KAT6A deficiency impairs cognitive functions through suppressing RSPO2/Wnt signaling in hippocampal CA3.

Intellectual disability (ID) affects ~2% of the population and ID-associated genes are enriched for epigenetic factors, including those encoding the largest family of histone lysine acetyltransferases (KAT5-KAT8). Among them is KAT6A, whose mutations cause KAT6A syndrome, with ID as a common clinical feature. However, the underlying molecular mechanism remains unknown. Here, we find that KAT6A deficiency impairs synaptic structure and plasticity in hippocampal CA3, but not in CA1 region, resulting in memory deficits in mice. We further identify a CA3-enriched gene Rspo2, encoding Wnt activator R-spondin 2, as a key transcriptional target of KAT6A. Deletion of Rspo2 in excitatory neurons impairs memory formation, and restoring RSPO2 expression in CA3 neurons rescues the deficits in Wnt signaling and learning-associated behaviors in Kat6a mutant mice. Collectively, our results demonstrate that KAT6A-RSPO2-Wnt signaling plays a critical role in regulating hippocampal CA3 synaptic plasticity and cognitive function, providing potential therapeutic targets for KAT6A syndrome and related neurodevelopmental diseases.

Comparison of root morphology and rhizosphere microbial communities form moso-bamboo in different forest types.

Moso-bamboo (Phyllostachys edulis), with the favor of human disturbance, rapidly invades adjacent forests to form monocultures in East Asia. Moso-bamboo not only intrudes the broadleaf forests but also the coniferous, and it could impact by above- and below-ground pathways. However, it still remains unclear whether the below-ground performance of moso-bamboo differs from broadleaf to coniferous forests, especially those differing in competitive and nutrient acquisition strategies. In this study, we investigated three types of forest stands in Guangdong, China, including a bamboo monoculture, a coniferous forest, and a broadleaf forest. We found that moso-bamboo may suffer stronger soil P limitation (soil N/P = 18.16) and may be infected by more AMF in coniferous than broadleaf forests (soil N/P = 16.17). Based on our PLS-path model analysis, soil P resource may be the key to differ moso-bamboo root morphology and rhizosphere microbe in different forests: in broadleaf forests with weaker soil P limitation, may be realized through increasing specific root length and specific surface area, whereas in coniferous forests with stronger soil, P limitation may be realized through combining more AMF. Our study highlights the importance of underground mechanisms about moso-bamboo expansion in different forest communities.

Leptin Promotes HTR-8/SVneo Cell Invasion via the Crosstalk between MTA1/WNT and PI3K/AKT Pathways.

The process of placental invasion is essential for a successful pregnancy. Leptin is involved in trophoblast invasiveness, and its dysregulation is connected with a series of diseases, including preeclampsia. However, the knowledge of the precise mechanisms in leptin-induced trophoblast invasiveness is still limited. According to the present research, transwell assay suggested that leptin is a dose- and time-dependent regulator in inducing HTR-8/SVneo cell invasion. Western blot analysis and immunofluorescence staining revealed that leptin-induced MMP9 expression is essential in the invasion process of HTR-8/SVneo cells. Mechanistically, we demonstrated that leptin activated ß-catenin via the crosstalk between the MTA1/WNT and PI3K/AKT pathways. Besides, we showed that downregulating the key molecules in the signaling pathways by siRNA can inhibit leptin-induced MMP9 expression and further suppress invasion of HTR-8/SVneo cells. In conclusion, our study revealed a new regulatory mechanism of leptin-induced HTR-8/SVneo cell invasiveness and will provide novel insights into the causes and potential therapeutic targets for diseases related to dysregulation of trophoblast invasion in the future.

JADE2 Is Essential for Hippocampal Synaptic Plasticity and Cognitive Functions in Mice.

