, ADP, and adenylyl-imidodiphosphate (non-hydrolysable analog of ATP). NeqB is ∼20 amino acids shorter at its C terminus than its homologs, but this does not impede its binding with NeqA to form the complex. The heterodimeric NeqAB complex assumes a closed, rigid conformation irrespective of nucleotide binding; this differs from its homologs, which require conformational changes for catalytic activity. Thus, although N. equitans possesses an ATP synthase core A3B3 hexameric complex, it might not function as a bona fide ATP synthase.

The bi-lobe-associated LRRP1 regulates Ran activity in Trypanosoma brucei.

Cilia and flagella are conserved eukaryotic organelles important for motility and sensory. The RanGTPase, best known for nucleocytoplasmic transport functions, may also play a role in protein trafficking into the specialized flagellar/ciliary compartments, although the regulatory mechanisms controlling Ran activity at the flagellum remain unclear. The unicellular parasite Trypanosoma brucei contains a single flagellum necessary for cell movement, division and morphogenesis. Correct flagellum functions require flagellar attachment to the cell body, which is mediated by a specialized flagellum attachment zone (FAZ) complex that is assembled together with the flagellum during the cell cycle. We have previously identified the leucine-rich-repeat protein 1 LRRP1 on a bi-lobe structure at the proximal base of flagellum and FAZ. LRRP1 is essential for bi-lobe and FAZ biogenesis, consequently affecting flagellum-driven cell motility and division. Here, we show that LRRP1 forms a complex with Ran and a Ran-binding protein, and regulates Ran-GTP hydrolysis in T. brucei. In addition to mitotic inhibition, depletion of Ran inhibits FAZ assembly in T. brucei, supporting the presence of a conserved mechanism that involves Ran in the regulation of flagellum functions in an early divergent eukaryote.

Annexin-A1 controls an ERK-RhoA-NFκB activation loop in breast cancer cells.

Wound healing is critical for normal development and pathological processes including cancer cell metastasis. MAPK, Rho-GTPases and NFκB are important regulators of wound healing, but mechanisms for their integration are incompletely understood. Annexin-A1 (ANXA1) is upregulated in invasive breast cancer cells resulting in constitutive activation of NFκB. We show here that silencing ANXA1 increases the formation of stress fibers and focal adhesions, which may inhibit wound healing. ANXA1 regulated wound healing is dependent on the activation of ERK1/2. ANXA1 increases the activation of RhoA, which is dependent on ERK activation. Furthermore, active RhoA is important in NF-κB activation, where constitutively active RhoA potentiates NFκB activation, while dominant negative RhoA inhibits NFκB activation in response to CXCL12 stimulation and active MEKK plasmids. These findings establish a central role for ANXA1 in the cell migration through the activation of NFκB, ERK1/2 and RhoA.

Structural basis for the allosteric inhibitory mechanism of human kidney-type glutaminase (KGA) and its regulation by Raf-Mek-Erk signaling in cancer cell metabolism.

Besides thriving on altered glucose metabolism, cancer cells undergo glutaminolysis to meet their energy demands. As the first enzyme in catalyzing glutaminolysis, human kidney-type glutaminase isoform (KGA) is becoming an attractive target for small molecules such as BPTES [bis-2-(5 phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide], although the regulatory mechanism of KGA remains unknown. On the basis of crystal structures, we reveal that BPTES binds to an allosteric pocket at the dimer interface of KGA, triggering a dramatic conformational change of the key loop (Glu312-Pro329) near the catalytic site and rendering it inactive. The binding mode of BPTES on the hydrophobic pocket explains its specificity to KGA. Interestingly, KGA activity in cells is stimulated by EGF, and KGA associates with all three kinase components of the Raf-1/Mek2/Erk signaling module. However, the enhanced activity is abrogated by kinase-dead, dominant negative mutants of Raf-1 (Raf-1-K375M) and Mek2 (Mek2-K101A), protein phosphatase PP2A, and Mek-inhibitor U0126, indicative of phosphorylation-dependent regulation. Furthermore, treating cells that coexpressed Mek2-K101A and KGA with suboptimal level of BPTES leads to synergistic inhibition on cell proliferation. Consequently, mutating the crucial hydrophobic residues at this key loop abrogates KGA activity and cell proliferation, despite the binding of constitutive active Mek2-S222/226D. These studies therefore offer insights into (i) allosteric inhibition of KGA by BPTES, revealing the dynamic nature of KGA's active and inhibitory sites, and (ii) cross-talk and regulation of KGA activities by EGF-mediated Raf-Mek-Erk signaling. These findings will help in the design of better inhibitors and strategies for the treatment of cancers addicted with glutamine metabolism.

