Loss Of Stress Fibers Research Articles

Impact of NSD1 Alternative Transcripts in Actin Filament Formation and Cellular Division Pathways in Fibroblasts.

Germline variants in the NSD1 gene are responsible for Sotos syndrome, while somatic variants promote neoplastic cell transformation. Our previous studies revealed three alternative RNA isoforms of NSD1 present in fibroblast cell lines (FBs): the canonical full transcript and 2 alternative transcripts, termed AT2 (NSD1 Δ5Δ7) and AT3 (NSD1 Δ19-23 at the 5' end). The precise molecular pathways affected by each specific isoform of NSD1 are uncharacterized to date. To elucidate the role of these isoforms, their expression was suppressed by siRNA knockdown in FBs and protein expression and transcriptome data was explored. We demonstrate that one gene target of NSD1 isoform AT2 is ARP3 actin-related protein 3 homolog B (ACTR3B). We show that loss of both canonical NSD1 and AT2 isoforms impaired the ability of fibroblasts to regulate the actin cytoskeleton, and we observed that this caused selective loss of stress fibers. Our findings provide novel insights into NSD1 function by distinguishing isoform function and demonstrating an essential role of NSD1 in regulating the actin cytoskeleton and stress fiber formation in fibroblasts.

Human septins organize as octamer-based filaments and mediate actin-membrane anchoring in cells.

Septins are cytoskeletal proteins conserved from algae and protists to mammals. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether septins organize as filaments in cells and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay, we show that all septins decorating actin stress fibers are octamer-containing filaments. Depleting octamers or preventing septins from polymerizing leads to a loss of stress fibers and reduced cell stiffness. Super-resolution microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that septin filaments are membrane bound and largely immobilized. Finally, reconstitution assays showed that septin filaments mediate actin-membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin filaments at the plasma membrane.

Keratinocyte Response to Infection with Sporothrix schenckii.

Sporotrichosis is a subacute, or chronic mycosis caused by traumatic inoculation of material contaminated with the fungus Sporothrix schenckii which is part of the Sporothrix spp. complex. The infection is limited to the skin, although its progression to more severe systemic or disseminated forms remains possible. Skin is the tissue that comes into contact with Sporothrix first, and the role of various cell lines has been described with regard to infection control. However, there is little information on the response of keratinocytes. In this study, we used the human keratinocyte cell line (HaCaT) and evaluated different aspects of infection from modifications in the cytoskeleton to the expression of molecules of the innate response during infection with conidia and yeast cells of Sporothrix schenckii. We found that during infection with both phases of the fungus, alterations of the actin cytoskeleton, formation of membrane protuberances, and loss of stress fibers were induced. We also observed an overexpression of the surface receptors MR, TLR6, CR3 and TLR2. Cytokine analysis showed that both phases of the fungus induced the production of elevated levels of the chemokines MCP-1 and IL-8, and proinflammatory cytokines IFN-α, IFN-γ and IL-6. In contrast, TNF-α production was significant only with conidial infection. In late post-infection, cytokine production was observed with immunoregulatory activity, IL-10, and growth factors, G-CSF and GM-CSF. In conclusion, infection of keratinocytes with conidia and yeast cells of Sporothrix schenckii induces an inflammatory response and rearrangements of the cytoskeleton.

The Plasma Membrane Ca2+ Pump PMCA4b Regulates Melanoma Cell Migration through Remodeling of the Actin Cytoskeleton.

