Characterization of a New Variant in ARHGAP31 Probably Involved in Adams-Oliver Syndrome in a Family with a Variable Phenotypic Spectrum.

Adams-Oliver syndrome is a rare inherited condition characterized by scalp defects and limb abnormalities. It is caused by variants in different genes such as ARHGAP31. Here, we used an interdisciplinary approach to study a family with lower limb anomalies. We identified a novel variant in the ARHGAP31 gene that is predicted to result in a truncated protein with a constitutively activated catalytic site due to the loss of 688 amino acids involved in the C-terminal domain, essential for protein auto-inhibition. Pathogenic variants in ARHGAP31 exon 12, leading to a premature protein termination, are associated with Adams-Oliver syndrome. Bioinformatic analysis was useful to elucidate the impact of the identified genetic variant on protein structure. To better understand the impact of the identified variant, 3D protein models were predicted for the ARHGAP31 wild type, the newly discovered variant, and other pathogenetic alterations already reported. Our study identified a novel variant probably involved in Adams-Oliver syndrome and increased the evidence on the phenotypic variability in patients affected by this syndrome, underlining the importance of translational research, including experimental and bioinformatics analyses. This strategy represents a successful model to investigate molecular mechanisms involved in syndrome occurrence.

Brain endothelial cells promote breast cancer cell extravasation to the brain via EGFR-DOCK4-RAC1 signalling.

The role of endothelial cells in promoting cancer cell extravasation to the brain during the interaction of cancer cells with the vasculature is not well characterised. We show that brain endothelial cells activate EGFR signalling in triple-negative breast cancer cells with propensity to metastasise to the brain. This activation is dependent on soluble factors secreted by brain endothelial cells, and occurs via the RAC1 GEF DOCK4, which is required for breast cancer cell extravasation to the brain in vivo. Knockdown of DOCK4 inhibits breast cancer cell entrance to the brain without affecting cancer cell survival or growth. Defective extravasation is associated with loss of elongated morphology preceding intercalation into brain endothelium. We also show that brain endothelial cells promote paracrine stimulation of mesenchymal-like morphology of breast cancer cells via DOCK4, DOCK9, RAC1 and CDC42. This stimulation is accompanied by EGFR activation necessary for brain metastatic breast cancer cell elongation which can be reversed by the EGFR inhibitor Afatinib. Our findings suggest that brain endothelial cells promote metastasis through activation of cell signalling that renders breast cancer cells competent for extravasation. This represents a paradigm of brain endothelial cells influencing the signalling and metastatic competency of breast cancer cells.

TBC9, an essential TBC-domain protein, regulates early vesicular transport and IMC formation in Toxoplasma gondii.

Apicomplexan parasites harbor a complex endomembrane system as well as unique secretory organelles. These complex cellular structures require an elaborate vesicle trafficking system, which includes Rab GTPases and their regulators, to assure the biogenesis and secretory of the organelles. Here we exploit the model apicomplexan organism Toxoplasma gondii that encodes a family of Rab GTPase Activating Proteins, TBC (Tre-2/Bub2/Cdc16) domain-containing proteins. Functional profiling of these proteins in tachyzoites reveals that TBC9 is the only essential regulator, which is localized to the endoplasmic reticulum (ER) in T. gondii strains. Detailed analyses demonstrate that TBC9 is required for normal distribution of proteins targeting to the ER, and the Golgi apparatus in the parasite, as well as for the normal formation of daughter inner membrane complexes (IMCs). Pull-down assays show a strong protein interaction between TBC9 and specific Rab GTPases (Rab11A, Rab11B, and Rab2), supporting the role of TBC9 in daughter IMC formation and early vesicular transport. Thus, this study identifies the only essential TBC domain-containing protein TBC9 that regulates early vesicular transport and IMC formation in T. gondii and potentially in closely related protists.

Exosome-transmitted circular RNA circ-LMO7 facilitates the progression of osteosarcoma by regulating miR-21-5p/ARHGAP24 axis.

