Motility Signaling Research Articles

Effects of microencapsulated phenethyl isothiocyanate on gastrointestinal cancer cells and pathogenic bacteria

Gastrointestinal cancers remain a global health burden, demanding more effective prevention and treatments. Phenethyl isothiocyanate (PEITC), a compound derived from cruciferous vegetables, stands out as a promising nutraceutical agent due to its chemopreventive and therapeutic properties. However, its therapeutic translation remains limited mainly due to its poor water solubility and rapid metabolism. Herein, we encapsulated PEITC into biocompatible chitosan-based microparticles with an extra virgin olive oil core to improve its bioavailability and stability. Pure PEITC's biocompatibility and microencapsulated PEITC's stability and antibacterial activity were evaluated. The antibacterial activity analysis showed microencapsulated PEITC as a promising antibacterial agent against gastrointestinal pathogenic bacteria (two Gram-positive and two Gram-negative). The impact of both pure and microencapsulated PEITC was assessed on gastrointestinal cancer cells (MKN45 gastric cancer and SW48 colon cancer cell lines). PEITC exhibited threshold or hormetic dose-dependent toxicity in colon fibroblasts and decreased gastric cancer cells' migration capacity, enhanced upon encapsulation into microparticles. In addition, microencapsulated PEITC induced downregulation of phosphorylated AKT, FAK, and ERK1/2 proteins, disrupting motility signaling pathways and tubulin expression. These findings suggest that the delivery of PEITC via chitosan-based microparticles holds promise as a nutraceutical delivery strategy against gastrointestinal disorders that predispose to cancer.

Shifts in keratin isoform expression activate motility signals during wound healing

Keratin intermediate filaments confer structural stability to epithelial tissues, but the reason this simple mechanical function requires a protein family with 54 isoforms is not understood. During skin wound healing, a shift in keratin isoform expression alters the composition of keratin filaments. If and how this change modulates cellular functions that support epidermal remodeling remains unclear. We report an unexpected effect of keratin isoform variation on kinase signal transduction. Increased expression of wound-associated keratin 6A, but not of steady-state keratin 5, potentiated keratinocyte migration and wound closure without compromising mechanical stability by activating myosin motors to increase contractile force generation. These results substantially expand the functional repertoire of intermediate filaments from their canonical role as mechanical scaffolds to include roles as isoform-tuned signaling scaffolds that organize signal transduction cascades in space and time to influence epithelial cell state.

Ion Signaling in Cell Motility and Development in Dictyostelium discoideum.

Cell-to-cell communication is fundamental to the organization and functionality of multicellular organisms. Intercellular signals orchestrate a variety of cellular responses, including gene expression and protein function changes, and contribute to the integrated functions of individual tissues. Dictyostelium discoideum is a model organism for cell-to-cell interactions mediated by chemical signals and multicellular formation mechanisms. Upon starvation, D. discoideum cells exhibit coordinated cell aggregation via cyclic adenosine 3',5'-monophosphate (cAMP) gradients and chemotaxis, which facilitates the unicellular-to-multicellular transition. During this process, the calcium signaling synchronizes with the cAMP signaling. The resulting multicellular body exhibits organized collective migration and ultimately forms a fruiting body. Various signaling molecules, such as ion signals, regulate the spatiotemporal differentiation patterns within multicellular bodies. Understanding cell-to-cell and ion signaling in Dictyostelium provides insight into general multicellular formation and differentiation processes. Exploring cell-to-cell and ion signaling enhances our understanding of the fundamental biological processes related to cell communication, coordination, and differentiation, with wide-ranging implications for developmental biology, evolutionary biology, biomedical research, and synthetic biology. In this review, I discuss the role of ion signaling in cell motility and development in D. discoideum.