Simple SummaryEarlier we demonstrated that the plasma membrane Ca2+ pump PMCA4b inhibits migration and metastatic activity of BRAF mutant melanoma cells, however, the exact mechanism has not been fully understood. Here we demonstrate that PMCA4b acted through actin cytoskeleton remodeling in generating a low migratory melanoma cell phenotype resulting in increased cell–cell connections, lamellipodia and stress fiber formation. Both proper trafficking and calcium transporting activity of the pump were essential to complete these tasks indicating that controlling Ca2+ concentration levels at specific plasma membrane locations such as the cell front played a role. Our findings suggest that PMCA4b downregulation is likely one of the mechanisms that leads to the perturbed cancer cell cytoskeleton organization resulting in enhanced melanoma cell migration and metastasis.We demonstrated that the plasma membrane Ca2+ ATPase PMCA4b inhibits migration and metastatic activity of BRAF mutant melanoma cells. Actin dynamics are essential for cells to move, invade and metastasize, therefore, we hypothesized that PMCA4b affected cell migration through remodeling of the actin cytoskeleton. We found that expression of PMCA4b in A375 BRAF mutant melanoma cells induced a profound change in cell shape, cell culture morphology, and displayed a polarized migratory character. Along with these changes the cells became more rounded with increased cell–cell connections, lamellipodia and stress fiber formation. Silencing PMCA4b in MCF-7 breast cancer cells had a similar effect, resulting in a dramatic loss of stress fibers. In addition, the PMCA4b expressing A375 cells maintained front-to-rear Ca2+ concentration gradient with the actin severing protein cofilin localizing to the lamellipodia, and preserved the integrity of the actin cytoskeleton from a destructive Ca2+ overload. We showed that both PMCA4b activity and trafficking were essential for the observed morphology and motility changes. In conclusion, our data suggest that PMCA4b plays a critical role in adopting front-to-rear polarity in a normally spindle-shaped cell type through F-actin rearrangement resulting in a less aggressive melanoma cell phenotype.

An AKT2-specific nanobody that targets the hydrophobic motif induces cell cycle arrest, autophagy and loss of focal adhesions in MDA-MB-231 cells

The AKT kinase family is a high-profile target for cancer therapy. Despite their high degree of homology the three AKT isoforms (AKT1, AKT2 and AKT3) are non-redundant and can even have opposing functions. Small-molecule AKT inhibitors affect all three isoforms which severely limits their usefulness as research tool or therapeutic. Using AKT2-specific nanobodies we examined the function of endogenous AKT2 in breast cancer cells. Two AKT2 nanobodies (Nb8 and Nb9) modulate AKT2 and reduce MDA-MB-231 cell viability/proliferation. Nb8 binds the AKT2 hydrophobic motif and reduces IGF-1-induced phosphorylation of this site. This nanobody also affects the phosphorylation and/or expression levels of a wide range of proteins downstream of AKT, resulting in a G0/G1 cell cycle arrest, the induction of autophagy, a reduction in focal adhesion count and loss of stress fibers. While cell cycle progression is likely to be regulated by more than one isoform, our results indicate that both the effects on autophagy and the cytoskeleton are specific to AKT2. By using an isoform-specific nanobody we were able to map a part of the AKT2 pathway. Our results confirm AKT2 and the hydrophobic motif as targets for cancer therapy. Nb8 can be used as a research tool to study AKT2 signalling events and aid in the design of an AKT2-specific inhibitor.

Synaptopodin Is Dispensable for Normal Podocyte Homeostasis but Is Protective in the Context of Acute Podocyte Injury

Synaptopodin (Synpo) is an actin-associated protein in podocytes and dendritic spines. Many functions in regulating the actin cytoskeleton via RhoA and other pathways have been ascribed to Synpo, yet no pathogenic mutations in the SYNPO gene have been discovered in patients. Naturally occurring Synpo isoforms are known (Synpo-short and -long), and a novel truncated version (Synpo-T) is upregulated in podocytes from Synpo mutant mice. Synpo-T maintains some Synpo functions, which may prevent a podocyte phenotype from emerging in unchallenged mutant mice. Novel mouse models were generated to further investigate the functions of Synpo. In one, CRISPR/Cas9 deleted most of the Synpo gene, preventing production of any detectable Synpo protein. Two other mutant strains made truncated versions of the protein. Adriamycin injections were used to challenge the mice, and Synpo functions were investigated in primary cultured podocytes. Mice that could not make detectable Synpo (Synpo -/- ) did not develop any kidney abnormalities up to 12 months of age. However, Synpo -/- mice were more susceptible to Adriamycin nephropathy. In cultured primary podocytes from mutant mice, the absence of Synpo caused loss of stress fibers, increased the number and size of focal adhesions, and impaired cell migration. Furthermore, loss of Synpo led to decreased RhoA activity and increased Rac1 activation. In contrast to previous findings, podocytes can function normally in vivo in the absence of any Synpo isoform. Synpo plays a protective role in the context of podocyte injury through its involvement in actin reorganization and focal adhesion dynamics.

