Reorganization Of Actin Cytoskeleton Research Articles

#1428 Rnd3, a novel gene involved in ADPKD

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is an adult-onset, multi-systemic disorder, that affects ∼12.5 million people worldwide. ADPKD is characterised by progressive kidney enlargement caused by uncontrollable growth of cysts, alongside extra-renal phenotypes such as cysts in the liver, pancreas, and hypertension. Truncated mutations in PKD1 and PKD2 that encode for PC1 and PC2, respectively, are responsible for 85% and 15% of reported cases. Mutations in PKD1 are associated with the more severe form of ADPKD and earlier mean age of onset. The phenotypic variability of ADPKD can be attributed to genic, allelic and gene modifier effects. Modifier genes influence the molecular and/or phenotypic expression of other genes, altering the range of symptoms and the severity of the disease. In mice with a single gene mutation, modifier genes reduce the penetrance, dominance modification, expressivity, and phenotypic pleiotropy. To understand the involvement of modifier genes in the ADPKD kidney phenotype, a novel mouse model Rnd3tm1b(EUCOMM)Hmgu was generated, and in adult and aged Rnd3tm1b/+ mice, a kidney phenotype characteristic of ADPKD was evident. Rnd3 is an atypical member of the Rnd subgroup of the Rho family of GTPases and has a role in actin cytoskeleton reorganisation, cell cycle progression, and cell migration. Method 5 and 9-month-old kidneys from Rnd3tm1b/+ and age-matched wildtype mice were studied. In the Rnd3tm1b (EUCOMM)Hmgu mouse model, the TB1B mutation causes an inactivation of Rnd3 via the deletion of Exon 2 in C57BL/6N mice. RT-qPCR and SDS-Page Westerns were performed as well as staining for markers associated with Polycystic Kidney Disease. Results The onset of cyst formation in the cortex is apparent in 5-month-old Rnd3tm1b/+ kidneys compared to age-matched wildtype kidneys (Fig. 1a). A statistically significant 10% (p < 0.0465) increase in the proliferative index was observed in 5-month-old Rnd3tm1b/+ mice (Fig. 1e,f). Vimentin expression was also increased in podocytes and confirmed by a statistically significant 25% (p < 0.0461) increase in Rnd3tm1b/+ kidneys (Fig. 1g,h). Aquaporin-2 (Aqp2) expression was decreased in collecting ducts and verified by a statistically significant 0.4-fold-change (p < 0.0451) decrease in protein level in kidneys of 5-month-old Rnd3tm1b/+ mice (Fig. 1i-k). A statistically significant 0.75-fold (p < 0.0006) and 1.5-fold-increase (p < 0.02) in Pkd1 and Pkd2 expression was observed in 5-month-old Rnd3tm1b/+ kidneys, respectively (Fig. 1c). Kidneys of 9-month-old Rnd3tm1b/+mice presented with multiple cysts in the cortex, mimicking the ADPKD kidney phenotype (Fig. 1b). Vimentin staining in podocytes was increased and confirmed by a statistically significant 14% (p < 0.0419) increase in Rnd3tm1b/+mice (Fig. 1l,m). Expression of Pkd1 and Pkd2 was downregulated by a statistically significant 0.39-fold (p < 0.0001) and 0.2-fold-change (p < 0.004) in Rnd3tm1b/+mice (Fig. 1d). Conclusion Together, these findings highlight the importance of Rnd3 as a potential modifier in the onset of ADPKD by mediating the expression of Pkd1 and Pkd2. Interestingly, there is a critical window where Pkd1 and Pkd2 expression is altered, exacerbating the phenotype between 5 and 9-month-old Rnd3tm1b/+ mice. In addition to this, by understanding how modifiers are involved in triggering the onset of phenotypes in ADPKD, potential diagnostic and therapeutic measures can be discovered to ultimately aid in diagnosing ADPKD at a much earlier stage.

Reorganization of Actin Cytoskeleton and Reduction of Cortical Elasticity in Podocytes Lacking Acid Ceramidase: Role of Sphingomyelinase-Like Phosphodiesterase 3b