BACKGROUND: Impairment of synaptic plasticity is closely correlated with a range of pathological conditions, such as cognitive deficits. However, how synaptic efficacy is regulated remains incompletely understood. Here, we report that the epigenetic factor JADE2 was indispensable for the maintenance of hippocampal synaptic plasticity and cognitive functions in mice. METHODS: We used the Morris water maze and the fear conditioning test to examine learning-related behaviors. In addition, Western blotting, viral-mediated JADE2 manipulations, RNA sequencing, and electrophysiological recordings were used to address our questions. RESULTS: JADE2 expression is increased upon enhanced neuronal activity in vitro and in vivo. Knockdown or genetic deletion of Jade2 in hippocampal CA1 results in impaired structural and functional synaptic plasticity, leading to memory impairment, whereas overexpression of JADE2 in CA1 neurons facilitates hippocampal-dependent learning and memory. Mechanistically, our data show that JADE2 modulates synaptic functions mainly by transcriptional activation of cytoskeletal regulator Rac1, and this activity is dependent on its interaction with histone acetyltransferase HBO1. Finally, we demonstrate that restoring RAC1 expression in Jade2 knockout mice could rescue the deficits in synaptic plasticity and learning-related behaviors. CONCLUSIONS: Our findings reveal that JADE2 plays a critical role in regulating synaptic plasticity and memory formation, suggesting that activity-dependent epigenetic regulation is an important molecular mechanism in controlling synaptic plasticity.

Hypoxia induced-disruption of lncRNA TUG1/PRC2 interaction impairs human trophoblast invasion through epigenetically activating Nodal/ALK7 signalling.

Inadequate trophoblastic invasion is considered as one of hallmarks of preeclampsia (PE), which is characterized by newly onset of hypertension (>140/90 mmHg) and proteinuria (>300 mg in a 24-h urine) after 20 weeks of gestation. Accumulating evidence has indicated that long noncoding RNAs are aberrantly expressed in PE, whereas detailed mechanisms are unknown. In the present study, we showed that lncRNA Taurine upregulated 1 (TUG1) were downregulated in preeclamptic placenta and in HTR8/SVneo cells under hypoxic conditions, together with reduced enhancer of zeste homolog2 (EZH2) and embryonic ectoderm development (EED) expression, major components of polycomb repressive complex 2 (PRC2), as well as activation of Nodal/ALK7 signalling pathway. Mechanistically, we found that TUG1 bound to PRC2 (EZH2/EED) in HTR8/SVneo cells and weakened TUG1/PRC2 interplay was correlated with upregulation of Nodal expression via decreasing H3K27me3 mark at the promoter region of Nodal gene under hypoxic conditions. And activation of Nodal signalling prohibited trophoblast invasion via reducing MMP2 levels. Overexpression of TUG1 or EZH2 significantly attenuated hypoxia-induced reduction of trophoblastic invasiveness via negative modulating Nodal/ALK7 signalling and rescuing expression of its downstream target MMP2. These investigations might provide some evidence for novel mechanisms responsible for inadequate trophoblastic invasion and might shed some light on identifying future therapeutic targets for PE.

Discovery of Novel and Potent N-Methyl-d-aspartate Receptor Positive Allosteric Modulators with Antidepressant-like Activity in Rodent Models.

N-Methyl-d-aspartate receptors (NMDARs) are glutamate-gated Na+ and Ca2+-permeable ion channels involved in excitatory synaptic transmission and synaptic plasticity. NMDAR hypofunction has long been implicated in the pathophysiology including major depressive disorders (MDDs). Herein, we report a series of furan-2-carboxamide analogues as novel NMDAR-positive allosteric modulators (PAMs). Through structure-based virtual screen and electrophysiological tests, FS2921 was identified as a novel NMDAR PAM with potential antidepressant effects. Further structure-activity relationship studies led to the discovery of novel analogues with increased potentiation. Compound 32h caused a significant increase in NMDAR excitability in vitro and impressive activity in the forced swimming test. Moreover, compound 32h showed no significant inhibition of hERG or cell viability and possessed a favorable PK/PD profile. Our study presented a series of novel NMDAR PAMs and provided potential opportunities for discovering of new antidepressants.

Leptin promotes endothelial dysfunction in chronic kidney disease by modulating the MTA1-mediated WNT/ß-catenin pathway.