The Vacuolating Autotransporter Toxin (Vat) of Escherichia coli Causes Cell Cytoskeleton Changes and Produces Non-lysosomal Vacuole Formation in Bladder Epithelial Cells.

Urinary tract infections (UTIs) affect more than 150 million people, with a cost of over 3.5 billion dollars, each year. Escherichia coli is associated with 70–80% of UTIs. Uropathogenic E. coli (UPEC) has virulence factors including adhesins, siderophores, and toxins that damage host cells. Vacuolating autotransporter toxin (Vat) is a member of serine protease autotransporter proteins of Enterobacteriaceae (SPATEs) present in some uropathogenic E. coli (UPEC) strains. Vat has been identified in 20–36% of UPEC and is present in almost 68% of urosepsis isolates. However, the mechanism of action of Vat on host cells is not well-known. Thus, in this study the effect of Vat in a urothelium model of bladder cells was investigated. Several toxin concentrations were tested for different time periods, resulting in 15–47% of cellular damage as measured by the LDH assay. Vat induced vacuole formation on the urothelium model in a time-dependent manner. Vat treatment showed loss of the intercellular contacts on the bladder cell monolayer, observed by Scanning Electron Microscopy. This was also shown using antibodies against ZO-1 and occludin by immunofluorescence. Additionally, changes in permeability of the epithelial monolayer was demonstrated with a fluorescence-based permeability assay. Cellular damage was also evaluated by the identification of cytoskeletal changes produced by Vat. Thus, after Vat treatment, cells presented F-actin distribution changes and loss of stress fibers in comparison with control cells. Vat also modified tubulin, but it was not found to affect Arp3 distribution. In order to find the nature of the vacuoles generated by Vat, the Lysotracker deep red fluorescent dye for the detection of acidic organelles was used. Cells treated with Vat showed generation of some vacuoles without acidic content. An ex vivo experiment with mouse bladder exposed to Vat demonstrated loss of integrity of the urothelium. In conclusion, Vat induced cellular damage, vacuole formation, and urothelial barrier dysregulation of bladder epithelial cells. Further studies are needed to elucidate the role of these vacuoles induced by Vat and their relationship with the pathogenesis of urinary tract infection.

