Arp2/3-dependent endocytosis ensures Cdc42 oscillations by removing Pak1-mediated negative feedback.

The GTPase Cdc42 regulates polarized growth in most eukaryotes. In the bipolar yeast Schizosaccharomyces pombe, Cdc42 activation cycles periodically at sites of polarized growth. These periodic cycles are caused by alternating positive feedback and time-delayed negative feedback loops. At each polarized end, negative feedback is established when active Cdc42 recruits the Pak1 kinase to prevent further Cdc42 activation. It is unclear how Cdc42 activation returns to each end after Pak1-dependent negative feedback. We find that disrupting branched actin-mediated endocytosis disables Cdc42 reactivation at the cell ends. Using experimental and mathematical approaches, we show that endocytosis-dependent Pak1 removal from the cell ends allows the Cdc42 activator Scd1 to return to that end to enable reactivation of Cdc42. Moreover, we show that Pak1 elicits its own removal via activation of endocytosis. These findings provide a deeper insight into the self-organization of Cdc42 regulation and reveal previously unknown feedback with endocytosis in the establishment of cell polarity.

CircCDC42-encoded CDC42-165aa regulates macrophage pyroptosis in Klebsiella pneumoniae infection through Pyrin inflammasome activation.

The circular RNA (circRNA) family is a group of endogenous non-coding RNAs (ncRNAs) that have critical functions in multiple physiological and pathological processes, including inflammation, cancer, and cardiovascular diseases. However, their roles in regulating innate immune responses remain unclear. Here, we define Cell division cycle 42 (CDC42)-165aa, a protein encoded by circRNA circCDC42, which is overexpressed in Klebsiella pneumoniae (KP)-infected alveolar macrophages. High levels of CDC42-165aa induces the hyperactivation of Pyrin inflammasomes and aggravates alveolar macrophage pyroptosis, while the inhibition of CDC42-165aa reduces lung injury in mice after KP infection by inhibiting Pyrin inflammasome-mediated pyroptosis. Overall, these results demonstrate that CDC42-165aa stimulates Pyrin inflammasome by inhibiting CDC42 GTPase activation and provides a potential clinical target for pathogenic bacterial infection in clinical practice.

LncRNA MALAT1 facilitates BM-MSCs differentiation into endothelial cells and ameliorates erectile dysfunction via the miR-206/CDC42/PAK1/paxillin signalling axis.

BACKGROUND: Erectile dysfunction (ED) is a common male sexual dysfunction, with an increasing incidence, and the current treatment is often ineffective. METHODS: Vascular endothelial growth factor (VEGFA) was used to treat bone marrow-derived mesenchymal stem cells (BM-MSCs), and their cell migration rates were determined by Transwell assays. The expression of the von Willebrand Factor (vWF)VE-cadherin, and endothelial nitric oxide synthase(eNOS) endothelial markers was determined by qRT‒PCR and Western blot analyses. The MALAT1-induced differentiation of BM-MCs to ECs via the CDC42/PAK1/paxillin pathway was explored by transfecting VEGFA-induced BM-MSC with si-MALAT1 and overexpressing CDC42 and PAK1. The binding capacity between CDC42, PAK1, and paxillin in VEGFA-treated and non-VEGFA-treated BM-MSCs was examined by protein immunoprecipitation. MiR-206 was overexpressed in VEGFA-induced BM-MSC, and the binding sites of MALAT1, miR-206, and CDC42 were identified using a luciferase assay. Sixty male Sprague‒Dawley rats were divided into six groups (n = 10/group). DMED modelling was demonstrated by APO experiments and was assessed by measuring blood glucose levels. Erectile function was assessed by measuring the intracavernosa pressure (ICP) and mean arterial pressure (MAP). Penile erectile tissue was analysed by qRT‒PCR, Western blot analysis, and immunohistochemical staining. RESULTS: MALAT1 under VEGFA treatment conditions regulates the differentiation of BM-MSCs into ECs by modulating the CDC42/PAK1/paxillin axis. In vitro experiments demonstrated that interference with CDC42 and MALAT1 expression inhibited the differentiation of BM-MSCs to ECs. CDC42 binds to PAK1, and PAK1 binds to paxillin. In addition, CDC42 in the VEGFA group had a greater ability to bind to PAK1, whereas PAK1 in the VEGFA group had a greater ability to bind to paxillin. Overexpression of miR-206 in VEGFA-induced BM-MSCs demonstrated that MALAT1 competes with the CDC42 3'-UTR for binding to miR-206, which in turn is involved in the differentiation of BM-MSCs to ECs. Compared to the DMED model group, the ICP/MAP ratio was significantly greater in the three BM-MSCs treatment groups. CONCLUSIONS: MALAT1 facilitates BM-MSC differentiation into ECs by regulating the miR-206/CDC42/PAK1/paxillin axis to improve ED. The present findings revealed the vital role of MALAT1 in the repair of BM-MSCs for erectile function and provided new mechanistic insights into the BM-MSC-mediated repair of DMED.