Podocytopathy and associated nephrotic syndrome have been reported in a mouse strain (Asah1fl/fl/Podocre) with a podocyte-specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (AC). Nevertheless, the pathogenic mechanism of podocytopathy in these mice remains poorly understood. Given that sphingomyelinase-like phosphodiesterase 3b (SMPDL3b) has been recently reported to play a vital role in maintaining the actin cytoskeleton and normal function of podocytes, the present study tested whether Asah1 gene deletion-induced podocytopathy is attributed to altered SMPDL3b activity and reorganized actin cytoskeleton in podocytes. By Western blot analysis and SMPDL3b enzyme activity assay, we demonstrated that Asah1 gene knockout remarkably decreased both expression and function of SMPDL3b in podocytes isolated from Asah1fl/fl/Podocre mice compared to podocytes of wild type (WT/WT) mice. As a monoclonal antibody which can bind to and stabilize SMPDL3b on podocytes, rituximab was found to significantly recover SMPDL3b expression and activity in podocytes lacking Asah1 gene. Correspondingly, Asah1 gene deletion reduced podocin expression in podocytes, which was attenuated by rituximab treatment. Phalloidin staining of F-actin showed elevation of cortical actin and reduction of stress fiber in podocytes lacking Asah1 gene. Such actin cytoskeleton redistribution was significantly attenuated by rituximab. Moreover, atom force microscopy was used to study the impact of actin cytoskeleton remodeling on mechanical properties of podocytes. Asah1 gene knockout was found to increase podocyte height but decrease cortical elasticity in these cells. These pathological changes were significantly inhibited by rituximab, suggesting the restoration of actin cytoskeleton in podocytes lacking Asah1 gene by rituximab. In vivo, intraperitoneal injection of rituximab markedly attenuated proteinuria and albuminuria in Asah1fl/fl/Podocre mice compared to control groups. In summary, our findings suggest that alteration of SMPDL3b activity and reorganization of actin cytoskeleton in podocytes may contribute to the pathogenesis of podocytopathy in mice with podocyte-specific Asah1 gene deletion. This study was supported by NIH grants DK054927 and DK120491. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

EFA6A, an Exchange Factor for Arf6, Regulates NGF-Dependent TrkA Recycling From Early Endosomes and Neurite Outgrowth in PC12 Cells.

Endosomal trafficking of TrkA is a critical process for nerve growth factor (NGF)-dependent neuronal cell survival and differentiation. The small GTPase ADP-ribosylation factor 6 (Arf6) is implicated in NGF-dependent processes in PC12 cells through endosomal trafficking and actin cytoskeleton reorganization. However, the regulatory mechanism for Arf6 in NGF signaling is largely unknown. In this study, we demonstrated that EFA6A, an Arf6-specific guanine nucleotide exchange factor, was abundantly expressed in PC12 cells and that knockdown of EFA6A significantly inhibited NGF-dependent Arf6 activation, TrkA recycling from early endosomes to the cell surface, prolonged ERK1/2 phosphorylation, and neurite outgrowth. We also demonstrated that EFA6A forms a protein complex with TrkA through its N-terminal region, thereby enhancing its catalytic activity for Arf6. Similarly, we demonstrated that EFA6A forms a protein complex with TrkA in cultured dorsal root ganglion (DRG) neurons. Furthermore, cultured DRG neurons from EFA6A knockout mice exhibited disturbed NGF-dependent TrkA trafficking compared with wild-type neurons. These findings provide the first evidence for EFA6A as a key regulator of NGF-dependent TrkA trafficking and signaling.

Proteomic Analysis of Prehypertensive and Hypertensive Patients: A Novel Role of Actin Cytoskeleton in Disease Progression

Background: Hypertension is a pervasive and widespread health condition that poses a significant risk factor for cardiovascular disease, which includes conditions such as heart attack, stroke, and heart failure. Despite its widespread occurrence, the exact cause of hypertension remains unknown, and the mechanisms underlying the progression from prehypertension to hypertension require further investigation. Methods: To uncover potential biomarkers related to disease development, proteomic profiling using SOMAscan proteomic platform was conducted on Qatari individuals with prehypertension and hypertension. In this study, proteomic data collected from the Qatar Biobank were analyzed to compare participants with prehypertension, hypertension, and normal blood pressure. Statistical analysis was performed to identify altered protein expression between the study groups and to elucidate the biological pathways contributing to this disease. Results: The results revealed a cluster of proteins, including the SRC family, CAMK2B and CAMK2D, TEC, GSK3, VAV, and RAC, that were markedly upregulated in patients with hypertension compared to those with prehypertension (fold change ≥ 1.6 or ≤ -1.6, area under the curve ≥ 0.8, and q-value < 0.05). Pathway analysis showed that the majority of these proteins play a role in actin cytoskeleton remodeling, a crucial pathological event involved in hypertension progression. Conclusion: Actin cytoskeleton reorganization affects various biological processes that contribute to the maintenance of blood pressure, including vascular tone, endothelial function, cellular signaling, inflammation, fibrosis, and mechanosensing. Therefore, the findings of this study suggest a potential novel role of actin cytoskeleton-related proteins in the progression from prehypertension to hypertension. The present study sheds light on the underlying pathological mechanisms involved in hypertension and could pave the way for new diagnostic and therapeutic approaches for the treatment of this disease. This study was made possible through the support of Qatar University. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Podocytopathy and Nephrotic Syndrome in Smpdl3b Gene Knockout Mice

