Rho Proteins Research Articles

Estimating SARS-CoV-2 Omicron XBB.1.5 Spike-Directed Functional Antibody Levels From an Anti-Receptor Binding Domain Wuhan-Hu-1-Based Commercial Immunoassay Results.

We investigated whether antibody concentrations measured in plasma using the Roche Elecsys® Anti-SARS-CoV-2 S assay (targeting the receptor binding domain, RBD) could estimate levels of Wuhan-Hu-1 and Omicron XBB.1.5 spike-directed antibodies with neutralizing ability (NtAb) or those mediating NK-cell activity. We analyzed 135 plasma samples from 39 vaccinated elderly nursing home residents. A strong correlation was found for NtAb against both Wuhan-Hu-1 (Rho = 0.73, p < 0.001) and Omicron XBB.1.5 (sub)variants (Rho = 0.73, p < 0.001). Moderate positive correlations were observed for NK-cell activity, based on lysosome-associated membrane protein 1 (LAMP1)-producing NK cells stimulated with Wuhan-Hu-1 (Rho = 0.43, p < 0.001) and Omicron XBB.1.5 spike proteins (Rho = 0.50, p < 0.001). Similarly, interferon-gamma (IFN-γ)-producing NK-cell frequencies showed moderate correlations (Wuhan-Hu-1: Rho = 0.43, p < 0.001; Omicron XBB.1.5: Rho = 0.50, p < 0.001). Random Forest models accurately predicted NtAb levels against Wuhan-Hu-1 (R2 = 0.72), though models for Omicron XBB.1.5 were less robust. Anti-RBD antibody concentrations of 4.73 and 5.02 log10 BAU/mL predicted high NtAb levels for Wuhan-Hu-1 and Omicron XBB.1.5, respectively. Antibody thresholds for predicting functional NK cell-mediated responses were 4.73 log10 and 4.54 log10 BAU/mL for Wuhan-Hu-1 and Omicron XBB.1.5, respectively. For LAMP1-producing NK cells, the thresholds were 4.94 and 4.75 log10 BAU/mL for Wuhan-Hu-1 and Omicron XBB.1.5, respectively. In summary, total anti-RBD antibody levels measured by the Roche assay may allow inference of NtAb levels and, to a lesser extent, Fc-mediated NK-cell responses against Omicron XBB.1.5.

Function of a complex of p-Y42 RhoA GTPase and pyruvate kinase M2 in EGF signaling pathway in glioma cells.

Epidermal growth factor (EGF) is known to be a critical stimulant for inducing the proliferation of glioma cancer cells. In our study, we observed that GST-RhoA binds to pyruvate kinase M2 (PKM2) invitro. While EGF reduced the levels of RhoA protein, it significantly increased p-Y42 RhoA, as well as PKM1 and PKM2 in LN18 glioma cell line. We determined that RhoA undergoes degradation through ubiquitination involving SCF1 and Smurf1. Interestingly, we observed that p-Y42 RhoA binds to PKM2, while the dephosphomimetic form, RhoA Y42F, did not. Additionally, our observation revealed that PKM2 stabilized both RhoA and p-Y42 RhoA. Importantly, RhoA, p-Y42 RhoA, and PKM2, but not RhoA-GTP, were localized in the nucleus upon EGF stimulation. Knockdown of RhoA with siRNA resulted in the reduced levels of phosphoglycerate kinase1 (PGK1) and microtubule affinity-regulating kinase 4 (MARK). Furthermore, we found that the promoter of PGK1 was associated with β-catenin and YAP. Notably, p-Y42 RhoA and PKM2 co-immunoprecipitated with β-catenin and YAP. Based on these findings, we proposed a novel mechanism by which p-Y42 RhoA and PKM2, in conjunction with β-catenin and YAP, regulate PGK1 expression, contributing to the progression of glioma upon EGF.

Functionally important components of the transcription elongation complex involved in Rho-dependent termination.