MO009NEUTRALIZATION OF UPAR WITH AN ANTI-UPAR ANTIBODY AMELIORATES RECURRENT FSGS SERA INDUCED PODOCYTE INJURY

Abstract Background and Aims The role of suPAR as a biomarker and/or causative factor in the pathogenesis of recurrent FSGS remains unclear (Harel E., et al. Transplantation 2020; 104:54-60). While anti-suPAR antibodies have been shown to block suPAR induced podocyte injury in mouse models of FSGS, this beneficial effect has not yet been demonstrated in FSGS patients. We report here the inhibitor effects of 2G10, a fully human anti-suPAR antibody that blocks the interaction of suPAR with the B3 integrin on podocytes. Method The immortalized podocyte cell line was developed by transfection with the temperature sensitive SV40 T gene. These cells proliferate at the “permissive” temperature (33°C) and are considered undifferentiated. After transferring to the “nonpermissive” temperature (37°C), they enter growth arrest and by day 10-14 express markers of differentiated podocytes in vivo, such as nephrin, podocin, CD2 associated protein (CD2AP), synaptopodin, and known molecules of the slit diaphragm ZO-1, α, β, and γ-catenin, and P-cadherin. The podocytes were cultured in RPMI medium supplemented with insulin, transferrin, selenium, sodium pyruvate (ITS-A, Gibco #513000), 10% FBS and penicillin/streptomycin. After differentiation for 14 days, cells were serum starved for 1h. Serum from rFSGS patients or from a control patient was added (4% final) and cells were cultured for an additional 24h. After fixation in PFA/sucrose. actin cytoskeleton was visualized by labeling with rhodamine-conjugated phalloidin. DAPI was used for nuclei staining. Cells were imaged by confocal microscopy at 40X magnification and the number of cells with intact stress fibers were counted. For rescue of stress fibers, podocytes were cultured in the presence of a fully humanized anti uPAR antibody (2G10, 1 ug/ml; Duriseti S, J Biol Chem, 2010, 285:26878-88) or an isotype IgG control antibody (1 ug/ml). Results Sera from three patients with recurrence of FSGS after transplant were used in the study. Podocyte culture in the presence of sera from all three patients caused significant depolarization of stress fibers as determined by number of stress fiber positive cells (30%, 59% and 49% reduction with respect to untreated podocytes respectively). Treatment of podocytes with control sera did not cause any significant changes (data not shown). Culture of podocytes with patient sera in the presence of 2G10 antibody against uPAR rescued stress fibers (Fig 1A and 1B). On the other hand, a control human IgG was unable to rescue the loss of stress fibers induced by sera from recurrent FSGS patients. Conclusion The therapeutic potential of a human anti-suPAR antibody in samples from patients with recurrent FSGS has not been previously demonstrated. The in vitro findings of 2G10 antibody on preserving the stress fibers in human podocytes from the disrupting effect of the sera of patients with recurrent FSGS suggest that antibodies that block suPAR could be effective in preventing recurrence of FSGS.

Colocation of Tpm3.1 and myosin IIa heads defines a discrete subdomain in stress fibres.

Co-polymers of tropomyosin and actin make up a major fraction of the actin cytoskeleton. Tropomyosin isoforms determine the function of an actin filament by selectively enhancing or inhibiting the association of other actin binding proteins, altering the stability of an actin filament and regulating myosin activity in an isoform-specific manner. Previous work has implicated specific roles for at least five different tropomyosin isoforms in stress fibres, as depletion of any of these five isoforms results in a loss of stress fibres. Despite this, most models of stress fibres continue to exclude tropomyosins. In this study, we investigate tropomyosin organisation in stress fibres by using super-resolution light microscopy and electron microscopy with genetically tagged, endogenous tropomyosin. We show that tropomyosin isoforms are organised in subdomains within the overall domain of stress fibres. The isoforms Tpm3.1 and 3.2 (hereafter Tpm3.1/3.2, encoded by TPM3) colocalise with non-muscle myosin IIa and IIb heads, and are in register, but do not overlap, with non-muscle myosin IIa and IIb tails. Furthermore, perturbation of Tpm3.1/3.2 results in decreased myosin IIa in stress fibres, which is consistent with a role for Tpm3.1 in maintaining myosin IIa localisation in stress fibres.

SUN-190 GFB-887, a small molecule inhibitor of TRPC5, protects against podocyte injury and attenuates proteinuria in models of FSGS