MDB-37. RAC1 INHIBITION FOR THE TREATMENT OF MEDULLOBLASTOMA

Abstract Despite considerable advances in identifying genetic and epigenetic abnormalities in medulloblastoma, many patients succumb to the disease. In addition, patients who respond to traditional therapy suffer from cognitive and intellectual deficits. Therefore, there is a dire need to identify novel therapies for treating medulloblastoma. Rho family GTPases are targets in multiple cancers including medulloblastoma. Rho, Rac1 and Cdc42 are essential mediators of cell-cell adhesion and cellular motility signaling, which are dysregulated in medulloblastoma. These GTPases are involved in the regulation of the cytoskeleton, cell migration, cellular proliferation, and developmental signaling. The small GTPase Rac1 has recently been reported as a possible therapeutic target in SHH-medulloblastoma due to its regulation of Hedgehog signaling via the GLI1 and GLI2 transcription factors. We have recently reported that GLI1/GLI2 are in a novel complex that contains the epigenetic regulators UHRF1 and DNMT1. Therefore, Rac1 may play a previously unappreciated role in epigenetic regulation of medulloblastoma by controlling the GLI1/GLI2/UHRF1/DNMT1 complex. To test this, we have utilized a novel brain penetrant Rac1 inhibitor termed GYS32661. GYS32661 treatment of medulloblastoma cells inhibits the GLI1-UHRF1 interaction. Importantly this inhibition of the interaction occurs rapidly suggesting that it is disrupting an early event in SHH signaling. GYS32661 also inhibits actin polymerization suggesting that utilizing this compound may provide a dual mechanism of inhibiting Shh signaling and cellular migration concurrently. GYS32661 is not toxic in animal models and is approximately 50% brain penetrant and therefore is an important clinical candidate for the treatment of medulloblastoma. Our studies identify an essential link between Rac1, Shh signaling, epigenetics, and cellular migration in medulloblastoma.

Abstract 6493: CRISPR-directed prime editing sensitizes EGFR inhibitor by correcting oncogenic KRAS mutants In NSCLC

Abstract EGFR inhibitor has been demonstrated with huge therapeutic potential in advanced non-small cell lung cancer (NSCLC), while the patients carrying KRAS mutation had poor response to EGFR inhibitor. KRAS mutation is the most frequent molecular alteration found in advanced NSCLC, which is associated with a poor prognosis without available targeted therapy. Previous research showed that CRISPR-directed prime editor could induce unconstrained correction for KRAS mutants (KRASG12D, KRASG12C, KRASG12V). Accordingly, it would be a promising strategy to combine PE-based genetic therapy with EGFR inhibitor for enhanced therapy in NSCLC. Here,we selected several PE variants with reported highly efficient reverse transcriptase, among which eEc48-based PE6a showed the highest editing efficiency toward KRAS mutation. The lentivirus-mediated PE6a editing inhibited cell proliferation and invasion in vitro and in vivo. After that, EGFR inhibitor combined with PE6a was employed in patient-derived organoid (PDO) model carrying diverse KRAS mutants. We found that PE6a editing significantly inhibited PDO proliferation compared to EGFR inhibitor alone and control. Correspondingly, the transcriptome sequencing also showed that the cell apoptosis, cell growth and cell motility signaling were inhibited upon PE6a editing. Our findings demonstrated that the PE-directed genetic therapy could sensitize EGFR inhibitor in NSCLC, providing an alternative choice for NSCLC patients carrying KRAS mutant. Citation Format: Qingxiao Fang, Chao Yang, Chongbiao Huang, Hongwei Wang, Jun Yu, Jihui Hao. CRISPR-directed prime editing sensitizes EGFR inhibitor by correcting oncogenic KRAS mutants In NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6493.

IL18 Receptor Signaling Inhibits Intratumoral CD8+ T-Cell Migration in a Murine Pancreatic Cancer Model.