NSUN4 mediated RNA 5-methylcytosine promotes the malignant progression of glioma through improving the CDC42 mRNA stabilization.

5-Methylcytosine (m5C) methylation is a significant post-transcriptional modification that play a crucial role in the development and progression of numerous cancers. Whereas the functions and molecular mechanisms underlying m5C methylation in gliomas remain unclear. This study dedicated to explore changes of m5C levels and the clinical significance of the m5C writer NSUN4 in gliomas. We found that high m5C levels were negatively related to prognosis of patients with glioma. Moreover, gain- and loss-of-function experiments revealed the role of NSUN4 in enhancing m5C modification of mRNA to promote the malignant progression of glioma. Mechanistically speaking, NSUN4-mediated m5C alterations regulated ALYREF binding to CDC42 mRNA, thereby impacting the mRNA stability of CDC42. We also demonstrated that CDC42 promoted glioma proliferation, migration, and invasion by activating the PI3K-AKT pathway. Additionally, rescue experiments proved that CDC42 overexpression weaken the inhibitory effect of NSUN4 knockdown on the malignant progression of gliomas in vitro and in vivo. Our findings elucidated that NSUN4-mediated high m5C levels promote ALYREF binding to CDC42 mRNA and regulate its stability, thereby driving the malignant progression of glioma. This provides theoretical support for targeted the treatment of gliomas.

Estrogen inhibits TGF­ß1­stimulated cardiac fibroblast differentiation and collagen synthesis by promoting Cdc42.

17ß­estradiol (E2) can inhibit cardiac fibrosis in female patients with heart failure (HF) and activate cell division cycle 42 (Cdc42), however it is unknown whether 17ß­estradiol (E2) can ameliorate differentiation and collagen synthesis in TGF­ß1­stimulated mouse cardiac fibroblasts (MCFs) by regulating cell division cycle 42 (Cdc42). The present study aimed to investigate the roles of estrogen and Cdc42 in preventing myocardial fibrosis and the underlying molecular mechanisms. An ELISA was used to measure the levels of E2 and Cdc42 in the serum of patients with heart failure (HF), and western blotting was used to measure the expression levels of Cdc42 in TGF­ß1­stimulated immortalized MCFs. MCFs were transfected with a Cdc42 overexpression (OE) lentivirus or small interfering RNA (siRNA), or treated with a Cdc42 inhibitor (MLS­573151), and the function of Cdc42 was assessed by western blotting, immunofluorescence staining, reverse transcription­quantitative PCR and dual­luciferase reporter assays. Western blotting and immunofluorescence staining were performed to verify the protective effect of E2 on TGF­ß1­stimulated MCFs, and the association between the protective effect and Cdc42. The results demonstrated that Cdc42 levels were increased in the serum of patients with HF and were positively correlated with the levels of E2; however, Cdc42 levels were decreased in TGF­ß1­stimulated MCFs. Cdc42 inhibited MCF differentiation and collagen synthesis, as indicated by the protein expression of α­smooth muscle actin, collagen I and collagen III. Mechanistically, Cdc42 inhibited the transcription of TGF­ß1 by promoting the expression of p21 (RAC1)­activated kinase 1 (Pak1)/JNK/c­Jun signaling pathway proteins and inhibiting the activity of the Tgfb1 gene promoter. In addition, E2 inhibited the differentiation and collagen synthesis of TGF­ß1­stimulated MCFs, and promoted the protein expression of Pak1, JNK and c­Jun, consistent with the effects of Cdc42, whereas the effects of E2 were abolished when Cdc42 was knocked down. The aforementioned findings suggested that E2 could inhibit differentiation and collagen synthesis in TGF­ß1­stimulated MCFs by regulating Cdc42 and the downstream Pak1/JNK/c­Jun signaling pathway.