Bacterial transcription terminator, Rho is an RNA-dependent ATPase that terminates transcription. Several structures of pre-termination complexes of the Rho-transcription elongation complex (EC) revealed a static picture of components of the EC that come close to the nascent RNA-bound Rho, where many of the residues of EC reside≤10Å from the Rho residues. However, the in vitro-formed Rho-EC complexes do not reveal the in vivo Rho-EC dynamic interaction patterns during the termination process. Here we report synthetic defect analyses of various combinations of the mutations in RNAP β, β' and ω-subunits, NusA, NusG, and Rho proteins to delineate the functional network of this process. Several mutations in the β-flap and β'-Zn-finger and -Clamp helices domains of RNAP are synthetically defective in the presence of Rho mutants indicating functional involvement of these domains. Mutations in the NusA RNA-binding domains were synthetically defective with the Rho mutants suggesting its involvement. Our genetic analyses also revealed functional antagonisms between the ω-subunit of RNAP and the NusG-CTD during termination. We concluded that the regions surrounding the RNA exit channel, the RNA-binding domains of NusA, the RNAP ω-subunit, and NusG-CTD constitute a functional network with Rho just before the onset of in vivo Rho-dependent termination.

Bortezomib induces Rho-dependent hyperpermeability of endothelial cells synergistically with inflammatory mediators

BackgroundBortezomib (BTZ), a selective 26 S proteasome inhibitor, is clinically useful in treating multiple myeloma and mantle cell lymphoma. BTZ exerts its antitumor effect by suppressing nuclear factor-B in myeloma cells, promoting endothelial cell apoptosis, and inhibiting angiogenesis. Despite its success, pulmonary complications, such as capillary leak syndrome of the vascular hyperpermeability type, were reported prior to its approval. Although the incidence of these complications has decreased with the use of steroids, the underlying mechanism remains unclear. This study aims to investigate how BTZ influences endothelial cell permeability.MethodsWe examined the impact of BTZ on vascular endothelial cells, focusing on its effects on RhoA and RhoC proteins. Stress fiber formation, a known indicator of increased permeability, was assessed through the Rho/ROCK pathway.ResultsBTZ was found to elevate the protein levels of RhoA and RhoC in vascular endothelial cells, leading to stress fiber formation via the Rho/ROCK pathway. This process resulted in enhanced vascular permeability in a Rho-dependent manner. Furthermore, the stress fiber formation induced by BTZ had synergistic effects with the inflammatory mediator histamine.ConclusionsOur findings suggest that BTZ accumulates RhoA and RhoC proteins in endothelial cells, amplifying the inflammatory mediator-induced increase in the active GTP-bound state of Rho, thereby exaggerating vascular permeability during pulmonary inflammation. This study provides novel insights into the molecular mechanism underlying the pulmonary complications of BTZ, suggesting that BTZ may enhance inflammatory responses in pulmonary endothelial cells by increasing RhoA and RhoC protein levels.

Rap1 and mTOR signaling pathways drive opposing immunotoxic effects of structurally similar aryl-OPFRs, TPHP and TOCP.

Aryl organophosphorus flame retardants (aryl-OPFRs), commonly used product additives with close ties to daily life, have been regrettably characterized by multiple well-defined toxicity risks. Triphenyl phosphate (TPHP) and tri-o-cresyl phosphate (TOCP), two structurally similar aryl-OPFRs, were observed in our previous study to exhibit contrasting immunotoxic effects on THP-1 macrophages, yet the underlying mechanisms remain unclear. This study sought to address the knowledge gap by integrating transcriptomic and metabolomic analyses to elucidate the intricate mechanisms. During individual omics analyses, we unfortunately only obtained highly similar results for both TPHP and TOCP, failing to identify the key reasons for their differences. These results revealed comparable disturbances induced by both compounds, including disruptions in nucleic acid synthesis and energy metabolism, blocking ADP to ATP conversion by reducing TCA cycle intermediates, consequently leading to ATP depletion. However, through integrative analysis, specific pathways affected by each compound were successfully identified, shedding light on their unique effects. TPHP reduced GTP levels necessary for Rap1 activation, thereby inhibiting phagocytosis and adhesion of THP-1 macrophages. Conversely, TOCP stimulated the mTOR signaling pathway, enhancing phosphorylation of downstream proteins S6K, RHOA, and PKC, consequently promoting immune responses. This study not only clarified the distinct immunotoxic mechanisms of TPHP and TOCP but also provided critical insights into how structural variations in aryl-OPFRs can lead to markedly different immune responses, thereby informing future risk assessments and regulatory strategies for these compounds.

Intercellular junction-driven stromal cell stacking in a confined 3D microcavity.