In pancreatic ductal adenocarcinoma (PDAC), the infiltration of CD8+ cytotoxic T cells (CTLs) is an important factor in determining prognosis. The migration pattern and interaction behavior of intratumoral CTLs are pivotal to tumor rejection. NLRP3-dependent proinflammatory cytokines IL-1β and IL-18 play a prominent role for CTL induction and differentiation. Here, we investigate the effects of T-cellular IL-1R and IL-18R signaling for intratumoral T-cell motility. Murine adenocarcinoma cell line Panc02 was stably transfected with ovalbumin (OVA) and fluorophore H2B-Cerulean to generate PancOVA H2B-Cerulean tumor cells. Dorsal skinfold chambers (DSFC) were installed on wild-type mice, and PancOVA H2B-Cerulean tumor cells were implanted into the chambers. PancOVA spheroids were formed using the Corning® Matrigel®-based 3D cell culture technique. CTLs were generated from OT-1 mice, Il1r-/- OT-1 mice, or Il18r-/- OT-1 mice and were marked with fluorophores. This was followed by the adoptive transfer of CTLs into tumor-bearing mice or the application into tumor spheroids. After visualization with multiphoton microscopy (MPM), Imaris software was used to perform T-cell tracking. Imaris analysis indicates a significantly higher accumulation of Il18r-/- CTLs in PancOVA tumors and a significant reduction in tumor volume compared to wild-type CTLs. Il18r-/- CTLs covered a longer distance (track displacement length) in comparison to wild-type (WT) CTLs, and had a higher average speed (mean track speed). The analysis of instantaneous velocity suggests a higher percentage of arrested tracks (arrests: <4 μm/min) for Il18r-/- CTLs. Our data indicate the contribution of IL-18R signaling to T-cell effector strength, warranting further investigation on phenomena such as intratumoral T-cell exhaustion.

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.

CapG promoted nasopharyngeal carcinoma cell motility involving Rho motility pathway independent of ROCK

BackgroundGelsolin-like capping actin protein (CapG) modulates actin dynamics and actin-based motility with a debatable role in tumorigenic progression. The motility-associated functions and potential molecular mechanisms of CapG in nasopharyngeal carcinoma (NPC) remain unclear.MethodsCapG expression was detected by immunohistochemistry in a cohort of NPC tissue specimens and by Western blotting assay in a variety of NPC cell lines. Loss of function and gain of function of CapG in scratch wound-healing and transwell assays were performed. Inactivation of Rac1 and ROCK with the specific small molecular inhibitors was applied to evaluate CapG’s role in NPC cell motility. GTP-bound Rac1 and phosphorylated-myosin light chain 2 (p-MLC2) were measured in the ectopic CapG overexpressing cells. Finally, CapG-related gene set enrichment analysis was conducted to figure out the significant CapG-associated pathways in NPC.ResultsCapG disclosed increased level in the poorly differentiated NPC tissues and highly metastatic cells. Knockdown of CapG reduced NPC cell migration and invasion in vitro, while ectopic CapG overexpression showed the opposite effect. Ectopic overexpression of CapG compensated for the cell motility loss caused by simultaneous inactivation of ROCK and Rac1 or inactivation of ROCK alone. GTP-bound Rac1 weakened, and p-MLC2 increased in the CapG overexpressing cells. Bioinformatics analysis validated a positive correlation of CapG with Rho motility signaling, while Rac1 motility pathway showed no significant relationship.ConclusionsThe present findings highlight the contribution of CapG to NPC cell motility independent of ROCK and Rac1. CapG promotes NPC cell motility at least partly through MLC2 phosphorylation and contradicts with Rac1 activation.

The impact of PrsA over-expression on the Bacillus subtilis transcriptome during fed-batch fermentation of alpha-amylase production.

The production of the alpha-amylase (AMY) enzyme in Bacillus subtilis at a high rate leads to the accumulation of unfolded AMY, which causes secretion stress. The over-expression of the PrsA chaperone aids enzyme folding and reduces stress. To identify affected pathways and potential mechanisms involved in the reduced growth, we analyzed the transcriptomic differences during fed-batch fermentation between a PrsA over-expressing strain and control in a time-series RNA-seq experiment. We observe transcription in 542 unannotated regions, of which 234 had significant changes in expression levels between the samples. Moreover, 1,791 protein-coding sequences, 80 non-coding genes, and 20 riboswitches overlapping UTR regions of coding genes had significant changes in expression. We identified putatively regulated biological processes via gene-set over-representation analysis of the differentially expressed genes; overall, the analysis suggests that the PrsA over-expression affects ATP biosynthesis activity, amino acid metabolism, and cell wall stability. The investigation of the protein interaction network points to a potential impact on cell motility signaling. We discuss the impact of these highlighted mechanisms for reducing secretion stress or detrimental aspects of PrsA over-expression during AMY production.

TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia.

The membrane protein TREM2 (Triggering Receptor Expressed on Myeloid cells 2) regulates key microglial functions including phagocytosis and chemotaxis. Loss-of-function variants of TREM2 are associated with increased risk of Alzheimer's disease (AD). Because abnormalities in Ca2+ signaling have been observed in several AD models, we investigated TREM2 regulation of Ca2+ signaling in human induced pluripotent stem cell-derived microglia (iPSC-microglia) with genetic deletion of TREM2. We found that iPSC-microglia lacking TREM2 (TREM2 KO) show exaggerated Ca2+ signals in response to purinergic agonists, such as ADP, that shape microglial injury responses. This ADP hypersensitivity, driven by increased expression of P2Y12 and P2Y13 receptors, results in greater release of Ca2+ from the endoplasmic reticulum stores, which triggers sustained Ca2+ influx through Orai channels and alters cell motility in TREM2 KO microglia. Using iPSC-microglia expressing the genetically encoded Ca2+ probe, Salsa6f, we found that cytosolic Ca2+ tunes motility to a greater extent in TREM2 KO microglia. Despite showing greater overall displacement, TREM2 KO microglia exhibit reduced directional chemotaxis along ADP gradients. Accordingly, the chemotactic defect in TREM2 KO microglia was rescued by reducing cytosolic Ca2+ using a P2Y12 receptor antagonist. Our results show that loss of TREM2 confers a defect in microglial Ca2+ response to purinergic signals, suggesting a window of Ca2+ signaling for optimal microglial motility.

Histone Methylation Regulates Gene Expression in the Round Spermatids to Set the RNA Payloads of Sperm.

Gene expression during spermatogenesis undergoes significant changes due to a demanding sequence of mitosis, meiosis, and differentiation. We investigated the contribution of H3 histone modifications to gene regulation in the round spermatids. Round spermatids were purified from rat testes using centrifugal elutriation and Percoll density-gradient centrifugation. After enzymatic chromatin shearing, immuno-precipitation using antibodies against histone marks H3k4me3 and H3K9me3 was undertaken. The immunoprecipitated DNA fragments were subjected to massive parallel sequencing. Gene expression in round spermatids and sperm was analyzed by transcriptome sequencing using next-generation sequencing methods. ChIP-seq analysis showed significant peak enrichment in H3K4me3 marks in active chromatin regions and H3K9me3 peak enrichment in repressive regions. We found 53 genes which showed overlapping peak enrichment in both H3K4me3 and H3K9me3 marks. Some of the top H3K4me3-enriched genes were involved in sperm tail formation (Odf1, Odf3, Odf4, Oaz3, Ccdc42, Ccdc63,andCcdc181), chromatin condensation (Dync1h1, Dynll1,andKdm3a), and sperm functions such as acrosome reaction (AcrbpandFabp9), energy generation (Gapdhs), and signaling for motility (Tssk1b, Tssk2,andTssk4). Transcriptome sequencing in round spermatids found 64% transcripts of the H3K4me3-enriched genes at high levels and of about 25% of H3K9me3-enriched genes at very low levels. Transcriptome sequencing in sperm found that more than 99% of the ChIP-seq corresponding transcripts were also present in sperm. H3K4me3 enrichment in the round spermatids correlates significantly with gene expression and H3K9me3 correlates with gene silencing that contribute to sperm differentiation and setting the RNA payloads of sperm.