NLRP4E regulates actin cap formation through SRC and CDC42 during oocyte meiosis.

BACKGROUND: Members of the nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing (NLRP) family regulate various physiological and pathological processes. However, none have been shown to regulate actin cap formation or spindle translocation during the asymmetric division of oocyte meiosis I. NLRP4E has been reported as a candidate protein in female fertility, but its function is unknown. METHODS: Immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR), and western blotting were employed to examine the localization and expression levels of NLRP4E and related proteins in mouse oocytes. small interfering RNA (siRNA) and antibody transfection were used to knock down NLRP4E and other proteins. Immunoprecipitation (IP)-mass spectrometry was used to identify the potential proteins interacting with NLRP4E. Coimmunoprecipitation (Co-IP) was used to verify the protein interactions. Wild type (WT) or mutant NLRP4E messenger RNA (mRNA) was injected into oocytes for rescue experiments. In vitro phosphorylation was employed to examine the activation of steroid receptor coactivator (SRC) by NLRP4E. RESULTS: NLRP4E was more predominant within oocytes compared with other NLRP4 members. NLRP4E knockdown significantly inhibited actin cap formation and spindle translocation toward the cap region, resulting in the failure of polar body extrusion at the end of meiosis I. Mechanistically, GRIN1, and GANO1 activated NLRP4E by phosphorylation at Ser429 and Thr430; p-NLRP4E is translocated and is accumulated in the actin cap region during spindle translocation. Next, we found that p-NLRP4E directly phosphorylated SRC at Tyr418, while p-SRC negatively regulated p-CDC42-S71, an inactive form of CDC42 that promotes actin cap formation and spindle translocation in the GTP-bound form. CONCLUSIONS: NLRP4E activated by GRIN1 and GANO1 regulates actin cap formation and spindle translocation toward the cap region through upregulation of p-SRC-Tyr418 and downregulation of p-CDC42-S71 during meiosis I.

Linkage of cell division cycle 42 with clinical features, treatment response and survival in adult Philadelphia chromosome negative acute lymphoblastic leukemia.

Cell division cycle 42 (CDC42) regulates the progression of leukemia via mediating proliferation and immune evasion of malignant cells. The study aimed to investigate the correlation of CDC42 with clinical features, treatment response, event-free survival (EFS) and overall survival (OS) in adult Philadelphia chromosome negative acute lymphoblastic leukemia (Ph- ALL) patients. CDC42 expression in bone marrow mononuclear cells was detected in 78 adult Ph- ALL patients and 10 donors using real-time reverse transcriptase-polymerase chain reaction. CDC42 was increased in adult Ph- ALL patients compared with donors (p < .001). Besides, elevated CDC42 was linked with pro-B ALL or early-T ALL (p = .038) and white blood cell (WBC) elevation at diagnosis (p = .025). Fifty (64.1%) and 23 (29.5%) patients had complete remission (CR) at 1 month and minimal residual disease (MRD) after CR, respectively. CDC42 was inversely associated with CR at 1 month (p = .034), but not MRD after CR (p = .066). Concerning survival, patients with CDC42 ≥ 3.310 (cut by median value in patients) showed a shortened EFS (p = .006) and OS (p = .036) compared to those with CDC42 < 3.310. In detail, patients with CDC42 ≥ 3.310 and CDC42 < 3.310 had 5-year EFS rate of 29.9% and 45.4%, and 5-year OS rate of 39.4% and 63.6%, correspondingly. Further multivariate Cox's regression analyses revealed that CDC42 ≥ 3.310 was independently related to shorter EFS (hazard ratio = 2.933, p = .005). Elevated CDC42 is related with pro-B ALL or early-T ALL, WBC elevation at diagnosis, unfavorable treatment response and worse survival in adult Ph- ALL patients.