Understanding the detailed mechanisms driving fibroblast migration within native tissue settings during pathophysiological events presents a critical research challenge. In this study, we elucidate how stromal cells migrate and contribute to the development of three-dimensional (3D) cellular aggregates within confined microcavities. Integrin α5β1 and β-catenin (β-cat) are central in guiding this collective migration and achieving optimal filling of the microcavity. When β-cat is suppressed, cells tend to migrate more sporadically, leading to less efficient cellular organization. Furthermore, we also detail the pivotal roles of Cx43 and N-cadherin (N-cad) in orchestrating collective migration and in shaping efficient cellular stacking. Suppressing gap junctions, especially Cx43, significantly impacts the extracellular matrix expression, integrin α5 and β1, and other elements in the 3D construct, emphasizing the importance of physicochemical cell-cell interactions. The distribution patterns of N-cad and focal adhesion kinase (FAK) further corroborate the essential roles in forming cell-cell junctions and FAK in establishing the foundational layer that underpins the cell stacking within the microcavity. Interestingly, neither Rho-associated protein kinase (ROCK) nor RhoA significantly alter the cell migration pattern toward microcavity. These findings provide fresh perspectives on fibroblast activities in 3D space, enriching our understanding and offering implications for advancements in wound healing and tissue engineering.

The Heterotaxy Gene CCDC11 Is Important for Cytokinesis via RhoA Regulation.

Mutations in CCDC11 (cfap53) have been identified in multiple patients with heterotaxy (Htx), a disorder of left-right (LR) patterning of the internal organs. In Xenopus, depletion of Ccdc11 causes defects in LR patterning, recapitulating the patient phenotype. Upon Ccdc11 depletion, monociliated cells of the Left-Right Organizer (LRO) exhibit multiple cilia per cell. Unexpectedly, we found that Ccdc11 is necessary for successful cytokinesis, explaining the multiciliation phenotype observed in Ccdc11-depleted cells. The small GTPase RhoA is critical for cytokinesis, and our Ccdc11 depletion phenotypes are reminiscent of RhoA loss of function. Here, we demonstrate that during cytokinesis CCDC11 is localized to the cytokinetic contractile ring overlapping with RhoA, and CCDC11 regulates total RhoA protein levels. Our results connect CCDC11 to cytokinesis and LR patterning via RhoA regulation, providing a potential mechanism for heterotaxy disease pathogenesis.

RHO subfamily of small GTPases in the development and function of hematopoietic cells.

RHOA, RHOB, and RHOC comprise a subfamily of RHO GTPase proteins famed for controlling cytoskeletal dynamics. RHO proteins operate downstream of multiple signals emerging from the microenvironment, leading to diverse cell responses, such as proliferation, adhesion, and migration. Therefore, RHO signaling has been centrally placed in the regulation of blood cells. Despite their high homology, unique roles of RHOA, RHOB, and RHOC have been described in hematopoietic cells. In this article, we overview the contribution of RHO proteins in the development and function of each blood cell lineage. Additionally, we highlight the aberrations of the RHO signaling pathways found in hematological malignancies, providing clues for the identification of new therapeutic targets.

Complex sporulation-specific expression of transcription termination factor Rho highlights its involvement in Bacillus subtilis cell differentiation

Termination factor Rho, responsible for the main factor-dependent pathway of transcription termination and the major inhibitor of antisense transcription, is an emerging regulator of various physiological processes in microorganisms. In Gram-positive bacterium Bacillus subtilis, Rho is involved in the control of cell adaptation to starvation and, in particular, in the control of sporulation, a complex differentiation program leading to formation of a highly resistant dormant spore. While the initiation of sporulation requires a decrease in Rho protein levels during the transition to stationary phase, the mechanisms regulating the expression of rho gene throughout the cell cycle remain largely unknown. Here we show that a drop in the activity of the vegetative SigA-dependent rho promoter causes the inhibition of rho expression in stationary phase. However, after the initiation of sporulation, rho gene is specifically reactivated in two compartments of the sporulating cell using distinct mechanisms. In the mother cell, rho expression occurs by read-through transcription initiated at the SigH-dependent promoter of the distal spo0F gene. In the forespore, rho gene is transcribed from the intrinsic promoter recognized by the alternative sigma factor SigF. These regulatory elements ensure the activity of Rho during sporulation, which appears important for the proper formation of spores. We provide experimental evidence that disruption of the spatiotemporal expression of rho during sporulation affects the resistance properties of spores, their morphology, and the ability to return to vegetative growth under favorable growth conditions.