Abstract PO-137: Comparative transcriptomic analysis of prostate cancer from African American and Caucasian American men by Gleason score and race

Abstract African American (AA) men exhibit 2-3 times higher mortality from prostate cancer compared with Caucasian American (CA) men. Factors contributing to the disparity include societal-, neighborhood- and institutional-level determinants of health. In addition, a number of studies have reported individual-level ancestry-related biological differences, including in mutations, copy number variation, aggregate gene expression and response to treatment between AA and CA prostate cancer patients. Previously, by comparing the transcriptome between 20 AA and 15 CA prostate cancer patients, we identified a large number of race-related Alternative RNA Splicing (ARS) events. Among these, we further demonstrated that an exon skipping event involving exon 20 within PIK3CD increased tumor growth rate, metastatic potential and drug resistance in prostate cancer. To expand our previous findings, we collected non-neoplastic and tumor-paired tissue from 37 AA and 40 CA prostate cancer patients with different Gleason score categories: 14 high grade (4 AA and 10 CA), 22 low grade (10 AA and 12 CA), and 41 intermediate grade (23 AA and 18 CA). DNA and RNA were isolated for ancestral genotyping and RNA seq analysis, respectively. To achieve RNA sequencing depth adequate for ARS analysis, we performed RNA seq of 150 bp paired-end and an average of 5 × 106 reads per sample. The read alignment was done using Star 2 TwoPass pipeline. The rMATS pipeline was used for ARS annotation and quantification. We identified 105,403 ARS events, including 60,657 exon skipping, 17,439 alternative acceptor, 12,737 alternative donor, 9,555 retained intron and 5,015 mutually exclusive exon. Using the Wilcoxon rank-sum test, we compared the Percent Spliced In (PSI) between AA and CA of the same Gleason score category and identified ARS events exhibiting ΔPSI &amp;gt; 15% and p-value &amp;lt; 0.05. Specifically, we identified 536 race-related ARS events in high grade, 492 race-related ARS events in low grade, and 447 race-related ARS events in intermediate grade. Gene Ontology analysis demonstrated that the genes undergoing race-related ARS events function in cellular processes relevant to cancer biology, including metabolic processes in low grade, NF-kappaB signaling in intermediate grade and cell motility in high grade. Specific examples of these genes include ERG and PARP2 in high grade, KLK2 and DNMT1 in intermediate grade, and AURKA and SEMA3A in low grade. These findings support a potential role for the ARS process in diversifying gene expression, potentially contributing to prostate cancer aggressiveness in AA patients. The race-related ARS events identified in our work represent potential biomarkers and/or therapeutic targets for precision oncology in the context of prostate cancer. Further analysis of the function of prioritized race-related ARS events and their association with local ancestry is ongoing. Funding: DoD Prostate Cancer Research Program Health Disparity Research Award. NIH Basic Research in Cancer Health Disparities R01 Award. Prostate Cancer Foundation Movember Challenge Award. Citation Format: Muthana Al Abo, Wen-Chi Foo, Daniel J. George, Steven R. Patierno, Jennifer A. Freedman. Comparative transcriptomic analysis of prostate cancer from African American and Caucasian American men by Gleason score and race [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-137.

Gα13 Mediates Transendothelial Migration of Neutrophils by Promoting Integrin-Dependent Motility without Affecting Directionality.

Neutrophil migration requires β2 integrins and chemoattractant receptor signaling for motility and directionality. G protein subunit Gα13 can facilitate cell migration by mediating RhoA activation induced by G protein-coupled receptors. However, the possible role of Gα13-integrin interaction in migration is unclear. In this study, we show that Gα13 -/- neutrophils are deficient in transendothelial migration and migration on β2 integrin ligand ICAM-1. However, unlike G protein-coupled receptors and integrin inside-out signaling pathways, Gα13 is important in migration velocity and neutrophil spreading but not in directionality nor cell adhesion. Importantly, neutrophil recruitment in vivo was also inhibited in Gα13 -/- mice, suggesting the importance of Gα13 in transendothelial migration of neutrophils in vitro and in vivo. Furthermore, a synthetic peptide (MB2mP6) derived from the Gα13 binding site of β2 inhibited Gα13-β2 interaction and Gα13-mediated transient RhoA inhibition in neutrophils, suggesting that this peptide inhibited integrin outside-in signaling. MB2mP6 inhibited migration of control neutrophils through endothelial cell monolayers or ICAM-1-coated filters, but was without further effect on Gα13 -/- neutrophils. It also inhibited integrin-dependent neutrophil migration velocity without affecting directionality. In vivo, MB2mP6 markedly inhibited neutrophil infiltration into the cardiac tissues induced by ischemia/reperfusion injury. Thus, Gα13-dependent outside-in signaling enables integrin-dependent neutrophil motility without affecting directionality and may be a new therapeutic target for inhibiting neutrophil trafficking but not adhesion.

