Inhibition Of Actin Polymerization Research Articles

Bcl11b Regulates Vascular Smooth Muscle Phenotype and Arterial Stiffness

BACKGROUNDArterial stiffness (AS) is the loss of compliance of elastic arteries known to increase the risk of cardiovascular events. A genome‐wide association study (GWAS) identified single nucleotide polymorphisms (SNPs) in a genetic locus downstream of BCL11B to be associated with increased AS, however a functional role for Bcl11b in the vasculature is unknown.OBJECTIVETo examine BCL11B's role in the aorta and vascular function including AS and blood pressure.METHODSTo study the role of BCL11B in the vasculature, we generated mice (SMMHCERCreT2‐Bcl11bfl/fl aka BSMKO) with tamoxifen‐inducible Bcl11b deletion in vascular smooth muscle (VSM). We measured pulse wave velocity (PWV), the gold standard in vivo measure of AS, by Doppler ultrasound, and blood pressure by telemetry in wild type (WT) and BSMKO mice. We measured isometric force, wall tension and stress in aortic rings of WT and BSMKO mice ex vivo in organ baths. We analyzed levels of filamentous (F) and globular (G) actin, smooth muscle myosin (MYH11), smooth muscle a‐actin (SMaA), phosphorylated and total VASP, and calcineurin by Western blot in WT and BSMKO VSM cell and aortic homogenates.RESULTSMice with Bcl11b deletion in VSM (BSMKO) exhibited higher PWV compared to WT littermates (3.1 ± 0.1 m/s in WT, n = 14 vs 3.8 ± 0.2 m/s in BSMKO, n = 17; p < 0.05), but had no detectable changes in blood pressure (systolic blood pressure, SBP: 121.8 ± 3.8 mmHg in WT, n = 7 vs 120.0 ± 2.3 mmHg in BSMKO, n = 8; NS). Compared to WT, BSMKO VSM cells had decreased mRNA and protein expression of VSM contractile proteins MYH11 (1.00 ± 0.05 A.U. in WT vs 0.42 ± 0.04 A.U. in BSMKO, n = 5 replicate experiments; p < 0.05) and SMA (1.00 ± 0.04 A.U. in WT vs 0.63 ± 0.06 A.U. in BSMKO, n = 4 replicate experiments; p < 0.05). Baseline force (1010 ± 96 mg in WT, n = 5 vs 1511 ± 106 mg in BSMKO, n = 7; p < 0.05), wall tension (1.72 ± 0.13 N/m in WT, n = 5 vs 2.60 ± 0.24 N/m in BSMKO, n = 7; p < 0.05) and stress (17.7 ± 1.5 kPa in WT, n = 5 vs 25.9 ± 2.2 kPa in BSMKO, n = 7; p < 0.05) generated by BSMKO aortic rings in organ baths were significantly increased compared to WT. In addition, BSMKO aortas exhibited increased F/G actin ratio indicating increased cytoskeletal actin polymerization (ratio of F to G actin: 4.82 ± 1.33 A.U. in WT, n = 6 vs 9.09 ± 1.02 A.U. in BSMKO, n = 7; p < 0.05). Phosphorylated VASP at S239, known to inhibit actin polymerization, was decreased (0.99 ± 0.04 A.U. in WT vs 0.19 ± 0.04 A.U. in BSMKO, n = 5 replicate experiments; p < 0.05), while PP2B, a phosphatase which regulates VASP phosphorylation, was increased in BSMKO aortas compared to WT. Treatment of BSMKO VSM cells with cyclosporine A (CSA, 10mM), an inhibitor of PP2B, reversed pVASP toward WT levels.CONCLUSIONSWe showed for the first time that BCL11B is present in VSM and regulates arterial stiffness at least in part by regulating contractile protein smooth muscle myosin and expression and cytoskeletal remodeling in VSM cells of the aorta via PP2B‐pVASP.Support or Funding InformationThis work was supported by NIH grant HL136311.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Actin filaments are dispensable for bulk autophagy in plants

ABSTRACTActin filament, also known as microfilament, is one of two major cytoskeletal elements in plants and plays important roles in various biological processes. Like in animal cells, actin filaments have been thought to participate in autophagy in plants. However, surprisingly, in this study we found that actin filaments are dispensable for the occurrence of autophagy in plants. Disruption of actin filaments by short term treatment with actin polymerization inhibitors, cytochalasin D and latrunculin B, or transient overexpression of Profilin 3 in Nicotiana benthamiana had no effect on basal autophagy as well as the upregulation of nocturnal autophagy and salt stress-induced autophagy. Furthermore, anti-microfilament drug treatment affected neither basal nor salt stress-induced autophagy in Arabidopsis. In addition, prolonged perturbation of actin filaments by silencing Actin7 or 24-h treatment with microfilament-disrupting agents in N. benthamiana caused endoplasmic reticulum (ER) disorganization and subsequent degradation via autophagy involving ATG2, 3, 5, 6 and 7. Our findings reveal that, unlike mammalian cells, actin filaments are unnecessary for bulk autophagy in plants.Abbreviations: ATG: autophagy-related; CD: cytochalasin D; Cvt pathway: cytoplasm to vacuole targeting pathway; DMSO: dimethyl sulfoxide; ER: endoplasmic reticulum; LatB: latrunculin B; Nb: Nicotiana benthamiana; PAS: phagophore assembly site; PRF3: Profilin 3; RER: rough ER; SER: smooth ER; TEM: transmission electron microscopy; TRV: Tobacco rattle virus; VIGS: virus-induced gene silencing; wpi: weeks post-agroinfiltration

Functional Fibronectin Adsorption on Aptamer-Doped Chitosan Modulates Cell Morphology by Integrin-Mediated Pathway.