Peripherally-restricted recurrent infection by engineered E. coli strain modulates hippocampal proteome promoting memory impairments in a rat model

Commensal bacteria that breach endothelial barrier has been reported to induce low grade chronic inflammation producing disease symptoms in major peripheral tissues. In this study, we investigated the role of genetically modified cellular invasive form of commensal E. coli K12 (SK3842) in cognitive impairment. Low-grade systemic infection model was developed using recurring peripheral inoculation of live bacteria in Wistar rats. To examine memory parameters, Novel object recognition test and Radial arm maze test were performed. Differential protein expression profiling of rat hippocampus was carried out using LC-MS/MS and subsequently quantified using SWATH. HBA1/2, NEFH, PFN1 and ATP5d were chosen for validation using quantitative RT-PCR. Results showed drastic decline in Recognition memory of the SK3842 infected rats. Reference and Working Memory of the infected group were also significantly reduced in comparison to control group. Proteome analysis using LC-MS/MS coupled with SWATH revealed differential expression of key proteins that are crucial for the maintenance of various neurological functions. Moreover, expression of NEFH and PFN1transcripts were found to be in line with the proteomics data. Protein interaction network of these validated proteins generated by STRING database converged to RhoA protein. Thus, the present study establishes an association between peripheral infection of a hippocampal protein network dysregulation and overall memory decline.

The Zpr-3 Antibody Recognizes the 320-354 Region of Rho and Labels Both Rods and Green Cones in Zebrafish.

Retinal markers with high quality and specificity are important for the observation of pathologic changes of retinal cells during retinal development, degeneration, and regeneration. The zpr-3 antibody is widely used to label rods in zebrafish, but the exact antigen is still unknown. In this study, we provided evidence to demonstrate that the antigen gene of zpr-3 is rho, which encodes the rod opsin, and the exact epitope of zpr-3 is the 320-354 region of Rho protein. More importantly, our immunofluorescence assays indicated that zpr-3 labels both the outer segments of rods and green cones on zebrafish retinal sections, probably due to the cross-reaction with the green-cone opsin. Our work is valuable for the scientific community to interpret the experimental data involving the zpr-3 antibody.

The role of the RHOA/ROCK pathway in the regulation of myometrial stages throughout pregnancy

BackgroundControlling uterine contractile activity is essential to regulate the duration of pregnancy. During most of the pregnancy, the uterus does not contract (i.e., myometrial quiescence). The myometrium recovers its contractile phenotype at around 36 weeks (i.e., myometrial activation) through several mechanisms. The RHOA/ROCK pathway plays a vital role in facilitating muscular contractions by calcium sensitization in humans. Yet, the role of this pathway during different myometrial stages, including quiescence, has not been elucidated. Objectivewe aimed to study the role of the RHOA/ROCK pathway in the regulation of the different myometrial stages throughout pregnancy. Specifically, we hypothesized that the inhibition of the components of the RHOA/ROCK pathway play an important role in maintaining uterine quiescence. Study designMyometrial samples were obtained from pregnant individuals who underwent cesarean section. Pregnant individuals who delivered preterm without labor (myometrial quiescence), preterm with labor (nonphysiological myometrial stimulation), term not in labor (activation), and term in labor (physiological myometrial stimulation) were included. The mRNA and protein expression of RHOA, ROCK I, ROCK II, RND1-3, and ROCK activity through pMYTP1 were evaluated. ResultsWe found that the human myometrium constitutively expressed RHOA/ROCK pathway components throughout pregnancy. No changes in the components of the RHOA/ROCK pathway were found during quiescence. Moreover, the RHOA protein and ROCK activity increased in the myometrium during labor, supporting the hypothesis that this pathway participates in maintaining the contractile activity of the myometrium. This study provides insight into the role of the RHOA/ROCK pathway in controlling myometrial contractile activity during pregnancy.

Filamin A regulates platelet shape change and contractile force generation via phosphorylation of the myosin light chain.