A DNA Molecular Robot that Autonomously Walks on the Cell Membrane to Drive Cell Motility

AbstractSynthetic molecular robots can execute sophisticated molecular tasks at nanometer resolution. However, a molecular robot capable of controlling cellular behavior remains unexplored. Herein, we report a self‐propelled DNA robot operating on the cell membrane to control the migration of a cell. Driven by DNAzyme catalytic activity, the DNA robot could autonomously and stepwise move on the membrane‐floating cell‐surface receptors in a stochastic manner and simultaneously trigger the receptor‐dimerization to activate downstream signaling for cell motility. The cell membrane‐associated continuous motion and operation of a DNA robot allowed for the ultrasensitive regulation of MET/AKT signaling and cytoskeleton remodeling to enhance cell migration. Finally, we designed distinct conditional DNA robots to orthogonally manipulate the cell migration in a coculture of mixed cell populations. We have developed a novel strategy to engineer a cell‐driving molecular robot, representing a promising avenue for precise cell manipulation with nanoscale resolution.

A DNA Molecular Robot that Autonomously Walks on the Cell Membrane to Drive Cell Motility.

Synthetic molecular robots can execute sophisticated molecular tasks at nanometer resolution. However, a molecular robot capable of controlling cellular behavior remains unexplored. Herein, we report a self-propelled DNA robot operating on the cell membrane to control the migration of a cell. Driven by DNAzyme catalytic activity, the DNA robot could autonomously and stepwise move on the membrane-floating cell-surface receptors in a stochastic manner and simultaneously trigger the receptor-dimerization to activate downstream signaling for cell motility. The cell membrane-associated continuous motion and operation of a DNA robot allowed for the ultrasensitive regulation of MET/AKT signaling and cytoskeleton remodeling to enhance cell migration. Finally, we designed distinct conditional DNA robots to orthogonally manipulate the cell migration in a coculture of mixed cell populations. We have developed a novel strategy to engineer a cell-driving molecular robot, representing a promising avenue for precise cell manipulation with nanoscale resolution.

FARP1, ARHGEF39, and TIAM2 are essential receptor tyrosine kinase effectors for Rac1-dependent cell motility in human lung adenocarcinoma.

Despite the undisputable role of the small GTPase Rac1 in the regulation of actin cytoskeleton reorganization, the Rac guanine-nucleotide exchange factors (Rac-GEFs) involved in Rac1-mediated motility and invasion in human lung adenocarcinoma cells remain largely unknown. Here, we identify FARP1, ARHGEF39, and TIAM2 as essential Rac-GEFs responsible for Rac1-mediated lung cancer cell migration upon EGFR and c-Met activation. Noteworthily, these Rac-GEFs operate in a non-redundant manner by controlling distinctive aspects of ruffle dynamics formation. Mechanistic analysis reveals a leading role of the AXL-Gab1-PI3K axis in conferring pro-motility traits downstream of EGFR. Along with the positive association between the overexpression of Rac-GEFs and poor lung adenocarcinoma patient survival, we show that FARP1 and ARHGEF39 are upregulated in EpCam+ cells sorted from primary human lung adenocarcinomas. Overall, our study reveals fundamental insights into the complex intricacies underlying Rac-GEF-mediated cancer cell motility signaling, hence underscoring promising targets for metastatic lung cancer therapy.

A Review on Environmental Contaminants-Related Fertility Threat in Male Fishes: Effects and Possible Mechanisms of Action Learned from Wildlife and Laboratory Studies.