Three-dimensional liquid metal-based neuro-interfaces for human hippocampal organoids.

Human hippocampal organoids (hHOs) derived from human induced pluripotent stem cells (hiPSCs) have emerged as promising models for investigating neurodegenerative disorders, such as schizophrenia and Alzheimer's disease. However, obtaining the electrical information of these free-floating organoids in a noninvasive manner remains a challenge using commercial multi-electrode arrays (MEAs). The three-dimensional (3D) MEAs developed recently acquired only a few neural signals due to limited channel numbers. Here, we report a hippocampal cyborg organoid (cyb-organoid) platform coupling a liquid metal-polymer conductor (MPC)-based mesh neuro-interface with hHOs. The mesh MPC (mMPC) integrates 128-channel multielectrode arrays distributed on a small surface area (~2*2 mm). Stretchability (up to 500%) and flexibility of the mMPC enable its attachment to hHOs. Furthermore, we show that under Wnt3a and SHH activator induction, hHOs produce HOPX+ and PAX6+ progenitors and ZBTB20+PROX1+ dentate gyrus (DG) granule neurons. The transcriptomic signatures of hHOs reveal high similarity to the developing human hippocampus. We successfully detect neural activities from hHOs via the mMPC from this cyb-organoid. Compared with traditional planar devices, our non-invasive coupling offers an adaptor for recording neural signals from 3D models.

Subcellular Force Imbalance in Actin Bundles Induces Nuclear Repositioning and Durotaxis.

Durotaxis is a phenomenon in which cells migrate toward substrates of increasing stiffness. However, how cells assimilate substrate stiffness as a directional cue remains poorly understood. In this study, we experimentally show that mouse embryonic fibroblasts can discriminate between different substrate stiffnesses and develop higher traction forces at regions of the cell adhering to the stiffer pillars. In this way, the cells generate a force imbalance between adhesion sites. It is this traction force imbalance that drives durotaxis by providing directionality for cell migration. Significantly, we found that traction forces are transmitted via LINC complexes to the cell nucleus, which serves to maintain the global force imbalance. In this way, LINC complexes play an essential role in anterograde nuclear movement and durotaxis. This conclusion is supported by the fact that LINC complex-deficient cells are incapable of durotaxis and instead migrate randomly on substrates featuring a stiffness gradient.

Deleterious Mechanical Deformation Selects Mechanoresilient Cancer Cells with Enhanced Proliferation and Chemoresistance.

Cancer cells in secondary tumors are found to form metastases more efficiently as compared to their primary tumor counterparts. This is partially due to the unfavorable microenvironments encountered by metastasizing cancer cells that result in the survival of a more metastatic phenotype from the original population. However, the role of deleterious mechanical stresses in this change of metastatic potential is unclear. Here, by forcing cancer cells to flow through small capillary-sized constrictions, it is demonstrated that mechanical deformation can select a tumor cell subpopulation that exhibits resilience to mechanical squeezing-induced cell death. Transcriptomic profiling reveals up-regulated proliferation and DNA damage response pathways in this subpopulation, which are further translated into a more proliferative and chemotherapy-resistant phenotype. These results highlight a potential link between the microenvironmental physical stresses and the enhanced malignancy of metastasizing cancer cells which may be utilized as a therapeutic strategy in preventing the metastatic spread of cancer cells.

CAMK2D serves as a molecular scaffold for RNF8-MAD2 complex to induce mitotic checkpoint in glioma.

MAD2 is a spindle assembly checkpoint protein that participates in the formation of mitotic checkpoint complex, which blocks mitotic progression. RNF8, an established DNA damage response protein, has been implicated in mitotic checkpoint regulation but its exact role remains poorly understood. Here, RNF8 proximity proteomics uncovered a role of RNF8-MAD2 in generating the mitotic checkpoint signal. Specifically, RNF8 competes with a small pool of p31comet for binding to the closed conformer of MAD2 via its RING domain, while CAMK2D serves as a molecular scaffold to concentrate the RNF8-MAD2 complex via transient/weak interactions between its p-Thr287 and RNF8's FHA domain. Accordingly, RNF8 overexpression impairs glioma stem cell (GSC) mitotic progression in a FHA- and RING-dependent manner. Importantly, low RNF8 expression correlates with inferior glioma outcome and RNF8 overexpression impedes GSC tumorigenicity. Last, we identify PLK1 inhibitor that mimics RNF8 overexpression using a chemical biology approach, and demonstrate a PLK1/HSP90 inhibitor combination that synergistically reduces GSC proliferation and stemness. Thus, our study has unveiled a previously unrecognized CAMK2D-RNF8-MAD2 complex in regulating mitotic checkpoint with relevance to gliomas, which is therapeutically targetable.