Recent clinical studies have shown that downregulation of sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) in podocytes is a hallmark of idiopathic nephrotic syndrome in children. Meanwhile, there is increasing evidence suggesting that changes of SMPDL3b level in podocytes determine the phenotypes of podocyte injury in glomerular diseases. However, it remains unknown whether SMPDL3b exclusively plays a crucial role in the pathogenesis of podocytopathy and nephrotic syndrome without any other pathogenic stimuli. In the present study, we tested whether Smpdl3b gene knockout induces podocyte injury and nephrotic syndrome in mice. By immunohistochemistry and confocal microscopy, we first confirmed that SMPDL3b was highly expressed in podocytes of wild type (WT) mice but undetectable in these cells of Smpdl3b knockout (KO) mice. Also, Western blot analysis showed that SMPDL3b was not detected in podocytes isolated from Smpdl3b KO mice compared to podocytes of WT mice. Although no significant morphologic changes in glomeruli were observed in Smpdl3b KO mice under light microscope, severe proteinuria and albuminuria were found in Smpdl3b KO mice compared to control littermates. Transmission electron microscopy showed that podocytes of Smpdl3b KO mice had distinctive foot process effacement and moderate microvillus formation and vacuolation. These functional and morphologic changes indicate the development of nephrotic syndrome in mice lacking Smpdl3b gene. In vitro, Smpdl3b gene silencing induced remarkable decrease in podocin levels in podocytes compared to control cells. Phalloidin staining of F-actin showed elevation of cortical actin and reduction of stress fiber in podocytes transfected with Smpdl3b shRNA. Such actin cytoskeleton rearrangement induced by Smpdl3b gene silencing was significantly attenuated by choline but exaggerated by CDP-choline, which are product and substrate of SMPDL3b, respectively. Furthermore, atom force microscopy was used to study mechanical properties of podocytes lacking Smpdl3b gene as the outcome of actin cytoskeleton reorganization. Smpdl3b gene deletion was found to increase podocyte height but decrease cortical elasticity in these cells. Moreover, these pathological changes were markedly inhibited by choline. Taken together, our findings suggest that SMPDL3b is essential for the maintenance of functional and structural integrity of podocytes and that Smpdl3b gene knockout may induce podocytopathy and nephrotic syndrome in mice. This study was supported by NIH grants DK054927 and DK120491. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Proteomic Analysis of Prehypertensive and Hypertensive Patients: Exploring the Role of the Actin Cytoskeleton.

Hypertension is a pervasive and widespread health condition that poses a significant risk factor for cardiovascular disease, which includes conditions such as heart attack, stroke, and heart failure. Despite its widespread occurrence, the exact cause of hypertension remains unknown, and the mechanisms underlying the progression from prehypertension to hypertension require further investigation. Recent proteomic studies have shown promising results in uncovering potential biomarkers related to disease development. In this study, serum proteomic data collected from Qatar Biobank were analyzed to identify altered protein expression between individuals with normal blood pressure, prehypertension, and hypertension and to elucidate the biological pathways contributing to this disease. The results revealed a cluster of proteins, including the SRC family, CAMK2B, CAMK2D, TEC, GSK3, VAV, and RAC, which were markedly upregulated in patients with hypertension compared to those with prehypertension (fold change ≥ 1.6 or ≤-1.6, area under the curve ≥ 0.8, and q-value < 0.05). Pathway analysis showed that the majority of these proteins play a role in actin cytoskeleton remodeling. Actin cytoskeleton reorganization affects various biological processes that contribute to the maintenance of blood pressure, including vascular tone, endothelial function, cellular signaling, inflammation, fibrosis, and mechanosensing. Therefore, the findings of this study suggest a potential novel role of actin cytoskeleton-related proteins in the progression from prehypertension to hypertension. The present study sheds light on the underlying pathological mechanisms involved in hypertension and could pave the way for new diagnostic and therapeutic approaches for the treatment of this disease.

Human restricted CHRFAM7A gene increases brain efficiency.