Simple SummaryPublic concern regarding environmental contaminants (ECs)-related reproductive disorders has increased due to increasing global rates of infertility. All kinds of ECs are on rise rapidly in developing and industrializing low- and middle-income countries. The aquatic environments throughout the world are repositories for enormous amounts of ECs. As the biology of the reproductive system is highly conserved in vertebrates, wildlife or laboratory studies on fish provide significant information to establish a detailed risk assessment, and to identify novel or more sensitive endpoints for ECs-related reproductive disorders. The adverse effects of ECs on endocrine regulation of reproduction in male fishes have been extensively studied and reviewed; however, our knowledge on the effects and mechanisms of action of ECs on determinants of male fertility is limited. The present study is a state-of-the-art comprehensive review on the ECs-related fertility threat in male fishes with emphasis on the ECs effects on sperm production, morphology, genome, and motility kinetics. After a brief introduction to reproductive biology, fertility indicators, and determinants of fertility in male fishes, wildlife evidences for reproductive disorders were reviewed in fishes from the polluted aquatic environment. The laboratory studies show that ECs detected in aquatic environment are capable of causing fertility threat at environmentally relevant concentrations associated with a decrease in fertility determinant(s). This study suggests an urgent need to better elucidate mechanisms through which ECs affect sperm functions to cause fertility threat.Increasing global rates of diminished fertility in males has been suggested to be associated with exposure to environmental contaminants (ECs). The aquatic environments are the final repository of ECs. As the reproductive system is conserved in vertebrates, studies on the effects of ECs on fertility endpoints in fishes provide us with valuable information to establish biomarkers in risk assessment of ECs, and to understand the ECs-related fertility threat. The aim of the present review was to evaluate associations between ECs and fertility determinants to better understand ECs-related male fertility threat in male fishes. Wildlife studies show that the reproductive system has been affected in fishes sampled from the polluted aquatic environment. The laboratory studies show the potency of ECs including natural and synthetic hormones, alkylphenols, bisphenols, plasticizers, pesticides, pharmaceutical, alkylating, and organotin agents to affect fertility determinants, resulting in diminished fertility at environmentally relevant concentrations. Both wildlife and laboratory studies reveal that ECs adverse effects on male fertility are associated with a decrease in sperm production, damage to sperm morphology, alternations in sperm genome, and decrease in sperm motility kinetics. The efficiency of ECs to affect sperm quality and male fertility highly depends on the concentration of the contaminants and the duration of exposure. Our review highlights that the number of contaminants examined over fertility tests are much lower than the number of contaminants detected in our environment. The ECs effects on fertility are largely unknown when fishes are exposed to the contaminants at early developmental stages. The review suggests the urgent need to examine ECs effects on male fertility when a fish is exposed at different developmental stages in a single or combination protocol. The ECs effects on the sperm genome are largely unknown to understand ECs-related inheritance of reproductive disorders transmitted to the progeny. To elucidate modes of action of ECs on sperm motility, it is needed to study functional morphology of the motility apparatus and to investigate ECs-disrupted motility signaling.

Suppression of α-catenin and adherens junctions enhances epithelial cell proliferation and motility via TACE-mediated TGF-α autocrine/paracrine signaling

Sustained cell migration is essential for wound healing and cancer metastasis. The epidermal growth factor receptor (EGFR) signaling cascade is known to drive cell migration and proliferation. While the signal transduction downstream of EGFR has been extensively investigated, our knowledge of the initiation and maintenance of EGFR signaling during cell migration remains limited. The metalloprotease TACE (tumor necrosis factor alpha converting enzyme) is responsible for producing active EGFR family ligands in the via ligand shedding. Sustained TACE activity may perpetuate EGFR signaling and reduce a cell’s reliance on exogenous growth factors. Using a cultured keratinocyte model system, we show that depletion of α-catenin perturbs adherens junctions, enhances cell proliferation and motility, and decreases dependence on exogenous growth factors. We show that the underlying mechanism for these observed phenotypical changes depends on enhanced autocrine/paracrine release of the EGFR ligand transforming growth factor alpha in a TACE-dependent manner. We demonstrate that proliferating keratinocyte epithelial cell clusters display waves of oscillatory extracellular signal–regulated kinase (ERK) activity, which can be eliminated by TACE knockout, suggesting that these waves of oscillatory ERK activity depend on autocrine/paracrine signals produced by TACE. These results provide new insights into the regulatory role of adherens junctions in initiating and maintaining autocrine/paracrine signaling with relevance to wound healing and cellular transformation.