Focal segmental glomerulosclerosis (FSGS) is a rare podocytopathy with a high likelihood of progression to end stage renal disease. The Ca2+-permeable transient receptor potential canonical 5 (TRPC5) channel is a critical mediator of proteinuria in FSGS. Activation of the TRPC5 pathway in podocytes culminates in activation of Rac1, which is the primary driver of proteinuria in many forms of familial and sporadic FSGS. Inhibition of TRPC5 channel activity has been shown to protect against proteinuria and podocyte loss in AT1R transgenic and Dahl salt-sensitive rats. We performed high throughput screening of a structurally diverse compound collection followed by lead optimization for the development of several potent TRPC5 inhibitors. We characterized TRPC5 inhibitors in vitrousing a mouse podocyte cell line and podocyte injury models derived from human inducible pluripotent stem cells (hiPSC), and in two in vivomodels of FSGS, the unilaterally nephrectomized deoxycorticosterone acetate (DOCA)-salt rat model and the puromycin aminonucleoside nephrosis (PAN) rat model Here we report the identification of GFB-887, a potent, selective and orally bioavailable small molecule inhibitor of TRPC5. Potency and selectivity across TRP channels were determined using FLIPR based assays and electrophysiology. We show that TRPC5 inhibition protects against loss of stress fibers induced by protamine sulfate, a known activator of TRPC5, and restores stress fibers in podocytes after knockdown of synaptopodin. We further show that inhibition of TRPC5 suppresses pathogenic podocyte motility in a scratch assay. Finally, we demonstrate that inhibition of TRPC5 attenuates proteinuria in hypertension-induced FSGS in DOCA-salt rat model without altering blood pressure, and in non-hypertensive FSGS in the PAN rat model suggesting direct glomerular protection in vivo. We have identified GFB-887 as a new selective small molecule inhibitor of TRPC5 for the treatment of proteinuria in FSGS

Disruption of podocyte cytoskeletal biomechanics by dasatinib leads to nephrotoxicity

Nephrotoxicity is a critical adverse event that leads to discontinuation of kinase inhibitor (KI) treatment. Here we show, through meta-analyses of FDA Adverse Event Reporting System, that dasatinib is associated with high risk for glomerular toxicity that is uncoupled from hypertension, suggesting a direct link between dasatinib and podocytes. We further investigate the cellular effects of dasatinib and other comparable KIs with varying risks of nephrotoxicity. Dasatinib treated podocytes show significant changes in focal adhesions, actin cytoskeleton, and morphology that are not observed with other KIs. We use phosphoproteomics and kinome profiling to identify the molecular mechanisms of dasatinib-induced injury to the actin cytoskeleton, and atomic force microscopy to quantify impairment to cellular biomechanics. Furthermore, chronic administration of dasatinib in mice causes reversible glomerular dysfunction, loss of stress fibers, and foot process effacement. We conclude that dasatinib induces nephrotoxicity through altered podocyte actin cytoskeleton, leading to injurious cellular biomechanics.

Group I metabotropic glutamate receptor activation induces TRPC6-dependent calcium influx and RhoA activation in cultured human kidney podocytes

Researches have shown that mice lacking the metabotropic glutamate receptor 1 (mGluR) showed albuminuria, remodeling of F-actin, with loss of stress fibers. Selective group I mGluRs agonist (S)-3,5-dihydroxyphenylglycine (DHPG) attenuated albuminuria in several rodent models of nephrotic syndrome. However, the molecular mechanism is obscure. Using a human podocyte cell line, we here investigated the molecular mechanisms of group I mGluRs-induced calcium influx and the formation of stress fibers. Our data showed that group I mGluRs activation by DHPG induced a significant calcium influx, and promoted cytoskeletal stress fiber formation and focal adhesions in podocytes. Pre-incubating podocytes with non-selective inhibitor of transient receptor potential channels (TRPC), or the knockdown of TRPC6 attenuated the calcium influx and the stress fiber formation induced by DHPG. Further, DHPG resulted in an increase of active RhoA expression. However, the knockdown of RhoA by siRNA abolished the DHPG-induced increase in stress fibers. Additionally, nonselective inhibitors of TRPC or TRPC6 knockdown clearly inhibited RhoA activation induced by DHPG, as assessed by Glutathione-S-transferase pull-down assay followed by Western blotting. Taken together, our findings suggest TRPC6 regulates actin stress fiber formation and focal adhesions via the RhoA pathway in response to group I mGluRs activation. Our data can potentially explain the mechanism of protective action of group I mGluRs in glomerular podocyte injury.