Endothelial dysfunction (ED) has a high incidence in chronic kidney disease (CKD) and is identified as a precursor to cardiovascular events. Recent studies suggest that leptin may be the missing link between ED and CKD. The objective of this study was to investigate the mechanism by which leptin causes ED and the connection with leptin and indicators of ED in CKD patients. Analysis of leptin-treated human umbilical vein endothelial cells (HUVECs) showed increased expression of interleukin 6 (IL-6), endothelin 1 (ET-1) and human monocyte chemoattractant protein 1 (MCP-1), resulting in F-actin recombination and vinculin aggregation as well as endothelial cell migration. In vitro studies have shown that leptin leads to increased WNT1 expression and the accumulation of ß-catenin. Metastasis-associated protein 1 (MTA1), a critical upstream modifier of WNT1 signaling, increased the expression level in leptin-mediated regulation. In contrast, opposite results were observed when cells are transfected with MTA1 or WNT1 shRNA lentivirus vectors. Among 160 patients with CKD and 160 healthy subjects, patients with CKD had significantly higher serum leptin levels than those of the control group, which were positively correlated with increased levels of IL-6, ET-1 and MCP-1. However, these levels were negatively correlated with flow-mediated dilatation (FMD). Hence, these investigations provided novel information on the increased serum leptin levels in CKD patients leading to ED via the MTA1-WNT/ß-catenin pathway.

LPS Induces Preeclampsia-Like Phenotype in Rats and HTR8/SVneo Cells Dysfunction Through TLR4/p38 MAPK Pathway.

Accumulating evidence has shown that preeclampsia (PE) was associated with an aberrant maternal-fetal inflammatory response. In the present study, we first found that in human PE placentas levels of toll-like receptor 4 (TLR4), phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) and inflammatory cytokines IL-6 and MCP-1 were significantly upregulated. Next, we demonstrated a notable increase in systolic blood pressure (SBP) and proteinuria in lipopolysaccharide (LPS)-treated pregnant rats and concomitant high levels of TLR4 and p-p38 in these PE-like rat placentas, which led to aberrant overexpression of both IL-6 and MCP-1, as well as deficient trophoblast invasion and spiral artery (SA) remodeling, and these abnormalities were ameliorated by SB203580, a reported inhibitor of p38. In vitro we further confirmed that LPS triggered the activation of TLR4/p38 signaling pathway, which promoted trophoblast apoptosis and damaged trophoblastic invasion via downstream effectors IL-6 and MCP-1; these mutations were rectified by silencing this signaling pathway. These findings elaborated potential mechanisms that aberrant TLR4/p38 signaling might contribute to PE and LPS-induced PE-like symptom by damaging trophoblast invasion and SA remodeling via activating inflammatory cytokines including IL-6 and MCP-1.

Identification and characterization of benzo[d]oxazol-2(3H)-one derivatives as the first potent and selective small-molecule inhibitors of chromodomain protein CDYL.

Chemical probes of epigenetic 'readers' of histone post-translational modifications (PTMs) have become powerful tools for mechanistic and functional studies of their target proteins in physiology and pathology. However, only limited 'reader' probes have been developed, which restricted our understanding towards these macromolecules and their roles in cells or animals. Here, we reported a structure-guided approach to develop and characterize benzo [d]oxazol-2(3H)-one analogs as the first potent and selective small-molecule inhibitors of chromodomain Y-like (CDYL), a histone methyllysine reader protein. The binding conformation between the chromodomain of CDYL and the modified peptidomimetics was studied via molecular docking and dynamic simulations, facilitating subsequent virtual screening of tens of hits from Specs chemical library validated by SPR technique (KD values: from 271.1 µM to 5.4 µM). Further design and synthesis of 43 compounds helped to interpret the structure-activity relationship (SAR) that lead to the discovery of novel small-molecule inhibitors of CDYL. Compound D03 (KD: 0.5 µM) was discovered and showed excellent selectivity among other chromodomain proteins, including CDYL2 (>140 folds), CDY1 (no observed binding) and CBX7 (>32 folds). Moreover, we demonstrated that D03 engaged with endogenous CDYL in a dose-dependent manner, and perturbed the recruitment of CDYL onto chromatin, resulting in transcriptional derepression of its target genes. Finally, the results showed that D03 promoted the development and branching of neurodendrites by inhibiting CDYL in hippocampal and cortical cultured neurons. This study not only discovers the first selective small-molecule inhibitors of CDYL, but provids a new chemical tool to intervene the dynamic nature of bio-macromolecules involved in epigenetic mechanism.

Chromodomain Y-like Protein-Mediated Histone Crotonylation Regulates Stress-Induced Depressive Behaviors.