Effect of plant extracts onPseudomonas syringaepv.actinidiaegene expression, motility and virulence

Pseudomonas syringae pv. actinidiae (Psa), the causative agent of bacterial canker of kiwi, was firstly isolated in 1992, but the second outbreak dating back to 2008 is responsible for the current pandemics that has led to major economic losses worldwide. Despite the high economic impact of Pseudomonas syringae pv. actinidiae (Psa) on kiwifruit industry, and the intense research efforts, the molecular mechanisms involved in pathogenesis are still not fully characterized. In particular, the precise mechanism of recognition/communication between Psa and its host plant has not been elucidated yet. Our findings show that plant crude extracts or xylem juices of Actinidia deliciosa and A. chinensis were able to increase Psa virulence in comparison with artificial growing media such as Luria Broth or Nutrient agar. The study of gene expression in response to plant-based or artificial growing medium was performed by qPCR and showed a differential expression of many virulence-related genes. In addition, genes related to biofilm formation, motility and inter-cell signaling (e.g. quorum sensing in the broad sense) were differentially expressed according to the type of growing media. Interestingly, the expression of many of the studied genes responded differentially both to plant extracts and Psa cell density. The molecular data were corroborated by phenological studies testing the effect of plant-based medium on biofilm formation, motility and symptom development in micro-cuttings of Actinidia spp. The present study demonstrates the existence of a not yet identified, host-specific group of molecules able to activate the pathogenesis machinery of Psa. Moreover, these molecules could lead to different physiological and molecular behavior depending on Psa population density. Finally, it was also demonstrated that these molecules were differentially present in leaves and xylem sap collected by different Actinidia species.

Sperm motility in fishes: (III) diversity of regulatory signals from membrane to the axoneme

Fish spermatozoa acquire potential for motility in the sperm duct where they are immotile. Osmolality of the seminal plasma is a key factor to maintain spermatozoa in the quiescent state in either freshwater or marine fishes. However, potassium (K+) ions prevent spermatozoa motility in salmonid and sturgeon fishes, while CO2 inhibits spermatozoa motility in flatfishes. Once, spermatozoa are released at spawning, their motility is initiated in hypo-osmotic and hyper-osmotic environments in freshwater and marine fishes, respectively. Some substances produced by the testes (a progestin), or released from oocytes (peptides) induce spermatozoa hypermotility in some marine fishes including the Atlantic croaker and Pacific herrings, respectively. Duration of spermatozoa motility is short, lasting for a few seconds to few minutes in most fishes due to rapid depletion of energy required for the beating of the motility apparatus called axoneme. In the osmotic-activated spermatozoa, K+ and water effluxes occur in freshwater and marine fishes, respectively, which trigger spermatozoa motility signaling. In general, initiation of axonemal beating is associated with an increase in intracellular calcium (Ca2+) ions in spermatozoa of both freshwater and marine fishes and a post- or pre-increase in intracellular pH, while cyclic adenosine monophosphate (cAMP) remains unchanged. However, axonemal beating is cAMP-dependent in demembranated spermatozoa of salmonid and sturgeon fishes. Calcium from extracellular environment or intracellular stores supply required Ca2+ concentration for axonemal beating. Several axonemal proteins have been so far identified in fishes that are activated by Ca2+ and cAMP, directly or mediated by protein kinase C and protein kinase A, respectively. The present study reviews differences and similarities in complex regulatory signals controlling spermatozoa motility initiation in fishes, and notes physiological mechanisms that await elucidation.