The potential function and mechanism of circRNAs in regulating malignant performances of Osteosarcoma (OS) cells have not been well investigated. The expression level of CircLMO7, miR-21-5p and ARHGAP24 were detected by RT-qPCR. The relationship between miR-21-5p and circ-LMO7, as well as between miR-21-5p and ARHGAP24, was predicted and examined through bioinformatics analysis and luciferase reporter gene experiments. Moreover, OS cell growth, invasion, migration, and apoptosis were detected using the cell counting kit-8 (CCK-8), transwell and flow cytometry assays, respectively. ARHGAP24 protein level was measured using western blotting. In present study, we choose to investigate the role and mechanism of circ-LOM7 on OS cell proliferation, migration and invasion. circ-LOM7 was found to be down-regulated in OS tissues and cell lines. Enforced expression of circ-LOM7 suppressed the growth, invasion, and migration of OS cells. In contrast, decreasing circ-LMO7 expression had opposite effects. Furthermore, miR-21-5p was predicted to be sponged by circ-LMO7, and had an opposite role of circ-LMO7 in OS. Moreover, ARHGAP24 served as miR-21-5p's downstream target. Mechanistically, circ-LMO7 was packed in exosomes and acted as a cancer-suppresser on OS by sponging miR-21-5p and upregulating the expression of ARHGAP24. The exosomal circ-LMO7 expression was significantly decreased in OS cell exosomes, and co-culture experiments showed that exosomal circ-LMO7 suppressed the proliferation ability of OS cells. Circ-LMO7 exerts as a tumor suppressor in OS, and the circ-LMO7/miR-21-5P/ARHGAP24 axis is involved in OS progression.

A comprehensive analysis of the oncogenic and prognostic role of TBC1Ds in human hepatocellular carcinoma.

Backgrounds: TBC1D family members (TBC1Ds) are a group of proteins that contain the Tre2-Bub2-Cdc16 (TBC) domain. Recent studies have shown that TBC1Ds are involved in tumor growth, but no analysis has been done of expression patterns and prognostic values of TBC1Ds in hepatocellular carcinoma (HCC). Methods: The expression levels of TBC1Ds were evaluated in HCC using the TIMER, UALCN and Protein Atlas databases. The correlation between the mRNA levels of TBC1Ds and the prognosis of patients with HCC in the GEPIA database was then analyzed. An enrichment analysis then revealed genes that potentially interact with TBC1Ds. The correlation between levels of TBC1Ds and tumor-infiltrating immune cells (TIICs) in HCC were studied using the TIMER 2.0 database. Finally, a series of in vitro assays verified the role of TBC1Ds in HCC progression. Results: This study revealed the upregulated expression of TBC1Ds in HCC and the strong positive correlation between the mRNA levels of TBC1Ds and poor prognosis of patients with HCC. The functions of TBC1Ds were mainly related to autophagy and the AMPK pathway. There was also a significant correlation between level of TBC1Ds and tumor-infiltrating immune cells (TIICs) in HCC. The promoting role of TBC1Ds in HCC progression was verified in vitro assays. Conclusion: The results of this analysis indicate that TBC1Ds may serve as new biomarkers for early diagnosis and treatment of HCC.

ARHGAP10, Transcriptionally Regulated by Sodium Butyrate, Promotes Ferroptosis of Ovarian Cancer Cells.