BACKGROUND: Major depressive disorder is a prevalent and life-threatening illness in modern society. The susceptibility to major depressive disorder is profoundly influenced by environmental factors, such as stressful lifestyle or traumatic events, which could impose maladaptive transcriptional program through epigenetic regulation. However, the underlying molecular mechanisms remain elusive. Here, we examined the role of histone crotonylation, a novel type of histone modification, and chromodomain Y-like protein (CDYL), a crotonyl-coenzyme A hydratase and histone methyllysine reader, in this process. METHODS: We used chronic social defeat stress and microdefeat stress to examine the depressive behaviors. In addition, we combined procedures that diagnose behavioral strategy in male mice with histone extraction, viral-mediated CDYL manipulations, RNA sequencing, chromatin immunoprecipitation, Western blot, and messenger RNA quantification. RESULTS: The results indicate that stress-susceptible rodents exhibit lower levels of histone crotonylation in the medial prefrontal cortex concurrent with selective upregulation of CDYL. Overexpression of CDYL in the prelimbic cortex, a subregion of the medial prefrontal cortex, increases microdefeat-induced social avoidance behaviors and anhedonia in mice. Conversely, knockdown of CDYL in the prelimbic cortex prevents chronic social defeat stress-induced depression-like behaviors. Mechanistically, we show that CDYL inhibits structural synaptic plasticity mainly by transcriptional repression of neuropeptide VGF nerve growth factor inducible, and this activity is dependent on its dual effect on histone crotonylation and H3K27 trimethylation on the VGF promoter. CONCLUSIONS: Our results demonstrate that CDYL-mediated histone crotonylation plays a critical role in regulating stress-induced depression, providing a potential therapeutic target for major depressive disorder.

ERG3 potassium channel-mediated suppression of neuronal intrinsic excitability and prevention of seizure generation in mice.

KEY POINTS: ERG3 channels have a high expression level in the central nervous system. Knockdown of ERG3 channels enhances neuronal intrinsic excitability (caused by decreased fast afterhyperpolarization, shortened delay time to the generation of an action potential and enhanced summation of somatic excitatory postsynaptic potentials) in hippocampal CA1 pyramidal neurons and dentate gyrus granule cells. The expression of ERG3 protein is reduced in human and mouse hippocampal epileptogenic foci. Knockdown of ERG3 channels in hippocampus enhanced seizure susceptibility, while mice treated with the ERG channel activator NS-1643 were less prone to epileptogenesis. The results provide strong evidence that ERG3 channels have a crucial role in the regulation of neuronal intrinsic excitability in hippocampal CA1 pyramidal neurons and dentate gyrus granule cells and are critically involved in the onset and development of epilepsy. ABSTRACT: The input-output relationship of neuronal networks depends heavily on the intrinsic properties of their neuronal elements. Profound changes in intrinsic properties have been observed in various physiological and pathological processes, such as learning, memory and epilepsy. However, the cellular and molecular mechanisms underlying acquired changes in intrinsic excitability are still not fully understood. Here, we demonstrate that ERG3 channels are critically involved in the regulation of intrinsic excitability in hippocampal CA1 pyramidal neurons and dentate gyrus granule cells. Knock-down of ERG3 channels significantly increases neuronal intrinsic excitability, which is mainly caused by decreased fast afterhyperpolarization, shortened delay time to the generation of an action potential and enhanced summation of somatic excitatory postsynaptic potentials. Interestingly, the expression level of ERG3 protein is significantly reduced in human and mouse brain tissues with temporal lobe epilepsy. Moreover, ERG3 channel knockdown in hippocampus significantly enhanced seizure susceptibility, while mice treated with the ERG channel activator NS-1643 were less prone to epileptogenesis. Taken together, our results suggest ERG3 channels play an important role in determining the excitability of hippocampal neurons and dysregulation of these channels may be involved in the generation of epilepsy. ERG3 channels may thus be a novel therapeutic target for the prevention of epilepsy.

Scale-Up Synthesis and Identification of GLYX-13, a NMDAR Glycine-Site Partial Agonist for the Treatment of Major Depressive Disorder.