CHRFAM7A, a uniquely human fusion gene, has been associated with neuropsychiatric disorders including Alzheimer's disease, schizophrenia, anxiety, and attention deficit disorder. Understanding the physiological function of CHRFAM7A in the human brain is the first step to uncovering its role in disease. CHRFAM7A was identified as a potent modulator of intracellular calcium and an upstream regulator of Rac1 leading to actin cytoskeleton reorganization and a switch from filopodia to lamellipodia implicating a more efficient neuronal structure. We performed a neurocognitive-MRI correlation exploratory study on 46 normal human subjects to explore the effect of CHRFAM7A on human brain. Dual locus specific genotyping of CHRFAM7A was performed on genomic DNA to determine copy number (TaqMan assay) and orientation (capillary sequencing) of the CHRFAM7A alleles. As only the direct allele is expressed at the protein level and affects α7 nAChR function, direct allele carriers and non-carriers are compared for neuropsychological and MRI measures. Subjects underwent neuropsychological testing to measure motor (Timed 25-foot walk test, 9-hole peg test), cognitive processing speed (Symbol Digit Modalities Test), Learning and memory (California Verbal Learning Test immediate and delayed recall, Brief Visuospatial Memory Test-Revised immediate and delayed recall) and Beck Depression Inventory-Fast Screen, Fatigue Severity Scale. All subjects underwent MRI scanning on the same 3 T GE scanner using the same protocol. Global and tissue-specific volumes were determined using validated cross-sectional algorithms including FSL's Structural Image Evaluation, using Normalization, of Atrophy (SIENAX) and FSL's Integrated Registration and Segmentation Tool (FIRST) on lesion-inpainted images. The cognitive tests were age and years of education-adjusted using analysis of covariance (ANCOVA). Age-adjusted analysis of covariance (ANCOVA) was performed on the MRI data. CHRFAM7A direct allele carrier and non-carrier groups included 33 and 13 individuals, respectively. Demographic variables (age and years of education) were comparable. CHRFAM7A direct allele carriers demonstrated an upward shift in cognitive performance including cognitive processing speed, learning and memory, reaching statistical significance in visual immediate recall (FDR corrected p = 0.018). The shift in cognitive performance was associated with smaller whole brain volume (uncorrected p = 0.046) and lower connectivity by resting state functional MRI in the visual network (FDR corrected p = 0.027) accentuating the cognitive findings. These data suggest that direct allele carriers harbor a more efficient brain consistent with the cellular biology of actin cytoskeleton and synaptic gain of function. Further larger human studies of cognitive measures correlated with MRI and functional imaging are needed to decipher the impact of CHRFAM7A on brain function.

Conditional deficiency of Rho-associated kinases disrupts endothelial cell junctions and impairs respiratory function in adult mice.

The Ras homology (Rho) family of GTPases serves various functions, including promotion of cell migration, adhesion, and transcription, through activation of effector molecule targets. One such pair of effectors, the Rho-associated coiled-coil kinases (ROCK1 and ROCK2), induce reorganization of actin cytoskeleton and focal adhesion through substrate phosphorylation. Studies on ROCK knockout mice have confirmed that ROCK proteins are essential for embryonic development, but their physiological functions in adult mice remain unknown. In this study, we aimed to examine the roles of ROCK1 and ROCK2 proteins in normal adultmice. Tamoxifen (TAM)-inducible ROCK1 and ROCK2 single and double knockout mice (ROCK1flox/flox and/or ROCK2flox/flox;Ubc-CreERT2) were generated and administered a 5-day course of TAM. No deaths occurred in either of the single knockout strains, whereas all of the ROCK1/ROCK2 double conditional knockout mice (DcKO) had died by Day 11 following the TAM course. DcKO mice exhibited increased lung tissue vascular permeability, thickening of alveolar walls, and a decrease in percutaneous oxygen saturation compared with noninducible ROCK1/ROCK2 double-floxed control mice. On Day 3 post-TAM, there was a decrease in phalloidin staining in the lungs in DcKO mice. On Day 5 post-TAM, immunohistochemical analysis also revealed reduced staining for vascular endothelial (VE)-cadherin, β-catenin, and p120-catenin at cell-cell contact sites in vascular endothelial cells in DcKO mice. Additionally, VE-cadherin/β-catenin complexes were decreased in DcKO mice, indicating that ROCK proteins play a crucial role in maintaining lung function by regulating cell-cell adhesion.

CHRFAM7A diversifies human immune adaption through Ca2+ signalling and actin cytoskeleton reorganization