BACKGROUND: Ovarian cancer is a highly lethal gynecologic malignancy. ARHGAP10, a member of Rho GTPase-activating proteins, is a potential tumor suppressor in ovarian cancer. However, its role and the involved mechanism need further examination. Here, we investigated whether ARHGAP10 is also associated with ferroptosis. METHODS: Lentivirus infection was used for gene overexpression or silencing. Real-time polymerase chain reaction (RT-PCR) and Western blot were used to assess mRNA and protein levels, respectively. Cell viability was assessed by Cell Counting Kit-8 (CCK-8) assay. Lipid reactive oxygen species level was measured by flow cytometry. A tumorigenicity assay was performed to evaluate tumor growth in vivo, and sections of mouse tumor tissues were examined by immunofluorescence microscopy. Chromatin Immunoprecipitation (ChIP) assay was used to assess the binding of H3K9ac to the promoter region of ARHGAP10. RESULTS: ARHGAP10 overexpression promoted ferroptosis in ovarian cancer cells, resulting in decreased cell viability, and increased lipid reactive oxygen species (ROS) level. Further, it decreased and increased GPX4 and PTGS2 expression, respectively, and also induced suppression of tumor growth in mice. Fer-1, a potent inhibitor of ferroptosis, suppressed the above effects of ARHGAP10. Contrarily, ARHGAP10 silencing alleviated ferroptosis in ovarian cancer cells, which was reversed by RSL3, a ferroptosis-inducing agent. Lastly, sodium butyrate (SB) was found to transcriptionally regulate ARHGAP10, thereby also contributing to the ferroptosis of ovarian cancer cells. CONCLUSIONS: Our results suggest that SB/ARHGAP10/GPX4 is a new signaling axis involved in inducing ferroptosis in ovarian cancer cells and suppressing tumor growth, which has potential clinical significance.

Deciphering the Role of miR-30a-5p and DLGAP1 Gene in Non-small Cell Lung Cancer.

BACKGROUND/AIM: Non-small cell lung cancer (NSCLC) is the deadliest form of cancer worldwide. Understanding the mechanisms of lung cancer development is vital for targeted therapy advancements. This article explores the little-known role of the guanylate kinase-associated protein (GKAP), encoded by the Disks large-associated protein 1 (DLGAP1) gene, in NSCLC along with assessing microRNA-30a-5p's influence on DLGAP1 gene expression in the A549 cell line. MATERIALS AND METHODS: Experiments were conducted on A549 cells transfected with synthetic oligonucleotides. The luciferase assay was employed to confirm the binding site of miR-30a-5p to the 3'UTR of DLGAP1 mRNA. The role of miRNA-30a-5p mimic in regulating potential target gene expression at the protein and mRNA levels was studied by performing RT-qPCR and western blot analyses. The effects of DLGAP1 knockdown and miRNA-30a-5p mimic on cell viability and the cell cycle were evaluated using the MTT test and flow cytometry with annexin/iodide cell staining. RESULTS: The luciferase assay indicated that miR-30a-5p has the ability to bind to the 3'UTR of DLGAP1 mRNA. RT-qPCR revealed that the overexpression of miR-30a-5p down-regulates DLGAP1 mRNA. Western blot analysis indicated that miR-30a-5p slightly reduces the level of the GKAP protein. Knockdown of DLGAP1 with synthetic oligonucleotides, as well as transfection with a miR-30a-5p mimic, significantly attenuates cell proliferation and increases the number of cells in the early and late stages of apoptosis. CONCLUSION: Our findings reveal the antiproliferative effect of miR-30a-5p and DLGAP1 gene knockdown on A549 cancer cells, implying that these elements could be considered as therapeutic targets for personalized medicine in NSCLC patients.

HIF2α-dependent Dock4/Rac1-signaling regulates formation of adherens junctions and cell polarity in normoxia.

Hypoxia-inducible factors (HIF) 1 and 2 regulate similar but distinct sets of target genes. Although HIFs are best known for their roles in mediating the hypoxia response accumulating evidence suggests that under certain conditions HIFs, particularly HIF2, may function also under normoxic conditions. Here we report that HIF2α functions under normoxic conditions in kidney epithelial cells to regulate formation of adherens junctions. HIF2α expression was required to induce Dock4/Rac1/Pak1-signaling mediating stability and compaction of E-cadherin at nascent adherens junctions. Impaired adherens junction formation in HIF2α- or Dock4-deficient cells led to aberrant cyst morphogenesis in 3D kidney epithelial cell cultures. Taken together, we show that HIF2α functions in normoxia to regulate epithelial morphogenesis.

Rab1b facilitates lipid droplet growth by ER-to-lipid droplet targeting of DGAT2.