Amplification of the Melanocortin-1 Receptor in Nephrotic Syndrome Identifies a Target for Podocyte Cytoskeleton Stabilization

The melanocortin-1 receptor (MC1R) in podocytes has been suggested as the mediator of the ACTH renoprotective effect in patients with nephrotic syndrome with the mechanism of action beeing stabilization of the podocyte actin cytoskeleton. To understand how melanocortin receptors are regulated in nephrotic syndrome and how they are involved in restoration of filtration barrier function, melanocortin receptor expression was evaluated in patients and a rat model of nephrotic syndrome in combination with cell culture analysis. Phosphoproteomics was applied and identified MC1R pathways confirmed using biochemical analysis. We found that glomerular MC1R expression was increased in nephrotic syndrome, both in humans and in a rat model. A MC1R agonist protected podocytes from protamine sulfate induced stress fiber loss with the top ranked phoshoproteomic MC1R activated pathway beeing actin cytoskeleton signaling. Actin stabilization through the MC1R consisted of ERK1/2 dependent phosphorylation and inactivation of EGFR signaling with stabilization of synaptopodin and stressfibers in podocytes. These results further explain how patients with nephrotic syndrome show responsiveness to MC1R receptor activation by decreasing EGFR signaling and as a consequence restore filtration barrier function by stabilizing the podocyte actin cytoskeleton.

The actin filament bundling protein α-actinin-4 actually suppresses actin stress fibers by permitting actin turnover

Cells organize actin filaments into contractile bundles known as stress fibers that resist mechanical stress, increase cell adhesion, remodel the extracellular matrix, and maintain tissue integrity. α-actinin is an actin filament bundling protein that is thought to be essential for stress fiber formation and stability. However, previous studies have also suggested that α-actinin might disrupt fibers, making the true function of this biomolecule unclear. Here we use fluorescence imaging to show that kidney epithelial cells depleted of α-actinin-4 via shRNA or CRISPR/Cas9, or expressing a disruptive mutant make more massive stress fibers that are less dynamic than those in WT cells, leading to defects in cell motility and wound healing. The increase in stress fiber mass and stability can be explained, in part, by increased loading of the filament component tropomyosin onto stress fibers in the absence of α-actinin, as monitored via immunofluorescence. We show using imaging and cosedimentation that α-actinin and tropomyosin compete for binding to F-actin and that tropomyosin shields actin filaments from cofilin-mediated disassembly in vitro and in cells. Perturbing tropomyosin in cells lacking α-actinin-4 results in a complete loss of stress fibers. Our results with α-actinin-4 on stress fiber organization are the opposite of what might have been predicted from previous in vitro biochemistry and further highlight how the complex interactions of multiple proteins competing for filament binding lead to unexpected functions for actin-binding proteins in cells.

Prolonged Activation of STAT3 Mediates the IL6‐Induced Loss of Stress Fibers and Increase in Endothelial Permeability

Interleukin 6 (IL6) promotes endothelial barrier breakdown in vitro, as well as, in vivo. However, the signaling mechanisms that mediate this response are not fully understood. IL6 trans‐signaling promotes a prolonged activation of JAK/STAT3 in human umbilical vein endothelial cells (HUVECs) that lasts for at least 24 hours, leading to a sustained increase in endothelial permeability. IL6‐induced STAT3 phosphorylation and loss of barrier function is prevented when cells are pre‐treated with the JAK inhibitor, ruxolitinib, suggesting that JAK activity is required for these responses. Furthermore, siRNA‐mediated STAT3 knockdown attenuates the loss of IL6‐induced loss of barrier function, demonstrating a critical role for STAT3 in this response. Immunofluorescence analysis showed that this change in barrier function correlates with a marked loss of junctional ZO‐1 (but not changes in ZO‐1 protein expression). Phalloidin staining demonstrates that IL‐6 also promotes a JAK, and STAT3, dependent loss of actin stress fibers and a marked reduction of the cortical actin signal. A prolonged activation of STAT3 is required for this response, because treatment of HUVECs with ruxolitinib up to 4 hours after activation of IL6 trans‐signaling, completely reverses the IL6‐induced loss of barrier function. STAT3 activation kinetics are regulated by a negative feedback loop that is mediated by the suppressor of cytokine signaling 3 (SOCS3). Consistently, siRNA‐mediated SOCS3 knockdown increases IL6‐induced STAT3 phosphorylation in HUVECs and exacerbates the IL6‐induced increase in permeability. Collectively, our data shows that prolonged activation of STAT3 is required for the IL6‐induced barrier breakdown, and loss of junctional ZO‐1, cortical actin and actin stress fibers in endothelial cells.Support or Funding InformationThis project was supported by an American Heart Association Science Development Grant 13SDG17100110 and funds from the Department of Molecular and Cellular Physiology, Albany Medical Center.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluation of Changes in Actin Filaments of RK13 Cells Infected with <i>Malassezia pachydermatis</i>