A transcribed ultraconserved noncoding RNA, uc.285+, promotes colorectal cancer proliferation through dual targeting of CDC42 by directly binding mRNA and protein.

The transcribed ultraconserved region (T-UCR) belongs to a new type of lncRNAs that are conserved in homologous regions of the rat, mouse and human genomes. A lot of research has reported that differential expression of T-UCRs can influence the development of various cancers, revealing the ability of T-UCRs as new therapeutic targets or potential cancer biomarkers. Most studies on the molecular mechanisms of T-UCRs in cancer have focused on ceRNA regulatory networks and interactions with target proteins, but the present study reveals an innovative dual-targeted regulatory approach in which T-UCRs bind directly to mRNAs and directly to proteins. We screened T-UCRs that may be related to colorectal cancer (CRC) by performing a whole-genome T-UCR gene microarray and further studied the functional mechanism of T-UCR uc.285+ in the development of CRC. Modulation of uc.285+ affected the proliferation of CRC cell lines and influenced the expression of the CDC42 gene. We also found that uc.285+ promoted the proliferation of CRC cells by directly binding to CDC42 mRNA and enhancing its stability while directly binding to CDC42 protein and affecting its stability. In short, our research on the characteristics of cell proliferation found that uc.285+ has a biological function in promoting CRC proliferation. uc.285+ may have considerable potential as a new diagnostic biomarker for CRC.

Myeloid Deletion of Cdc42 Protects Liver From Hepatic Ischemia-Reperfusion Injury via Inhibiting Macrophage-Mediated Inflammation in Mice.

BACKGROUND & AIMS: Hepatic ischemia-reperfusion injury (HIRI) often occurs in liver surgery, such as partial hepatectomy and liver transplantation, in which myeloid macrophage-mediated inflammation plays a critical role. Cell division cycle 42 (Cdc42) regulates cell migration, cytoskeleton rearrangement, and cell polarity. In this study, we explore the role of myeloid Cdc42 in HIRI. METHODS: Mouse HIRI models were established with 1-hour ischemia followed by 12-hour reperfusion in myeloid Cdc42 knockout (Cdc42mye) and Cdc42flox mice. Myeloid-derived macrophages were traced with RosamTmG fluorescent reporter under LyzCre-mediated excision. The experiments for serum or hepatic enzymic activities, histologic and immunologic analysis, gene expressions, flow cytometry analysis, and cytokine antibody array were performed. RESULTS: Myeloid deletion of Cdc42 significantly alleviated hepatic damages with the reduction of hepatic necrosis and inflammation, and reserved hepatic functions following HIRI in mice. Myeloid Cdc42 deficiency suppressed the infiltration of myeloid macrophages, reduced the secretion of proinflammatory cytokines, restrained M1 polarization, and promoted M2 polarization of myeloid macrophages in livers. In addition, inactivation of Cdc42 promoted M2 polarization via suppressing the phosphorylation of STAT1 and promoting phosphorylation of STAT3 and STAT6 in myeloid macrophages. Furthermore, pretreatment with Cdc42 inhibitor, ML141, also protected mice from hepatic ischemia-reperfusion injury. CONCLUSIONS: Inhibition or deletion of myeloid Cdc42 protects liver from HIRI via restraining the infiltration of myeloid macrophages, suppressing proinflammatory response, and promoting M2 polarization in macrophages.

The distinct localization of CDC42 isoforms is responsible for their specific functions during migration.