Human restricted genes contribute to human specific traits in the immune system. CHRFAM7A, a uniquely human fusion gene, is a negative regulator of the α7 nicotinic acetylcholine receptor (α7 nAChR), the highest Ca2+ conductor of the ACh receptors implicated in innate immunity. Understanding the mechanism of how CHRFAM7A affects the immune system remains unexplored. Two model systems are used, human induced pluripotent stem cells (iPSC) and human primary monocytes, to characterize α7 nAChR function, Ca2+ dynamics and decoders to elucidate the pathway from receptor to phenotype. CHRFAM7A/α7 nAChR is identified as a hypomorphic receptor with mitigated Ca2+ influx and prolonged channel closed state. This shifts the Ca2+ reservoir from the extracellular space to the endoplasmic reticulum (ER) leading to Ca2+ dynamic changes. Ca2+ decoder small GTPase Rac1 is then activated, reorganizing the actin cytoskeleton. Observed actin mediated phenotypes include cellular adhesion, motility, phagocytosis and tissue mechanosensation. CHRFAM7A introduces an additional, human specific, layer to Ca2+ regulation leading to an innate immune gain of function. Through the actin cytoskeleton it drives adaptation to the mechanical properties of the tissue environment leading to an ability to invade previously immune restricted niches. Human genetic diversity predicts profound translational significance as its understanding builds the foundation for successful treatments for infectious diseases, sepsis, and cancer metastasis. This work is supported in part by the Community Foundation for Greater Buffalo (Kinga Szigeti) and in part by NIH grant R01HL163168 (Yongho Bae).

Actin-Dependent Mechanism of Tumor Progression Induced by a Dysfunction of p53 Tumor Suppressor.

Cancer cell aggressiveness, marked by actin cytoskeleton reconfiguration critical for metastasis, may result from an imbalanced ratio favoring γ-actin. Dysfunctional p53 emerges as a key regulator of invasiveness and migration in various cancer cells, both in vitro and in vivo. P53 inactivation (via mutants R175H, R248W, R273H, or TP53 repression) significantly enhanced the migration, invasion, and proliferation of human lung adenocarcinoma A549 cells in vitro and in vivo, facilitating intrapulmonary xenograft metastasis in athymic mice. Conversely, wild-type TP53 (TP53 WT) overexpression in p53-deficient non-small- cell lung cancer (NSCLC) H1299 cells substantially reduced proliferation and migration in vitro, effectively curbing orthotopic tumorigenicity and impeding in vivo metastasis. These alterations in cell motility were closely associated with actin cytoskeleton restructuring, favoring γ-actin, and coincided with ERK1/2-mediated signaling activation, unveiling an innovative regulatory mechanism in malignancy progression. Cancer cell aggressiveness, driven by actin cytoskeleton reorganization and a shift towards γ-actin predominance, may be regulated by p53 dysfunction, thereby providing novel insight into tumor progression mechanisms.

SSPH I, A Novel Anti-cancer Saponin, Inhibits EMT and Invasion and Migration of NSCLC by Suppressing MAPK/ERK1/2 and PI3K/AKT/mTOR Signaling Pathways.

Saponin of Schizocapsa plantaginea Hance I (SSPH I)，a bioactive saponin found in Schizocapsa plantaginea, exhibits significant anti-proliferation and antimetastasis in lung cancer. To explore the anti-metastatic effects of SSPH I on non-small cell lung cancer (NSCLC) with emphasis on epithelial-mesenchymal transition (EMT) both in vitro and vivo. The effects of SSPH I at the concentrations of 0, 0.875,1.75, and 3.5 μM on A549 and PC9 lung cancer cells were evaluated using colony formation assay, CCK-8 assay, transwell assay and wound-healing assay. The actin cytoskeleton reorganization of PC9 and A549 cells was detected using the FITC-phalloidin fluorescence staining assay. The proteins related to EMT (N-cadherin, E-cadherin and vimentin), p- PI3K, p- AKT, p- mTOR and p- ERK1/2 were detected by Western blotting. A mouse model of lung cancer metastasis was established by utilizing 95-D cells, and the mice were treated with SSPH I by gavage. The results suggested that SSPH I significantly inhibited the migration and invasion of NSCLC cells under a non-cytotoxic concentration. Furthermore, SSPH I at a non-toxic concentration of 0.875 μM inhibited F-actin cytoskeleton organization. Importantly, attenuation of EMT was observed in A549 cells with upregulation in the expression of epithelial cell marker E-cadherin and downregulation of the mesenchymal cell markers vimentin as well as Ncadherin. Mechanistic studies revealed that SSPH I inhibited MAPK/ERK1/2 and PI3K/AKT/mTOR signaling pathways. SSPH I inhibited EMT, migration, and invasion of NSCLC cells by suppressing MAPK/ERK1/2 and PI3K/AKT/mTOR signaling pathways, suggesting that the natural compound SSPH I could be used for inhibiting metastasis of NSCLC.

Zwitterionic Polysulfobetaine Inhibits Cancer Cell Migration Owing to Actin Cytoskeleton Dynamics.