The scaffold RhoGAP protein ARHGAP8/BPGAP1 synchronizes Rac and Rho signaling to facilitate cell migration.

Rho GTPases regulate cell morphogenesis and motility under the tight control of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). However, the underlying mechanism(s) that coordinate their spatiotemporal activities, whether separately or together, remain unclear. We show that a prometastatic RhoGAP, ARHGAP8/BPGAP1, binds to inactive Rac1 and localizes to lamellipodia. BPGAP1 recruits the RacGEF Vav1 under epidermal growth factor (EGF) stimulation and activates Rac1, leading to polarized cell motility, spreading, invadopodium formation, and cell extravasation and promotes cancer cell migration. Importantly, BPGAP1 down-regulates local RhoA activity, which influences Rac1 binding to BPGAP1 and its subsequent activation by Vav1. Our results highlight the importance of BPGAP1 in recruiting Vav1 and Rac1 to promote Rac1 activation for cell motility. BPGAP1 also serves to control the timing of Rac1 activation with RhoA inactivation via its RhoGAP activity. BPGAP1, therefore, acts as a dual-function scaffold that recruits Vav1 to activate Rac1 while inactivating RhoA to synchronize both Rho and Rac signaling in cell motility. As epidermal growth factor receptor (EGFR), Vav1, RhoA, Rac1, and BPGAP1 are all associated with cancer metastasis, BPGAP1 could provide a crucial checkpoint for the EGFR-BPGAP1-Vav1-Rac1-RhoA signaling axis for cancer intervention.

Active Mek2 as a regulatory scaffold that promotes Pin1 binding to BPGAP1 to suppress BPGAP1-induced acute Erk activation and cell migration.

BPGAP1 is a multidomain Rho GTPase-activating protein (RhoGAP) that promotes Erk activation and cell motility. However, the molecular mechanism of how these two processes are linked and regulated remains unclear. Here, we show that the RhoGAP domain of BPGAP1 interacts with the peptidyl-prolyl cis/trans isomerase (PPI) Pin1, leading to enhanced GAP activity towards RhoA. BPGAP1 also interacted with wild-type and constitutively active Mek2, but not with its kinase-dead mutant. However, only active Mek2 could bind Pin1, acting as a scaffold to bridge Pin1 and BPGAP1 in a manner that involves the release of an autoinhibited proline-rich motif, 186-PPLP-189, proximal to the RhoGAP domain. This allows the non-canonical 186-PPLP-189 and 256-DDYGD-260 motifs of the proline-rich region and RhoGAP domain of BPGAP1 to become accessible to concerted binding by the WW and PPI domains of Pin1, respectively. Interestingly, Pin1 knockdown led to 'super-induction' of BPGAP1-induced acute, but not chronic, Erk activation upon epidermal growth factor stimulation, in a process independent of GAP modulation. Reintroducing Pin1, but not its catalytic or non-binding mutants, reversed the effect and inhibited cell migration induced by coexpression of BPGAP1 and active Mek2. Thus, Pin1 regulates BPGAP1 function in Rho and Erk signalling, with active Mek2 serving as a novel regulatory scaffold that promotes crosstalk between RhoGAP, Pin1 and Erk in the regulation of cell migration.

Lung Cancer Induces NK Cell Contractility and Cytotoxicity Through Transcription Factor Nuclear Localization.

Actomyosin-mediated cellular contractility is highly conserved for mechanotransduction and signalling. While this phenomenon has been observed in adherent cell models, whether/how contractile forces regulate the function of suspension cells like natural killer (NK) cells during cancer surveillance, is unknown. Here, we demonstrated in coculture settings that the evolutionarily conserved NK cell transcription factor, Eomes, undergoes nuclear shuttling during lung cancer cell surveillance. Biophysical and biochemical analyses revealed mechanistic enhancement of NK cell actomyosin-mediated contractility, which is associated with nuclear flattening, thus enabling nuclear entry of Eomes associated with enhanced NK cytotoxicity. We found that NK cells responded to the presumed immunosuppressive TGFß in the NK-lung cancer coculture medium to sustain its intracellular contractility through myosin light chain phosphorylation, thereby promoting Eomes nuclear localization. Therefore, our results demonstrate that lung cancer cells provoke NK cell contractility as an early phase activation mechanism and that Eomes is a plausible mechano-responsive protein for increased NK cytotoxicity. There is scope for strategic application of actomyosin-mediated contractility modulating drugs ex vivo, to reinvigorate NK cells prior to adoptive cancer immunotherapy in vivo (177 words).