Background: Malassezia pachydermatis is the main causative agent of canine otitis and also of a myriad of dermatological problems in companion animals; its interaction mechanisms with host cells are still unclear. Objectives: To establish an in vitro infection model of M. pachydermatis-exposed RK13 cells in order to evaluate cell morphological changes as well as changes in the structure of actin filaments. Methods: Cultures of RK13 cells were infected with M. pachydermatis, alterations caused by the yeast were evaluated by optical and fluorescence microscopy. Results: M. pachydermatis adheres itself to the cell and produces the formation of multiple agglomerates that cause changes in cell morphology, formation of cell aggregates in overlays, presence of syncytia and destruction of cell culture structure. The damaged cells presented changes in the actin filaments consisting of thickening of the cell cortex and loss of stress fibers. On the other hand, the formation of perinuclear actin rings in the yeasts was observed. Conclusions: An in vitro infection model was established with M. pachydermatis and alterations in cell morphology were observed consisting of changes in the structure of the actin filaments, overgrowth of the cells and the presence of syncytia.

Synaptopodin Is a Coincidence Detector of Tyrosine versus Serine/Threonine Phosphorylation for the Modulation of Rho Protein Crosstalk in Podocytes.

Tyrosine and serine/threonine signal-transduction pathways influence many aspects of cell behavior, including the spatial and temporal regulation of the actin cytoskeleton. However, little is known about how input from diverse tyrosine and serine/threonine kinases is integrated to control Rho protein crosstalk and actin remodeling, which are critically important in podocyte health and disease. Here we unveil the proteolytically-regulated, actin organizing protein synaptopodin as a coincidence detector of tyrosine versus serine/threonine phosphorylation. We show that serine/threonine and tyrosine kinases duel for synaptopodin stability versus degradation. EGFR/Src-mediated tyrosine phosphorylation of synaptopodin in podocytes promotes binding to the serine/threonine phosphatase calcineurin. This leads to the loss of 14-3-3 binding, resulting in synaptopodin degradation, Vav2 activation, enhanced Rac1 signaling, and ultimate loss of stress fibers. Our studies reveal how synaptopodin, a single proteolytically-controlled protein, integrates antagonistic tyrosine versus serine/threonine phosphorylation events for the dynamic control of the actin cytoskeleton in podocytes.

RhoA deficiency disrupts podocyte cytoskeleton and induces podocyte apoptosis by inhibiting YAP/dendrin signal.