Cell migration is an essential dynamic process for most living cells, mainly driven by the reorganization of actin cytoskeleton. To control actin dynamics, a molecular architecture that can serve as a nucleator has been designed by polymerizing sulfobetaine methacrylate. The synthesized zwitterionic polymer, poly(sulfobetaine methacrylate) (PZI), effectively nucleates the polymerization process of G-actin and substantially accelerates the rate of polymerization. Isothermal titration calorimetry (ITC) and bioinformatics analysis indicated binding between PZI and monomeric G-actin. Thus, in vitro actin dynamics was studied by dynamic light scattering (DLS), pyrene-actin polymerization assay, and total internal reflection fluorescence microscopy (TIRFM). Furthermore, a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophore-containing monomeric unit was incorporated into the sulfobetaine zwitterionic architecture to visualize the effect of polymer in the cellular environment. The BODIPY-containing zwitterionic sulfobetaine polymer (PZI-F) successfully penetrated the cell and remained in the lysosome with minimal cytotoxicity. Confocal microscopy revealed the influence of this polymer on the cellular actin cytoskeleton dynamics. The PZI-F polymer was successfully able to inhibit the collective migration of the human cervical cancer cell line (HeLa cell) and breast cancer cell line (MDA-MB-231 cell), as confirmed by a wound healing assay. Therefore, polyzwitterionic sulfobetaine could be explored as an inhibitor of cancer cell migration.

The Actin Bundling Protein L-Plastin Mediates Platelet Force Generation and Thrombosis

Within minutes of activation, platelets dramatically change their shape, forming filopodia and lamellipodia, aggregate with other platelets or different cells, and can increase their surface area up to 4 times. This relies on a dynamic actin cytoskeleton, regulated by many actin binding proteins in a time- and spatial-specific manner. Deficiency or inhibition of several actin binding proteins in platelets have been shown with impaired platelet spreading and reduced thrombosis. Recently, we found that an actin bundling protein, L-plastin, is expressed in platelets. Its function in platelets has not been studied. In megakaryocytes, we showed that L-plastin is responsible for maintaining cytoskeletal stiffness; in this study we sought to determine L-plastin's role in platelet functions, including thrombosis. We examined the role of L-plastin in platelets in vivo and ex vivo using L-plastin deficient ( Lcp1 -/-) male and female mice. First, we used the collagen/epinephrine induced pulmonary embolism (PE) model and found that survival times were significantly shorter in Lcp1 -/- mice than in their littermate wildtype (WT) controls (Mean±SEM WT 202±12 sec, Lcp1 -/- 139.5±14 sec, P=0.004), suggesting accelerated thrombosis upon L-plastin deficiency in vivo. Second, we examined L-plastin's role in arterial thrombosis in vivo using the laser-induced cremaster arteriole thrombosis mode. Platelets were labeled with anti-GPIb IgG derivative X488 and the thrombosis formation after laser injury was recorded. Preliminary data indicate that thrombi in Lcp1 -/- mice are larger than those in WT mice. Third, we examined L-plastin's role in platelet adhesion using a microfluidics assay with DiOC6-labelled whole blood. At a shear rate of 200/s, the Lcp1 -/- mice had significantly increased platelet adhesion to both collagen and fibrinogen matrices. Fourth, to evaluate the effect of L-plastin on platelet aggregation in vitro, we stimulated washed platelets with 3 different concentrations of thrombin and measured the platelet aggregation (i.e., 0.1, 0.5 and 1 U/mL). When platelets are stimulated with 0.5U/mL or 1U/mL thrombin, platelets from WT and Lcp1 -/- mice showed comparable aggregation kinetics. At 0.1U/mL thrombin, however, preliminary data showed increased aggregation in the Lcp1 -/- group. The difference between the low and high concentration of thrombin indicates potential regulation of the GPIba mediated actin cytoskeleton rearrangement during platelet aggregation. We next examined under low thrombin concentration, the platelet actin cytoskeleton reorganization and platelet spreading. We seeded washed platelets on a fibrinogen matrix with the presence of thrombin (0.1U/mL). Platelets were then fixed and stained for F-actin and imaged using immunofluorescent confocal microscopy. The spreading area per platelet was calculated using FIJI software. Platelets from Lcp1 -/- mice showed significantly increased average membrane area at 30 min (Median WT 689 pixels, Lcp1 -/- 866 pixels, P&amp;lt;0.0001). This suggests that the actin rearrangement in platelets upon fibrinogen activation and thrombin stimulation is inhibited by L-plastin. Lastly, we examined whether L-plastin is involved in platelet force generation during the actin reorganization. We used a recently developed “black dot” assay. Briefly, a fluorescent pattern (i.e., black dots) was microcontact printed onto a flexible polydimethylsiloxane substrate and coated with fibrinogen. Platelets were seeded onto the matrix and fixed after 30 min. The contractile forces of individual platelets were measured based on the deformation of the fluorescent micropattern as illustrated in Fig 1A. The contractile force in platelets from Lcp1 -/- mice is decreased with altered actin distribution pattern compared to WT mice (Fig 1. Contractile force per platelet, Mean±SEM WT 14.78±0.86nN, Lcp1 -/- 12.03±0.67nN, P=0.01). In summary, we found accelerated thrombosis in Lcp1 -/- mice, associated with accelerated platelet adhesion and spreading ex vivo. Mechanistically, our preliminary data suggest this is due to impaired actin bundle formation and decreased platelet contractile force. L-plastin may be unique among actin binding proteins.