Lipid droplets (LDs) comprise a triglyceride core surrounded by a lipid monolayer enriched with proteins, many of which function in LD homeostasis. How proteins are targeted to the growing LD is still unclear. Rab1b, a GTPase regulating secretory transport, was recently associated with targeting proteins to LDs in a Drosophila RNAi screen. LD formation was prevented in human hepatoma cells overexpressing dominant-negative Rab1b. We thus hypothesized that Rab1b recruits lipid-synthesizing enzymes, facilitating LD growth. Here, FRET between diacylglycerol acyltransferase 2 (DGAT2) and Rab1b and activity mutants of the latter demonstrated that Rab1b promotes DGAT2 ER to the LD surface redistribution. Last, alterations in LD metabolism and DGAT2 redistribution, consistent with Rab1b activity, were caused by mutations in the Rab1b-GTPase activating protein TBC1D20 in Warburg Micro syndrome (WARBM) model mice fibroblasts. These data contribute to our understanding of the mechanism of Rab1b in LD homeostasis and WARBM, a devastating autosomal-recessive disorder caused by mutations in TBC1D20.

Integrated analysis identified the role of three family members of ARHGAP in pancreatic adenocarcinoma.

The Rho GTPase activating protein family (ARHGAPs) is expressed in pancreatic adenocarcinoma (PAAD) but its function is unclear. The aim of this study was to explore the role and potential clinical value of ARHGAPs in PAAD. Using TCGA and GEO databases to analyze expression of ARHGAPs in PAAD and normal tissues. Survival curve was drawn by Kaplan-Meier. ARHGAPs were integrated analyzed by GEPIA2, TIMER, UCLCAN, cBioPortal and R language. Protein level and prognostic value were evaluated via IHC staining or survival analysis. We totally identify 18 differentially expressed (DE) ARHGAPs in PAAD. Among the 18 DE genes, 8 were positively correlated with tumor grade; abnorrmal expression of 5 was positively correlated with copy number variation; expression of 4 was positively correlated with promoter hypomethylation. Multivariate Cox regression identified ARHGAP5, ARHGAP11A, and ARHGAP12 as independent prognostic factors of PAAD. The function of ARHGAPs was mainly related to GTPase activity and signaling, axon guidance, proteoglycans in cancer and focal adhesion. Expression of 7 ARHGAPs was strongly correlated with immune infiltration. Immunohistochemistry showed increased protein levels of ARHGAP5, ARHGAP11A, and ARHGAP12 in PAAD tissues. Survival analysis confirmed a negative correlation between ARHGAP5, ARHGAP11A, and ARHGAP12 expression and patient prognosis. Multivariate Cox regression proved ARHGAP5, ARHGAP11A, and ARHGAP12 could serve as independent prognostic indicators for PAAD. Finally, this study verified ARHGAP5, ARHGAP11A, and ARHGAP12 as independent prognostic factors in PAAD, suggesting their significance for the diagnosis and treatment of PAAD.

Functional synergy of a human-specific and an ape-specific metabolic regulator in human neocortex development.

Metabolism has recently emerged as a major target of genes implicated in the evolutionary expansion of human neocortex. One such gene is the human-specific gene ARHGAP11B. During human neocortex development, ARHGAP11B increases the abundance of basal radial glia, key progenitors for neocortex expansion, by stimulating glutaminolysis (glutamine-to-glutamate-to-alpha-ketoglutarate) in mitochondria. Here we show that the ape-specific protein GLUD2 (glutamate dehydrogenase 2), which also operates in mitochondria and converts glutamate-to-αKG, enhances ARHGAP11B's ability to increase basal radial glia abundance. ARHGAP11B + GLUD2 double-transgenic bRG show increased production of aspartate, a metabolite essential for cell proliferation, from glutamate via alpha-ketoglutarate and the TCA cycle. Hence, during human evolution, a human-specific gene exploited the existence of another gene that emerged during ape evolution, to increase, via concerted changes in metabolism, progenitor abundance and neocortex size.

Identification of m6A-related lncRNAs LINC02471 and DOCK9-DT as potential biomarkers for thyroid cancer.