Platelets are critical mediators of hemostasis and thrombosis. Platelets circulate as discs in their resting form but change shape rapidly upon activation by vascular damage and/or soluble agonists such as thrombin. Platelet shape change is driven by a dynamic remodeling of the actin cytoskeleton. Actin filaments interact with the protein myosin, which is phosphorylated on the myosin light chain (MLC) upon platelet activation. Actin-myosin interactions trigger contraction of the actin cytoskeleton, which drives platelet spreading and contractile force generation. Filamin A (FLNA) is an actin cross-linking protein that stabilizes the attachment between subcortical actin filaments and the cell membrane. In addition, FLNA binds multiple proteins and serves as a critical intracellular signaling scaffold. Here, we used platelets from mice with a megakaryocyte/platelet-specific deletion of FLNA to investigate the role of FLNA in regulating platelet shape change. Relative to controls, FLNA-null platelets exhibited defects in stress fiber formation, contractile force generation, and MLC phosphorylation in response to thrombin stimulation. Blockade of Rho kinase (ROCK) and protein kinase C (PKC) with the inhibitors Y27632 and bisindolylmaleimide (BIM), respectively, also attenuated MLC phosphorylation; our data further indicate that ROCK and PKC promote MLC phosphorylation through independent pathways. Notably, the activity of both ROCK and PKC was diminished in the FLNA-deficient platelets. We conclude that FLNA regulates thrombin-induced MLC phosphorylation and platelet contraction, in a ROCK- and PKC-dependent manner.

Interleukin receptor therapeutics attenuate inflammation in canine synovium following cruciate ligament injury

ObjectiveIn the knee, synovial fibrosis after ligamentous injury is linked to progressive joint pain and stiffness. The objective of this study was to evaluate changes in synovial architecture, mechanical properties, and transcriptional profiles following naturally occurring cruciate ligament injury in canines and to test potential therapeutics that target drivers of synovial inflammation and fibrosis. DesignSynovia from canines with spontaneous cruciate ligament tears and from healthy knees were assessed via histology (n=10/group) and micromechanical testing (n=5/group) to identify changes in tissue architecture and stiffness. Additional samples (n=5/group) were subjected to RNA-sequencing to define the transcriptional response to injury. Finally, synovial tissue samples from injured animals (n=6 (IL1) or n=8 (IL6)/group) were assessed in vitro for response to therapeutic molecules directed against interleukin (IL) signaling (IL1 or IL6). ResultsCruciate injury resulted in increased synovial fibrosis, vascularity, inflammatory cell infiltration, and intimal hyperplasia. Additionally, the stiffness of both the intima and subintima regions were higher in diseased compared to healthy tissue. Differential gene expression analysis showed that diseased synovium had an upregulation of immune response and cell adhesion pathways and a downregulation of Rho protein transduction pathways. In vitro application of small molecule therapeutics targeting IL1 (anakinra) or IL6 (tocilizumab) dampened expression of inflammatory and matrix deposition mediators. ConclusionSpontaneous cruciate ligament injury in canines is associated with synovial inflammation and fibrosis in a relevant model for testing emerging intra-articular treatments. Small molecule therapeutics targeting IL pathways may be ideal interventions for delivery to the joint space after injury.

Recurrent RhoGAP gene fusion CLDN18-ARHGAP26 promotes RHOA activation and focal adhesion kinase and YAP-TEAD signalling in diffuse gastric cancer

ObjectiveGenomic studies of gastric cancer have identified highly recurrent genomic alterations impacting RHO signalling, especially in the diffuse gastric cancer (DGC) histological subtype. Among these alterations are interchromosomal translations leading...

Diversification of the Rho transcription termination factor in bacteria.

Correct termination of transcription is essential for gene expression. In bacteria, factor-dependent termination relies on the Rho factor, that classically has three conserved domains. Some bacteria also have a functional insertion region. However, the variation in Rho structure among bacteria has not been analyzed in detail. This study determines the distribution, sequence conservation, and predicted features of Rho factors with diverse domain architectures by analyzing 2730 bacterial genomes. About half (49.8%) of the species analyzed have the typical Escherichia coli like Rho while most of the other species (39.8%) have diverse, atypical forms of Rho. Besides conservation of the main domains, we describe a duplicated RNA-binding domain present in specific species and novel variations in the bicyclomycin binding pocket. The additional regions observed in Rho proteins exhibit remarkable diversity. Commonly, however, they have exceptional amino acid compositions and are predicted to be intrinsically disordered, to undergo phase separation, or have prion-like behavior. Phase separation has recently been shown to play roles in Rho function and bacterial fitness during harsh conditions in one species and this study suggests a more widespread role. In conclusion, diverse atypical Rho factors are broadly distributed among bacteria, suggesting additional cellular roles.