Rap1 small GTPase is essential for maintaining pulmonary endothelial barrier function in mice.

Vascular permeability is dynamically but tightly controlled by vascular endothelial (VE)-cadherin-mediated endothelial cell-cell junctions to maintain homeostasis. Thus, impairments of VE-cadherin-mediated cell adhesions lead to hyperpermeability, promoting the development and progression of various disease processes. Notably, the lungs are a highly vulnerable organ wherein pulmonary inflammation and infection result in vascular leakage. Herein, we showed that Rap1, a small GTPase, plays an essential role for maintaining pulmonary endothelial barrier function in mice. Endothelial cell-specific Rap1a/Rap1b double knockout mice exhibited severe pulmonary edema. They also showed vascular leakage in the hearts, but not in the brains. En face analyses of the pulmonary arteries and 3D-immunofluorescence analyses of the lungs revealed that Rap1 potentiates VE-cadherin-mediated endothelial cell-cell junctions through dynamic actin cytoskeleton reorganization. Rap1 inhibits formation of cytoplasmic actin bundles perpendicularly binding VE-cadherin adhesions through inhibition of a Rho-ROCK pathway-induced activation of cytoplasmic nonmuscle myosin II (NM-II). Simultaneously, Rap1 induces junctional NM-II activation to create circumferential actin bundles, which anchor and stabilize VE-cadherin at cell-cell junctions. We also showed that the mice carrying only one allele of either Rap1a or Rap1b out of the two Rap1 genes are more vulnerable to lipopolysaccharide (LPS)-induced pulmonary vascular leakage than wild-type mice, while activation of Rap1 by administration of 007, an activator for Epac, attenuates LPS-induced increase in pulmonary endothelial permeability in wild-type mice. Thus, we demonstrate that Rap1 plays an essential role for maintaining pulmonary endothelial barrier functions under physiological conditions and provides protection against inflammation-induced pulmonary vascular leakage.

Ephexin3/ARHGEF5 Together with Cell Migration Signaling Partners within the Tumor Microenvironment Define Prognostic Transcriptional Signatures in Multiple Cancer Types.

Cancer cell migration involves a repertoire of signaling proteins that lead cytoskeleton reorganization as a critical step in metastatic dissemination. RhoGEFs are multidomain effectors that integrate signaling inputs to activate the molecular switches that orchestrate actin cytoskeleton reorganization. Ephexins, a group of five RhoGEFs, play oncogenic roles in invasive and metastatic cancer, leading to a mechanistic hypothesis about their function as signaling nodes assembling functional complexes that guide cancer cell migration. To identify clinically significant Ephexin signaling partners, we applied three systematic data mining strategies, based on the screening of essential Ephexins in multiple cancer cell lines and the identification of coexpressed signaling partners in the TCGA cancer patient datasets. Based on the domain architecture of encoded proteins and gene ontology criteria, we selected Ephexin signaling partners with a role in cytoskeletal reorganization and cell migration. We focused on Ephexin3/ARHGEF5, identified as an essential gene in multiple cancer cell types. Based on significant coexpression data and coessentiality, the signaling repertoire that accompanies Ephexin3 corresponded to three groups: pan-cancer, cancer-specific and coessential. To further select the Ephexin3 signaling partners likely to be relevant in clinical settings, we first identified those whose high expression was statistical linked to shorter patient survival. The resulting Ephexin3 transcriptional signatures represent significant accumulated risk, predictive of shorter survival, in 17 cancer types, including PAAD, LUAD, LGG, OSC, AML, KIRC, THYM, BLCA, LIHC and UCEC. The signaling landscape that accompanies Ephexin3 in various cancer types included the tyrosine kinase receptor MET and the tyrosine phosphatase receptor PTPRF, the serine/threonine kinases MARK2 and PAK6, the Rho GTPases RHOD, RHOF and RAC1, and the cytoskeletal regulator DIAHP1. Our findings set the basis to further explore the role of Ephexin3/ARHGEF5 as an essential effector and signaling hub in cancer cell migration.

ITRAQ-Based Phosphoproteomic Analysis Exposes Molecular Changes in the Small Intestinal Epithelia of Cats after Toxoplasma gondii Infection.