The small G-protein CDC42 is an evolutionary conserved polarity protein and a key regulator of polarized cell functions, including directed cell migration. In vertebrates, alternative splicing gives rise to two CDC42 proteins: the ubiquitously expressed isoform (CDC42u) and the brain isoform (CDC42b), which only differ in their carboxy-terminal sequence, including the CAAX motif essential for their association with membranes. We show that these divergent sequences do not directly affect the range of CDC42's potential binding partners but indirectly influence CDC42-driven signaling by controlling the subcellular localization of the two isoforms. In astrocytes and neural precursors, which naturally express both variants, CDC42u associates with the leading-edge plasma membrane of migrating cells, where it recruits the Par6-PKCζ complex to fulfill its polarity function. In contrast, CDC42b mainly localizes to intracellular membrane compartments, where it regulates N-WASP-mediated endocytosis. Both CDC42 isoforms contribute their specific functions to promote the chemotaxis of neural precursors, demonstrating that their expression pattern is decisive for tissue-specific cell behavior.

RhoU forms homo-oligomers to regulate cellular responses.

RhoU is an atypical member of the Rho family of small G-proteins, which has N- and C-terminal extensions compared to the classic Rho GTPases RhoA, Rac1 and Cdc42, and associates with membranes through C-terminal palmitoylation rather than prenylation. RhoU mRNA expression is upregulated in prostate cancer and is considered a marker for disease progression. Here, we show that RhoU overexpression in prostate cancer cells increases cell migration and invasion. To identify RhoU targets that contribute to its function, we found that RhoU homodimerizes in cells. We map the region involved in this interaction to the C-terminal extension and show that C-terminal palmitoylation is required for self-association. Expression of the isolated C-terminal extension reduces RhoU-induced activation of p21-activated kinases (PAKs), which are known downstream targets for RhoU, and induces cell morphological changes consistent with inhibiting RhoU function. Our results show for the first time that the activity of a Rho family member is stimulated by self-association, and this is important for its activity.

Salmonella engages CDC42 effector protein 1 for intracellular invasion.

Human enterocytes are primary targets of infection by invasive bacterium Salmonella Typhimurium, and studies using nonintestinal epithelial cells established that S. Typhimurium activates Rho family GTPases, primarily CDC42, to modulate the actin cytoskeletal network for invasion. The host intracellular protein network that engages CDC42 and influences the pathogen's invasive capacity are relatively unclear. Here, proteomic analyses of canonical and variant CDC42 interactomes identified a poorly characterized CDC42 interacting protein, CDC42EP1, whose intracellular localization is rapidly redistributed and aggregated around the invading bacteria. CDC42EP1 associates with SEPTIN-7 and Villin, and its relocalization and bacterial engagement depend on host CDC42 and S. Typhimurium's capability of activating CDC42. Unlike CDC42, CDC42EP1 is not required for S. Typhimurium's initial cellular entry but is found to associate with Salmonella-containing vacuoles after long-term infections, indicating a contribution to the pathogen's intracellular growth and replication. These results uncover a new host regulator of enteric Salmonella infections, which may be targeted to restrict bacterial load at the primary site of infection to prevent systemic spread.

Rho GTPase activity crosstalk mediated by Arhgef11 and Arhgef12 coordinates cell protrusion-retraction cycles.

Rho GTPases play a key role in the spatio-temporal coordination of cytoskeletal dynamics during cell migration. Here, we directly investigate crosstalk between the major Rho GTPases Rho, Rac and Cdc42 by combining rapid activity perturbation with activity measurements in mammalian cells. These studies reveal that Rac stimulates Rho activity. Direct measurement of spatio-temporal activity patterns show that Rac activity is tightly and precisely coupled to local cell protrusions, followed by Rho activation during retraction. Furthermore, we find that the Rho-activating Lbc-type GEFs Arhgef11 and Arhgef12 are enriched at transient cell protrusions and retractions and recruited to the plasma membrane by active Rac. In addition, their depletion reduces activity crosstalk, cell protrusion-retraction dynamics and migration distance and increases migration directionality. Thus, our study shows that Arhgef11 and Arhgef12 facilitate exploratory cell migration by coordinating cell protrusion and retraction by coupling the activity of the associated regulators Rac and Rho.

PI3K Functions Downstream of Cdc42 to Drive Cancer phenotypes in a Melanoma Cell Line.