Thyroid cancer (THCA) is the most common endocrine malignancy worldwide and has been rising at the fastest rate in recent years. Long-stranded non-coding RNAs (lncRNAs) and N6-methyladenosine (m6A) have been associated with immunotherapy efficacy and cancer prognosis. However, how m6A-associated lncRNAs (mrlncRNAs) affect the prognosis of patients with thyroid cancer is unclear. Therefore, this study utilized The Cancer Genome Atlas (TCGA) database to provide thyroid cancer-related transcriptomic data and related clinical data. The R program was used to identify m6A-related lncRNAs, and a risk model consisting of two lncRNAs (LINC02471 and DOCK9-DT) was obtained using least absolute shrinkage and selection operator (LASSO) Cox regression analysis. Kaplan-Meier survival analysis and transient subject operating characteristics (ROC) were used for analysis. The results showed a substantial association between immune cell infiltration and risk scores. Independent analyses confirmed that the expression of LINC02471 and DOCK9-DT was significantly higher in thyroid cancer tissues than in normal tissues, suggesting that they may be useful biomarkers for thyroid cancer.

Phosphorylation of AS160-serine 704 is not essential for exercise-increase in insulin-stimulated glucose uptake by skeletal muscles from female or male rats.

One exercise session can increase subsequent insulin-stimulated glucose uptake (ISGU) by skeletal muscle from rodents and humans of both sexes. We recently found that concurrent mutation of three key sites to prevent their phosphorylation (Ser588, Thr642, and Ser704) on Akt substrate of 160 kDa (AS160; also known as TBC1D4) reduced the magnitude of the enhancement of postexercise ISGU (PEX-ISGU) by muscle from male, but not female rats. However, we did not test the role of individual phosphorylation sites on PEX-ISGU. Accordingly, our current aim was to test whether AS160 Ser704 phosphorylation (pSer704) is required for elevated PEX-ISGU by muscle. AS160-knockout (AS160-KO) rats (female and male) were studied when either in sedentary or 3 h after acute exercise. Adeno-associated virus (AAV) vectors were used to enable muscle expression of wild-type AS160 (AAV-WT-AS160) or AS160 mutated Ser704 to alanine to prevent phosphorylation (AAV-1P-AS160). Paired epitrochlearis muscles from each rat were injected with AAV-WT-AS160 or AAV-1P-AS160. We discovered that regardless of sex 1) AS160 abundance in AS160-KO rats was similar in paired muscles expressing WT-AS160 versus 1P-AS160; 2) muscles from exercised versus sedentary rats had greater ISGU, and PEX-ISGU was slightly greater for muscles expressing 1P-AS160 versus contralateral muscles expressing WT-AS160; and 3) pAS160Thr642 was lower in muscles expressing 1P-AS160 versus paired muscles expressing WT-AS160. These results indicate that pAS160Ser704 was not essential for elevated PEX-ISGU by skeletal muscle from rats of either sex. Furthermore, elimination of the postexercise increase in pAS160Thr642 did not lessen the postexercise effect on ISGU.NEW & NOTEWORTHY The current study evaluated the role of Akt substrate of 160 kDa (AS160) phosphorylation on Ser704 in increased insulin-stimulated glucose uptake by skeletal muscle after exercise. Adeno-associated virus vectors were engineered to express either wild-type-AS160 or AS160 mutated so that it could not be phosphorylated on Ser704 in paired muscles from AS160-knockout rats. The results demonstrated that AS160 phosphorylation on Ser704 was not essential for exercise-induced elevation in insulin-stimulated glucose uptake by rats of either sex.

A RabGAP negatively regulates plant autophagy and immune trafficking.

Plants rely on autophagy and membrane trafficking to tolerate stress, combat infections, and maintain cellular homeostasis. However, the molecular interplay between autophagy and membrane trafficking is poorly understood. Using an AI-assisted approach, we identified Rab3GAP-like (Rab3GAPL) as a key membrane trafficking node that controls plant autophagy negatively. Rab3GAPL suppresses autophagy by binding to ATG8, the core autophagy adaptor, and deactivating Rab8a, a small GTPase essential for autophagosome formation and defense-related secretion. Rab3GAPL reduces autophagic flux in three model plant species, suggesting that its negative regulatory role in autophagy is conserved in land plants. Beyond autophagy regulation, Rab3GAPL modulates focal immunity against the oomycete pathogen Phytophthora infestans by preventing defense-related secretion. Altogether, our results suggest that Rab3GAPL acts as a molecular rheostat to coordinate autophagic flux and defense-related secretion by restraining Rab8a-mediated trafficking. This unprecedented interplay between a RabGAP-Rab pair and ATG8 sheds new light on the intricate membrane transport mechanisms underlying plant autophagy and immunity.