GLYX-13, a NMDAR glycine-site partial agonist, was discovered as a promising antidepressant with rapidly acting effects but no ketamine-like side effects. However, the reported synthetic process route had deficiencies of low yield and the use of unfriendly reagents. Here, we report a scaled-up synthesis of GLYX-13 with an overall yield of 30% on the hectogram scale with a column chromatography-free strategy, where the coupling and deprotection reaction conditions were systematically optimized. Meanwhile, the absolute configuration of precursor compound of GLYX-13 was identified by X-ray single crystal diffraction. Finally, the activity of GLYX-13 was verified in the cortical neurons of mice through whole-cell voltage-clamp technique.

Podocyte-specific Rac1 deficiency ameliorates podocyte damage and proteinuria in STZ-induced diabetic nephropathy in mice.

Activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) has been implicated in diverse kidney diseases, yet its in vivo significance in diabetic nephropathy (DN) is largely unknown. In the present study, we demonstrated a podocyte-specific Rac1-deficient mouse strain and showed that specific inhibition of Rac1 was able to attenuate diabetic podocyte injury and proteinuria by the blockade of Rac1/PAK1/p38/ß-catenin signaling cascade, which reinstated the integrity of podocyte slit diaphragms (SD), rectified the effacement of foot processes (FPs), and prevented the dedifferentiation of podocytes. In vitro, we showed Rac1/PAK1 physically bound to ß-catenin and had a direct phosphorylation modification on its C-terminal Ser675, leading to less ubiquitylated ß-catenin, namely more stabilized ß-catenin, and its nuclear migration under high-glucose conditions; further, p38 activation might be responsible for ß-catenin nuclear accumulation via potentiating myocyte-specific enhancer factor 2C (MEF2c) phosphorylation. These findings provided evidence for a potential renoprotective and therapeutic strategy of cell-specific Rac1 deficiency for DN and other proteinuric diseases.

LncRNA MALAT1 is dysregulated in diabetic nephropathy and involved in high glucose-induced podocyte injury via its interplay with ß-catenin.

Metastasis associated lung adenocarcinoma transcript 1(MALAT1) is a long non-coding RNA, broadly expressed in mammalian tissues including kidney and up-regulated in a variety of cancer cells. To date, its functions in podocytes are largely unknown. ß-catenin is a key mediator in the canonical and non-canonical Wnt signalling pathway; its aberrant expression promotes podocyte malfunction and albuminuria, and contributes to kidney fibrosis. In this study, we found that MALAT1 levels were increased in kidney cortices from C57BL/6 mice with streptozocin (STZ)-induced diabetic nephropathy, and dynamically regulated in cultured mouse podocytes stimulated with high glucose, which showed a trend from rise to decline. The decline of MALAT1 levels was accompanied with ß-catenin translocation to the nuclei and enhanced expression of serine/arginine splicing factor 1 (SRSF1), a MALAT1 RNA-binding protein. Further we showed early interference with MALAT1 siRNA partially restored podocytes function and prohibited ß-catenin nuclear accumulation and SRSF1 overexpression. Intriguingly, we showed that ß-catenin was involved in MALAT1 transcription by binding to the promotor region of MALAT1; ß-catenin knock-down also decreased MALAT1 levels, suggesting a novel feedback regulation between MALAT1 and ß-catenin. Notably, ß-catenin deletion had limited effects on SRSF1 expression, demonstrating ß-catenin might serve as a downstream signal of SRSF1. These findings provided evidence for a pivotal role of MALAT1 in diabetic nephropathy and high glucose-induced podocyte damage.

FAK contributes to proteinuria in hypercholesterolaemic rats and modulates podocyte F-actin re-organization via activating p38 in response to ox-LDL.