Toxoplasma gondii, an obligate intracellular parasite, has the ability to invade and proliferate within most nucleated cells. The invasion and destruction of host cells by T. gondii lead to significant changes in the cellular signal transduction network. One important post-translational modification (PTM) of proteins is phosphorylation/dephosphorylation, which plays a crucial role in cell signal transmission. In this study, we aimed to investigate how T. gondii regulates signal transduction in definitive host cells. We employed titanium dioxide (TiO2) affinity chromatography to enrich phosphopeptides in the small intestinal epithelia of cats at 10 days post-infection with the T. gondii Prugniuad (Pru) strain and quantified them using iTRAQ technology. A total of 4998 phosphopeptides, 3497 phosphorylation sites, and 1805 phosphoproteins were identified. Among the 705 differentially expressed phosphoproteins (DEPs), 68 were down-regulated and 637 were up-regulated. The bioinformatics analysis revealed that the DE phosphoproteins were involved in various cellular processes, including actin cytoskeleton reorganization, cell necroptosis, and MHC immune processes. Our findings confirm that T. gondii infection leads to extensive changes in the phosphorylation of proteins in the cat intestinal epithelial cells. The results of this study provide a theoretical foundation for understanding the interaction between T. gondii and its definitive host.

Human amniotic membrane inhibits migration and invasion of muscle-invasive bladder cancer urothelial cells by downregulating the FAK/PI3K/Akt/mTOR signalling pathway

Bladder cancer is the 10th most commonly diagnosed cancer with the highest lifetime treatment costs. The human amniotic membrane (hAM) is the innermost foetal membrane that possesses a wide range of biological properties, including anti-inflammatory, antimicrobial and anticancer properties. Despite the growing number of studies, the mechanisms associated with the anticancer effects of human amniotic membrane (hAM) are poorly understood. Here, we reported that hAM preparations (homogenate and extract) inhibited the expression of the epithelial–mesenchymal transition markers N-cadherin and MMP-2 in bladder cancer urothelial cells in a dose-dependent manner, while increasing the secretion of TIMP-2. Moreover, hAM homogenate exerted its antimigratory effect by downregulating the expression of FAK and proteins involved in actin cytoskeleton reorganisation, such as cortactin and small RhoGTPases. In muscle-invasive cancer urothelial cells, hAM homogenate downregulated the PI3K/Akt/mTOR signalling pathway, the key cascade involved in promoting bladder cancer. By using normal, non-invasive papilloma and muscle-invasive cancer urothelial models, new perspectives on the anticancer effects of hAM have emerged. The results identify new sites for therapeutic intervention and are prompt encouragement for ongoing anticancer drug development studies.

Thiol sulfenylation plays a role on actin cytoskeleton reorganization during vascular contraction

Thiol sulfenylation plays a role on actin cytoskeleton reorganization during vascular contraction

Expression of Concern for Meconi et al., "Activation of Protein Tyrosine Kinases by Coxiella burnetii: Role in Actin Cytoskeleton Reorganization and Bacterial Phagocytosis".

Expression of Concern for Meconi et al., "Activation of Protein Tyrosine Kinases by Coxiella burnetii: Role in Actin Cytoskeleton Reorganization and Bacterial Phagocytosis".

Hydrogen sulfide promotes migration of trophoblast cells by a Rho GTPase mediated actin cytoskeleton reorganization

IntroductionPreeclampsia (PE) arises due to defective spiral artery remodelling which may be due to deficient migration of trophoblast cells. Migration of human endothelial cells has been shown to be promoted via Hydrogen sulphide(H2S)/Rho GTPase Rac1 axis. This novel role of H2S and its downstream processes have not yet been studied in the development and function of the placental trophoblast cells. MethodsPlacental tissues were obtained post-delivery from consented preeclamptic and normotensive mothers (n = 60). The protein expression levels of cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS) along with its downstream migratory molecules were compared in both the arms. The pro-migratory role of H2S was investigated in a first trimester placental cell line. ResultsH2S promoted the migration of trophoblast cells in a Rho GTPase dependent manner mediated by actin cytoskeleton reorganization. The reduced levels of H2S producing enzymes in the PE placentae along with decreased levels of Rho GTPases (Rac1 and Rho A) corroborate the results of PAG and AOAA treatment in down regulating the Rho GTPases in the in vitro grown placental cultures. Reduction of the migratory potential of trophoblastic cells caused due to hypoxia/reoxygenation was rescued by upregulating the H2S expression with the use of NaHS as a H2S donor. DiscussionExogenous H2S increases the migratory potential of the placental cells in culture conditions and also post hypoxia/reoxygenation injury. H2S as a gaso-transmitter holds a great potential as a therapeutic agent. Its long-term effects need to be investigated using model systems (rat/mouse) of PE following it up with clinical regulatory trials.