Change in RhoGAP and RhoGEF availability drives transitions in cortical patterning and excitability in Drosophila.

Actin cortex patterning and dynamics are critical for cell shape changes. These dynamics undergo transitions during development, often accompanying changes in collective cell behavior. Although mechanisms have been established for individual cells' dynamic behaviors, the mechanisms and specific molecules that result in developmental transitions in vivo are still poorly understood. Here, we took advantage of two developmental systems in Drosophila melanogaster to identify conditions that altered cortical patterning and dynamics. We identified a Rho guanine nucleotide exchange factor (RhoGEF) and Rho GTPase activating protein (RhoGAP) pair required for actomyosin waves in egg chambers. Specifically, depletion of the RhoGEF, Ect2, or the RhoGAP, RhoGAP15B, disrupted actomyosin wave induction, and both proteins relocalized from the nucleus to the cortex preceding wave formation. Furthermore, we found that overexpression of a different RhoGEF and RhoGAP pair, RhoGEF2 and Cumberland GAP (C-GAP), resulted in actomyosin waves in the early embryo, during which RhoA activation precedes actomyosin assembly by ∼4 s. We found that C-GAP was recruited to actomyosin waves, and disrupting F-actin polymerization altered the spatial organization of both RhoA signaling and the cytoskeleton in waves. In addition, disrupting F-actin dynamics increased wave period and width, consistent with a possible role for F-actin in promoting delayed negative feedback. Overall, we showed a mechanism involved in inducing actomyosin waves that is essential for oocyte development and is general to other cell types, such as epithelial and syncytial cells.

DLC1 promotes mechanotransductive feedback for YAP via RhoGAP-mediated focal adhesion turnover.

Angiogenesis is a tightly controlled dynamic process demanding a delicate equilibrium between pro-angiogenic signals and factors that promote vascular stability. The spatiotemporal activation of the transcriptional co-factors YAP (herein referring to YAP1) and TAZ (also known WWTR1), collectively denoted YAP/TAZ, is crucial to allow for efficient collective endothelial migration in angiogenesis. The focal adhesion protein deleted-in-liver-cancer-1 (DLC1) was recently described as a transcriptional downstream target of YAP/TAZ in endothelial cells. In this study, we uncover a negative feedback loop between DLC1 expression and YAP activity during collective migration and sprouting angiogenesis. In particular, our study demonstrates that signaling via the RhoGAP domain of DLC1 reduces nuclear localization of YAP and its transcriptional activity. Moreover, the RhoGAP activity of DLC1 is essential for YAP-mediated cellular processes, including the regulation of focal adhesion turnover, traction forces, and sprouting angiogenesis. We show that DLC1 restricts intracellular cytoskeletal tension by inhibiting Rho signaling at the basal adhesion plane, consequently reducing nuclear YAP localization. Collectively, these findings underscore the significance of DLC1 expression levels and its function in mitigating intracellular tension as a pivotal mechanotransductive feedback mechanism that finely tunes YAP activity throughout the process of sprouting angiogenesis.

Case report of a novel mutation in the TNC gene in Chinese patients with nonsyndromic hearing loss.