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that regulates cell adhesion, proliferation and differentiation. In the present study, a rat model of high fat diet-induced hypercholesterolaemia was established to investigate the involvement of FAK in lipid disorder-related kidney diseases. We showed focal fusion of podocyte foot process that occurred at as early as 4 weeks in rats consuming high fat diet, preceding the onset of proteinuria when aberrant phosphorylation of FAK was found. These abnormalities were ameliorated by dietary intervention of TAE226, a reported inhibitor of FAK. FAK is also an adaptor protein initiating cascades of intracellular signals including c-Src, Rho GTPase and mitogen-activated protein kinase (MAPK). P38 MAPK belongs to the latter and is centrally involved in kidney diseases. Our cell culture data revealed oxidized low-density lipoprotein (ox-LDL) triggered hyper-phosphorylation of FAK and p38, ectopic expression of cellular markers (manifested as decreased WT1, podocin and NEPH1, and increased vimentin and mmp9), and re-arrangement of F-actin filaments with enhanced cell motility; these mutations were significantly rectified by FAK shRNA. Notably, pre-treatment of p38 inhibitor did not alter FAK activation, albeit its deletion of p38 hyper-activity and attenuation of cellular abnormalities, demonstrating that p38 acted as a downstream effector of FAK signalling and ox-LDL damaged podocytes in a FAK/p38-dependent manner. This was further identified by animal data that p38 activation was also abrogated by TAE226 treatment in hypercholesterolaemic rats, suggesting that FAK/p38 axis might also be involved in in vivo events. These findings provided a potential early mechanism of hypercholesterolaemia-related podocyte damage and proteinuria.

Rac1/ß-Catenin Signalling Pathway Contributes to Trophoblast Cell Invasion by Targeting Snail and MMP9.

BACKGROUND/AIMS: Preeclampsia is an idiopathic and serious complication during gestation in which placental trophoblast cells differentiate into several functional subtypes, including highly invasive extravillous trophoblasts (EVTs). Although the cause and pathogenesis of preeclampsia have remained unclear, numerous studies have suggested that the inadequacy of EVT invasion leads to imperfect uterine spiral artery remodelling, which plays a crucial role in the development of preeclampsia. Rac1, or Ras-related C3 botulinum toxin substrate 1, was found to be a key regulator of the migration, invasion uand apoptosis of various tumour cells. Because EVTs share similar invasive and migratory biological behaviours with malignant cells, this study aimed to determine whether the Rac1 signalling pathway affects trophoblast invasion and is thus involved in the pathogenesis of preeclampsia. METHODS: We measured the activity of Rac1 and its downstream targets, ß-catenin, Snail and MMP9 in placental tissues from patients experiencing a normal pregnancy and those with preeclampsia. Furthermore, we treated HTR-8/SVneo cells with a shRNA Rac1 vector and the ß-catenin inhibitor IWP-2 and explored Rac1 signalling pathway activation as well as the effects of Snail and ß-catenin on trophoblast invasion. RESULTS: In placental samples from patients experiencing a normal pregnancy and those with preeclampsia, active Rac1 levels and MMP9 protein and mRNA levels were significantly decreased in term pregnancy samples compared to early pregnancy samples. Lower levels were found in preeclampsia samples than in normal term pregnancy samples, and these levels significantly declined in severe preeclampsia samples compared with mild preeclampsia samples. Further analyses demonstrated that both Rac1 shRNA and the ß-catenin inhibitor significantly suppressed MMP9 and Snail activation in trophoblasts, thus impairing trophoblast invasion. Notably, silencing Rac1 down-regulated the expression of ß-catenin in HTR-8/SVneo cells, demonstrating that ß-catenin is a downstream effector of Rac1 in trophoblast invasion. CONCLUSION: Our data suggest that Rac1-mediated activation of ß-catenin might regulate Snail and MMP9 expression subsequently promoting trophoblast invasion in pregnancy.

Fyn Mediates High Glucose-Induced Actin Cytoskeleton Reorganization of Podocytes via Promoting ROCK Activation In Vitro.

Fyn, a member of the Src family of tyrosine kinases, is a key regulator in cytoskeletal remodeling in a variety of cell types. Recent studies have demonstrated that Fyn is responsible for nephrin tyrosine phosphorylation, which will result in polymerization of actin filaments and podocyte damage. Thus detailed involvement of Fyn in podocytes is to be elucidated. In this study, we investigated the potential role of Fyn/ROCK signaling and its interactions with paxillin. Our results presented that high glucose led to filamentous actin (F-actin) rearrangement in podocytes, accompanied by paxillin phosphorylation and increased cell motility, during which Fyn and ROCK were markedly activated. Gene knockdown of Fyn by siRNA showed a reversal effect on high glucose-induced podocyte damage and ROCK activation; however, inhibition of ROCK had no significant effects on Fyn phosphorylation. These observations demonstrate that in vitro Fyn mediates high glucose-induced actin cytoskeleton remodeling of podocytes via promoting ROCK activation and paxillin phosphorylation.