Substrate stress relaxation regulates neural stem cell fate commitment

Adult neural stem cells (NSCs) reside in the dentate gyrus of the hippocampus, and their capacity to generate neurons and glia plays a role in learning and memory. In addition, neurodegenerative diseases are known to be caused by a loss of neurons and glial cells, resulting in a need to better understand stem cell fate commitment processes. We previously showed that NSC fate commitment toward a neuronal or glial lineage is strongly influenced by extracellular matrix stiffness, a property of elastic materials. However, tissues in vivo are not purely elastic and have varying degrees of viscous character. Relatively little is known about how the viscoelastic properties of the substrate impact NSC fate commitment. Here, we introduce a polyacrylamide-based cell culture platform that incorporates mismatched DNA oligonucleotide-based cross-links as well as covalent cross-links. This platform allows for tunable viscous stress relaxation properties via variation in the number of mismatched base pairs. We find that NSCs exhibit increased astrocytic differentiation as the degree of stress relaxation is increased. Furthermore, culturing NSCs on increasingly stress-relaxing substrates impacts cytoskeletal dynamics by decreasing intracellular actin flow rates and stimulating cyclic activation of the mechanosensitive protein RhoA. Additionally, inhibition of motor-clutch model components such as myosin II and focal adhesion kinase partially or completely reverts cells to lineage distributions observed on elastic substrates. Collectively, our results introduce a unique system for controlling matrix stress relaxation properties and offer insight into how NSCs integrate viscoelastic cues to direct fate commitment.

Growth Differentiation Factor 15 and Matrix Metalloproteinase 3 in Plasma as Biomarkers for Neuropathy and Nephropathy in Type 1 Diabetes.

Diabetic neuropathy and nephropathy are common complications of type 1 diabetes (T1D). The symptoms are often elusive in the early stages, and available diagnostic methods can be improved using biomarkers. Matrix metalloproteinase 3 (MMP-3) has been identified in the kidneys and is thought to be involved in diabetic nephropathy. Growth differentiation factor 15 (GDF-15) has been suggested to have positive effects in diabetes, but is otherwise associated with adverse effects such as cardiovascular risk, declined kidney function, and neurodegeneration. This study aims to investigate plasma MMP-3 and GDF-15 as systemic biomarkers for diabetic neuropathy and nephropathy in T1D. The study involves patients with childhood-onset T1D (n = 48, age 38 ± 4 years) and a healthy control group (n = 30, age 38 ± 5 years). Neurophysiology tests, evaluations of albuminuria, and measurements of routine biochemical markers were conducted. The neuropathy impairment assessment (NIA) scoring system, where factors such as loss of sensation and weakened reflexes are evaluated, was used to screen for symptoms of neuropathy. MMP-3 and GDF-15 concentrations were determined in heparinized plasma using ELISA kits. In total, 9 patients (19%) had albuminuria, and 25 (52%) had diabetic neuropathy. No significant differences were found in MMP-3 concentrations between the groups. GDF-15 levels were higher in T1D, with median and interquartile range (IQR) of 358 (242) pg/mL in T1D and 295 (59) in controls (p < 0.001). In the merged patient group, a positive correlation was found between MMP-3 and plasma creatinine, a negative correlation was found between MMP-3 and estimated glomerular filtration rate (eGFR; rho = -0.358, p = 0.012), and there was a positive correlation between GDF-15 and NIA (rho = 0.723, p < 0.001) and high-sensitive C-reactive protein (rho = 0.395, p = 0.005). MMP-3 was increased in macroalbuminuria and correlated positively with NIA only in the nine T1D patients with albuminuria (rho = 0.836, p = 0.005). The present study indicates that high MMP-3 is associated with low eGFR, high plasma creatinine, and macroalbuminuria, and that GDF-15 can be a biomarker for diabetic neuropathy in T1D. MMP-3 may be useful as biomarker for neuropathy in T1D with albuminuria.