BackgroundPodocyte apoptosis is a major mechanism that leads to proteinuria in many kidney diseases. However, the concert mechanisms that cause podocyte apoptosis in these kidney diseases are not fully understood. RhoA is one of Rho GTPases that has been well studied and plays a key role in regulating cytoskeletal architecture. Previous study showed that insufficient RhoA could result in rat aortic smooth muscle cell apoptosis. However, whether RhoA is involved in podocyte apoptosis remains unknown.MethodsCulture podocytes were treated with LPS, ADR or siRNA for 48 h before harvest. Subcellular immunoblotting, qRT-PCR, immunofluorescence and flow cytometry were used to exam the expression and function of RhoA or YAP in podocytes.ResultsWe found that the expression of RhoA and its activity were significantly decreased in LPS or ADR-injured podocytes, accompanying loss of stress fibers and increased cell apoptosis. Knocking down RhoA or its downstream effector mDia expression by siRNA also caused loss of stress fibers and podocyte apoptosis. Moreover, our results further demonstrated that RhoA deficiency could reduce the mRNA and protein expression of YAP, which had been regarded as an anti-apoptosis protein in podocyte. Silenced dendrin expression significantly abolished RhoA, mDia or YAP deficiency-induced podocyte apoptosis.ConclusionRhoA deficiency could disrupt podocyte cytoskeleton and induce podocyte apoptosis by inhibiting YAP/dendrin signal. RhoA/mDia/YAP/dendrin signal pathway may potentially play an important role in regulating podocyte apoptosis. Maintaining necessary RhoA would be one potent way to prevent proteinuria kidney diseases.

Cdc42 deficiency induces podocyte apoptosis by inhibiting the Nwasp/stress fibers/YAP pathway.

Podocyte apoptosis is a major mechanism that leads to proteinuria in many chronic kidney diseases. However, the concert mechanisms that cause podocyte apoptosis in these kidney diseases are not fully understood. The Rho family of small GTPases has been shown to be required in maintaining podocyte structure and function. Recent studies have indicated that podocyte-specific deletion of Cdc42 in vivo, but not of RhoA or Rac1, leads to congenital nephrotic syndrome and glomerulosclerosis. However, the underlying cellular events in podocyte controlled by Cdc42 remain unclear. Here, we assessed the cellular mechanisms by which Cdc42 regulates podocyte apoptosis. We found that the expression of Cdc42 and its activity were significantly decreased in high glucose-, lipopolysaccharide- or adriamycin-injured podocytes. Reduced Cdc42 expression in vitro and in vivo by small interfering RNA and selective Cdc42 inhibitor ML-141, respectively, caused podocyte apoptosis and proteinuria. Our results further demonstrated that insufficient Cdc42 or Nwasp, its downstream effector, could decrease the mRNA and protein expression of YAP, which had been regarded as an anti-apoptosis protein in podocyte. Moreover, our data indicated that the loss of stress fibers caused by Cdc42/Nwasp deficiency also decreased Yes-associated protein (YAP) mRNA and protein expression, and induced podocyte apoptosis. Podocyte apoptosis induced by Cdc42/Nwasp/stress fiber deficiency was significantly inhibited by overexpressing-active YAP. Thus, the Cdc42/Nwasp/stress fibers/YAP signal pathway may potentially play an important role in regulating podocyte apoptosis. Maintaining necessary Cdc42 would be one potent way to prevent proteinuria kidney diseases.

Rnd3-induced cell rounding requires interaction with Plexin-B2.

ABSTRACTRnd proteins are atypical members of the Rho GTPase family that induce actin cytoskeletal reorganization and cell rounding. Rnd proteins have been reported to bind to the intracellular domain of several plexin receptors, but whether plexins contribute to the Rnd-induced rounding response is not known. Here we show that Rnd3 interacts preferentially with plexin-B2 of the three plexin-B proteins, whereas Rnd2 interacts with all three B-type plexins, and Rnd1 shows only very weak interaction with plexin-B proteins in immunoprecipitations. Plexin-B1 has been reported to act as a GAP for R-Ras and/or Rap1 proteins. We show that all three plexin-B proteins interact with R-Ras and Rap1, but Rnd proteins do not alter this interaction or R-Ras or Rap1 activity. We demonstrate that plexin-B2 promotes Rnd3-induced cell rounding and loss of stress fibres, and enhances the inhibition of HeLa cell invasion by Rnd3. We identify the amino acids in Rnd3 that are required for plexin-B2 interaction, and show that mutation of these amino acids prevents Rnd3-induced morphological changes. These results indicate that plexin-B2 is a downstream target for Rnd3, which contributes to its cellular function.