Rho proteins are part of the Ras superfamily, which function to modulate cytoskeletal dynamics including cell adhesion and motility. Recently, an activating mutation in Cdc42, a Rho family GTPase, was found in a patient sample of melanoma. Previously, our work had shown the PI3K was important downstream of mutationally active Cdc42. Our present study sought to determine whether PI3K was a crucial downstream partner for Cdc42 in a melanoma cells line with a BRAF mutation, which is the most common mutation in cutaneous melanoma. In this work we were able to show that Cdc42 contributes to proliferation, anchorage-independent growth, cell motility and invasion. Treatment with a pan-PI3K inhibitor was able to effectively ameliorate all these cancer phenotypes. These data suggest that PI3K may be an important target downstream of Cdc42 in melanoma.

"From molecular to clinic": The pivotal role of CDC42 in pathophysiology of human papilloma virus related cancers and a correlated sensitivity of afatinib.

Background: Human papilloma virus (HPV)-related cancers are global health challenge. Insufficient comprehension of these cancers has impeded the development of novel therapeutic interventions. Bioinformatics empowered us to investigate these cancers from new entry points. Methods: DNA methylation data of cervical squamous cell carcinoma (CESC) and anal squamous cell carcinoma (ASCC) were analyzed to identify the significantly altered pathways. Through analyses integrated with RNA sequencing data of genes in these pathways, genes with strongest correlation to the TNM staging of CESC was identified and their correlations with overall survival in patients were assessed. To find a potential promising drug, correlation analysis of gene expression levels and compound sensitivity was performed. In vitro experiments were conducted to validate these findings. We further performed molecular docking experiments to explain our findings. Results: Significantly altered pathways included immune, HPV infection, oxidative stress, ferroptosis and necroptosis. 10 hub genes in these pathways (PSMD11, RB1, SAE1, TAF15, TFDP1, CORO1C, JOSD1, CDC42, KPNA2 and NUP62) were identified, in which only CDC42 high expression was statistically significantly correlated with overall survival (Hazard Ratio: 1.6, P = 0.045). Afatinib was then screened out to be tested. In vitro experiments exhibited that the expression level of CDC42 was upregulated in HaCaT/A431 cells transfected with HPV E6 and E7, and the inhibitory effect of afatinib on proliferation was enhanced after transfection. CDC42-GTPase-effector interface-EGFR-afatinib was found to be a stable complex with a highest ZDOCK score of 1264.017. Conclusion: We identified CDC42 as a pivotal gene in the pathophysiology of HPV-related cancers. The upregulation of CDC42 could be a signal for afatinib treatment and the mechanism in which may be an increased affinity of EGFR to afatinib, inferred from a high stability in the quaternary complex of CDC42-GTPase-effector interface-EGFR-afatinib.

ARHGAP17 regulates the spatiotemporal activity of Cdc42 at invadopodia.

Invadopodia formation is regulated by Rho GTPases. However, the molecular mechanisms that control Rho GTPase signaling at invadopodia remain poorly understood. Here, we have identified ARHGAP17, a Cdc42-specific RhoGAP, as a key regulator of invadopodia in breast cancer cells and characterized a novel ARHGAP17-mediated signaling pathway that controls the spatiotemporal activity of Cdc42 during invadopodia turnover. Our results show that during invadopodia assembly, ARHGAP17 localizes to the invadopodia ring and restricts the activity of Cdc42 to the invadopodia core, where it promotes invadopodia growth. Invadopodia disassembly starts when ARHGAP17 translocates from the invadopodia ring to the core, in a process that is mediated by its interaction with the Cdc42 effector CIP4. Once at the core, ARHGAP17 inactivates Cdc42 to promote invadopodia disassembly. Our results in invadopodia provide new insights into the coordinated transition between the activation and inactivation of Rho GTPases.

Negative cooperativity underlies dynamic assembly of the Par complex regulators Cdc42 and Par-3.