Morphologic, cytometric, quantitative transcriptomic and functional characterisation provide insights into the haemocyte immune responses of Pacific abalone (Haliotis discus hannai).

In recent years, the abalone aquaculture industry has been threatened by the bacterial pathogens. The immune responses mechanisms underlying the phagocytosis of haemocytes remain unclear in Haliotis discus hannai. It is necessary to investigate the immune mechanism in response to these bacterial pathogens challenges. In this study, the phagocytic activities of haemocytes in H. discus hannai were examined by flow cytometry combined with electron microscopy and transcriptomic analyses. The results of Vibrio parahaemolyticus, Vibrio alginolyticus and Staphylococcus aureu challenge using electron microscopy showed a process during phagosome formation in haemocytes. The phagocytic rate (PP) of S. aureus was higher than the other five foreign particles, which was about 63%. The PP of Vibrio harveyi was about 43%, the PP peak of V. alginolyticus in haemocyte was 63.7% at 1.5h. After V. parahaemolyticus and V. alginolyticus challenge, acid phosphatase, alkaline phosphatase, total superoxide dismutase, lysozyme, total antioxidant capacity, catalase, nitric oxide synthase and glutathione peroxidase activities in haemocytes were measured at different times, differentially expressed genes (DEGs) were identified by quantitative transcriptomic analysis. The identified DEGs after V. parahaemolyticus challenge included haemagglutinin/amebocyte aggregation factor-like, supervillin-like isoform X4, calmodulin-like and kyphoscoliosis peptidase-like; the identified DEGs after V. alginolyticus challenge included interleukin-6 receptor subunit beta-like, protein turtle homolog B-like, rho GTPase-activating protein 6-like isoform X2, leukocyte surface antigen CD53-like, calponin-1-like, calmodulin-like, troponin C, troponin I-like isoform X4, troponin T-like isoform X18, tumor necrosis factor ligand superfamily member 10-like, rho-related protein racA-like and haemagglutinin/amebocyte aggregation factor-like. Some immune-related KEGG pathways were significantly up-regulated or down-regulated after challenge, including thyroid hormone synthesis, Th17 cell differentiation signalling pathway, focal adhesion, melanogenesis, leukocyte transendothelial migration, inflammatory mediator regulation of TRP channels, ras signalling pathway, rap1 signalling pathway. This study is the first step towards understanding the H. discus hannai immune system by adapting several tools to gastropods and providing a first detailed morpho-functional study of their haemocytes.

RhoB plays a central role in hyperosmolarity-induced cell shrinkage in renal cells.

The small Rho GTP-binding proteins are important cell morphology, function, and apoptosis regulators. Unlike other Rho proteins, RhoB can be subjected to either geranylgeranylation (RhoB-GG) or farnesylation (RhoB-F), making that the only target of the farnesyltransferase inhibitor (FTI). Fluorescence resonance energy transfer experiments revealed that RhoB is activated by hyperosmolarity. By contrast, hyposmolarity did not affect RhoB activity. Interestingly, treatment with farnesyltransferase inhibitor-277 (FTI-277) decreased the cell size. To evaluate whether RhoB plays a role in volume reduction, renal collecting duct MCD4 cells and Human Kidney, HK-2 were transiently transfected with RhoB-wildtype-Enhance Green Fluorescence Protein (RhoB-wt-EGFP) and RhoB-CLLL-EGFP which cannot undergo farnesylation. A calcein-based fluorescent assay revealed that hyperosmolarity caused a significant reduction of cell volume in mock and RhoB-wt-EGFP-expressing cells. By contrast, cells treated with FTI-277 or expressing the RhoB-CLLL-EGFP mutant did not properly respond to hyperosmolarity with respect to mock and RhoB-wt-EGFP expressing cells. These findings were further confirmed by 3D-LSCM showing that RhoB-CLLL-EGFP cells displayed a significant reduction in cell size compared to cells expressing RhoB-wt-EGFP. Moreover, flow cytometry analysis revealed that RhoB-CLLL-EGFP expressing cells as well as FTI-277-treated cells showed a significant increase in cell apoptosis. Together, these data suggested that: (i) RhoB is sensitive to hyperosmolarity and not to hyposmolarity; (ii) inhibition of RhoB farnesylation associates with an increase in cell apoptosis, likely suggesting that RhoB might be a paramount player controlling apoptosis by interfering with responses to cell volume change.