RATIONALE: Hereditary hearing loss is known to exhibit a significant degree of genetic heterogeneity. Herein, we present a case report of a novel mutation in the tenascin-C (TNC) gene in Chinese patients with nonsyndromic hearing loss (NSHL). PATIENT CONCERNS: This includes a young deaf couple and their 2-year-old baby. DIAGNOSES: Based on the clinical information, hearing test, metagenomic next-generation sequencing (mNGS), Sanger sequencing, protein function and structure analysis, and model prediction, in our case, the study results revealed 2 heterozygous mutations in the TNC gene (c.2852C>T, p.Thr951Ile) and the TBC1 domain family member 24 (TBC1D24) gene (c.1570C>T, p.Arg524Trp). These mutations may be responsible for the hearing loss observed in this family. Notably, the heterozygous mutations in the TNC gene (c.2852C>T, p.Thr951Ile) have not been previously reported in the literature. INTERVENTIONS: Avoid taking drugs that can cause deafness, wearing hearing AIDS, and cochlear implants. OUTCOMES: Regular follow-up of family members is ongoing. LESSONS: The genetic diagnosis of NSHL holds significant importance as it helps in making informed treatment decisions, providing prognostic information, and offering genetic counseling for the patient's family.

The Ras GTPase-activating protein UvGap1 orchestrates conidiogenesis and pathogenesis in the rice false smut fungus Ustilaginoidea virens.

Ras GTPase-activating proteins (Ras GAPs) act as negative regulators for Ras proteins and are involved in various signalling processes that influence cellular functions. Here, the function of four Ras GAPs, UvGap1 to UvGap4, was identified and analysed in Ustilaginoidea virens, the causal agent of rice false smut disease. Disruption of UvGAP1 or UvGAP2 resulted in reduced mycelial growth and an increased percentage of larger or dumbbell-shaped conidia. Notably, the mutant ΔUvgap1 completely lost its pathogenicity. Compared to the wild-type strain, the mutants ΔUvgap1, ΔUvgap2 and ΔUvgap3 exhibited reduced tolerance to H2 O2 oxidative stress. In particular, the ΔUvgap1 mutant was barely able to grow on the H2 O2 plate, and UvGAP1 was found to influence the expression level of genes involved in reactive oxygen species synthesis and scavenging. The intracellular cAMP level in the ΔUvgap1 mutant was elevated, as UvGap1 plays an important role in maintaining the intracellular cAMP level by affecting the expression of phosphodiesterases, which are linked to cAMP degradation in U. virens. In a yeast two-hybrid assay, UvRas1 and UvRasGef (Ras guanyl nucleotide exchange factor) physically interacted with UvGap1. UvRas2 was identified as an interacting partner of UvGap1 through a bimolecular fluorescence complementation assay and affinity capture-mass spectrometry analysis. Taken together, these findings suggest that the UvGAP1-mediated Ras pathway is essential for the development and pathogenicity of U. virens.

C. elegans Afadin is required for epidermal morphogenesis and functionally interfaces with the cadherin-catenin complex and RhoGAP PAC-1/ARHGAP21.

During epithelial morphogenesis, the apical junctions connecting cells must remodel as cells change shape and make new connections with their neighbors. In the C. elegans embryo, new apical junctions form when epidermal cells migrate and seal with one another to encase the embryo in skin ('ventral enclosure'), and junctions remodel when epidermal cells change shape to squeeze the embryo into a worm shape ('elongation'). The junctional cadherin-catenin complex (CCC), which links epithelial cells to each other and to cortical actomyosin, is essential for C. elegans epidermal morphogenesis. RNAi genetic enhancement screens have identified several genes encoding proteins that interact with the CCC to promote epidermal morphogenesis, including the scaffolding protein Afadin (AFD-1), whose depletion alone results in only minor morphogenesis defects. Here, by creating a null mutation in afd-1, we show that afd-1 provides a significant contribution to ventral enclosure and elongation on its own. Unexpectedly, we find that afd-1 mutant phenotypes are strongly modified by diet, revealing a previously unappreciated parental nutritional input to morphogenesis. We identify functional interactions between AFD-1 and the CCC by demonstrating that E-cadherin is required for the polarized distribution of AFD-1 to cell contact sites in early embryos. Finally, we show that afd-1 promotes the enrichment of polarity regulator, and CCC-interacting protein, PAC-1/ARHGAP21 to cell contact sites, and we identify genetic interactions suggesting that afd-1 and pac-1 regulate epidermal morphogenesis at least in part through parallel mechanisms. Our findings reveal that C. elegans AFD-1 makes a significant contribution to epidermal morphogenesis and functionally interfaces with core and associated CCC proteins.