The Par complex polarizes diverse animal cells through the concerted action of multiple regulators. Binding to the multi-PDZ domain containing protein Par-3 couples the complex to cortical flows that construct the Par membrane domain. Once localized properly, the complex is thought to transition from Par-3 to the Rho GTPase Cdc42 to activate the complex. While this transition is a critical step in Par-mediated polarity, little is known about how it occurs. Here, we used a biochemical reconstitution approach with purified, intact Par complex and qualitative binding assays and found that Par-3 and Cdc42 exhibit strong negative cooperativity for the Par complex. The energetic coupling arises from interactions between the second and third PDZ protein interaction domains of Par-3 and the aPKC Kinase-PBM (PDZ binding motif) that mediate the displacement of Cdc42 from the Par complex. Our results indicate that Par-3, Cdc42, Par-6, and aPKC are the minimal components that are sufficient for this transition to occur and that no external factors are required. Our findings provide the mechanistic framework for understanding a critical step in the regulation of Par complex polarization and activity.

The Cdc42 GAP Rga6 promotes monopolar outgrowth of spores.

The molecular mechanisms underlying the establishment of the monopolar growth of fission yeast spores have been less characterized. Here, we report that the Cdc42 GTPase-activating protein (GAP) Rga6 is required for promoting monopolar growth during spore germination. The absence of Rga6 increases the number of spores that grow in a bipolar fashion. Rga6 decorates the non-growing cortical region, binds phosphatidylinositol 4,5-bisphosphate, and colocalizes with the phosphatidylinositol 4,5-bisphosphate-binding protein Opy1. Overexpression of Opy1 diminishes the cortical localization of Rga6. The characteristic localization of Rga6 on the cell cortex depends on the C-terminal PBR region of Rga6. Moreover, engineered chimera composed of the Rga6 C-terminal PBR region fused to the GAP domain of Rga3 or Rga4 are sufficient to rescue the spore growth phenotype caused by the absence of Rga6. Hence, our work establishes a paradigm in which the lipid composition of the plasma membrane directs polarized cell growth by specifying the cortical localization of a GAP protein.

Stress-induced cell depolarization through the MAP kinase-Cdc42 axis.

General stress responses, which sense environmental or endogenous signals, aim at promoting cell survival and fitness during adverse conditions. In eukaryotes, mitogen-activated protein (MAP) kinase-driven cascades trigger a shift in the cell's gene expression program as a cellular adaptation to stress. Here, we review another aspect of activated MAP kinase cascades reported in fission yeast: the transient inhibition of cell polarity in response to oxidative stress. The phosphorylation by a stress-activated MAP kinase of regulators of the GTPase cell division cycle 42 (Cdc42) causes a transient inhibition of polarized cell growth. The formation of growth sites depends on limiting and essential polarity components. We summarize here some processes in which inhibition of Cdc42 may be a general mechanism to regulate polarized growth also under physiological conditions.

In silico identification and characterization of small-molecule inhibitors specific to RhoG/Rac1 signaling pathway.

Rho family GTPases serve as molecular switches in numerous cellular processes, and their overexpression is involved in disease conditions. RhoG is one of the less explored Rho GTPases with significant sequential and structural homology with Rac1. Experimental mutations in RhoG (i.e., RhoGG12V and RhoGQ61L) are shown to dysregulate cell migration. Thus, targeting upstream activators of RhoG, such as guanine nucleotide exchange factors (GEFs), maybe an important strategy for inhibiting RhoG activation. In the current study, we have modelled the 3D structure of RhoG with greater accuracy as confirmed through PROCHECK, ProSA, and Verify3D. Our results indicate that 90.4% of residues are in the Ramachandran plots favoured region, with the Z-score of -6.46, and 87.96% of residues had an averaged 3D-1D score ≥0.2. Further, we have evaluated and binding dynamics of ten Rac1 inhibitors to investigate their potential to inhibit RhoG by targeting GEFs binding grooves. To this end, the binding energy of the docked complexes of the wild-type (WT) RhoG and its mutant proteins with inhibitor molecules was calculated using the MM/PBSA method. Our results from docking studies showed that macrolide1 binds efficiently with the GEF site of WT RhoG and the mutants mentioned above. However, an extensive analysis using MD simulations (200 ns) showed that the Rac1 based inhibitor, EHop-016, and NSC23766 might bind with greater affinity to GEF sites of mutants and WT RhoG. Thus, the results from the study indicate that Rac1 inhibitors have the potential for use as therapeutics in conditions involving dysregulation of RhoG.Communicated by Ramaswamy H. Sarma.