A decisive step in cell-biomaterial interaction is represented by the adsorption of proteins at the interface, whose fine control may be useful to trigger proper cell response. To this purpose, we can selectively control protein adsorption on biomaterials by means of aptamers. Aptamers selected to recognize fibronectin dramatically enhance chitosan ability to promote cell proliferation and adhesion, but the underlying biological mechanism remains unknown. We supposed that aptamers contributed to ameliorate the adsorption of fibronectin in an advantageous geometrical conformation for cells, thus regulating their morphology by the proper activation of the integrin-mediated pathway. We investigated this possibility by culturing epithelial cells on chitosan enriched with increasing doses of aptamers in the presence or in the absence of cytoskeleton pharmacological inhibitors. Our results showed that aptamers control cell morphology in a dose dependent manner (p < 0.0001). Simultaneously, when the inhibition of actin polymerization was induced, the control of cell morphology was attenuated (p < 0.0001), while no differences were detected when cells contractility was challenged (p > 0.05). Altogether, our data provide evidence that aptamers contribute to control fibronectin adsorption on biomaterials by preserving its conformation and thus function. Furthermore, our work provides a new insight into a new way to accurately tailor material surface bioactivity.

Congenital myopathy-related mutations in tropomyosin disrupt regulatory function through altered actin affinity and tropomodulin binding.

Tropomyosin (Tpm) binds along actin filaments and regulates myosin binding to control muscle contraction. Tropomodulin binds to the pointed end of a filament and regulates actin dynamics, which maintains the length of a thin filament. To define the structural determinants of these Tpm functions, we examined the effects of two congenital myopathy mutations, A4V and R91C, in the Tpm gene, TPM3, which encodes the Tpm3.12 isoform, specific for slow-twitch muscle fibers. Mutation A4V is located in the tropomodulin-binding, N-terminal region of Tpm3.12. R91C is located in the actin-binding period 3 and directly interacts with actin. The A4V and R91C mutations resulted in a 2.5-fold reduced affinity of Tpm3.12 homodimers for F-actin in the absence and presence of troponin, and a two-fold decrease in actomyosin ATPase activation in the presence of Ca2+ . Actomyosin ATPase inhibition in the absence of Ca2+ was not affected. The Ca2+ sensitivity of ATPase activity was decreased by R91C, but not by A4V. Invitro, R91C altered the ability of tropomodulin 1 (Tmod1) to inhibit actin polymerization at the pointed end of the filaments, which correlated with the reduced affinity of Tpm3.12-R91C for Tmod1. Molecular dynamics simulations of Tpm3.12 in complex with F-actin suggested that both mutations reduce the affinity of Tpm3.12 for F-actin binding by perturbing the van der Waals energy, which may be attributable to two different molecular mechanisms-a reduced flexibility of Tpm3.12-R91C and an increased flexibility of Tpm3.12-A4V.

HLA class II antibodies induce necrotic cell death in human endothelial cells via a lysosomal membrane permeabilization-mediated pathway

Antibody-mediated rejection (AMR) is the major cause of allograft loss after solid organ transplantation. Circulating donor-specific antibodies against human leukocyte antigen (HLA), in particular HLA class II antibodies are critical for the pathogenesis of AMR via interactions with endothelial cells (ECs). To investigate the effects of HLA class II antibody ligation to the graft endothelium, a model of HLA-DR antibody-dependent stimulation was utilized in primary human ECs. Antibody ligation of HLA class II molecules in interferon-γ-treated ECs caused necrotic cell death without complement via a pathway that was independent of apoptosis and necroptosis. HLA-DR-mediated cell death was blocked by specific neutralization of antibody ligation with recombinant HLA class II protein and by lentiviral knockdown of HLA-DR in ECs. Importantly, HLA class II-mediated cytotoxicity was also induced by relevant native allele-specific antibodies from human allosera. Necrosis of ECs in response to HLA-DR ligation was mediated via hyperactivation of lysosomes, lysosomal membrane permeabilization (LMP), and release of cathepsins. Notably, LMP was caused by reorganization of the actin cytoskeleton. This was indicated by the finding that LMP and actin stress fiber formation by HLA-DR antibodies were both downregulated by the actin polymerization inhibitor cytochalasin D and inhibition of Rho GTPases, respectively. Finally, HLA-DR-dependent actin stress fiber formation and LMP led to mitochondrial stress, which was revealed by decreased mitochondrial membrane potential and generation of reactive oxygen species in ECs. Taken together, ligation of HLA class II antibodies to ECs induces necrotic cell death independent of apoptosis and necroptosis via a LMP-mediated pathway. These findings may enable novel therapeutic approaches for the treatment of AMR in solid organ transplantation.

Endothelin-1 increases CHSY-1 expression in aortic endothelial cells via transactivation of transforming growth factor β type I receptor induced by type B receptor endothelin-1.

TGF-β through hyperelongation of glycosaminoglycan (GAG) chains leads to binding of low-density lipoproteins to the proteoglycans. The vasoactive peptide, endothelin-1 (ET-1), plays a key role in the development of atherosclerosis. This study addressed the question whether ET-1 by activating the Rho kinase and cytoskeletal rearrangement can transactivate the TGF-β receptor leading to phosphorylation of the transcription factor Smad2 and increased expression of the GAG chain synthesizing enzyme such as chondroitin synthase-1 (CHSY-1) in bovine aortic endothelial cells (BAECs). In this study, intermediates in ET-1-induced Smad2C phosphorylation and the protein level of CHSY-1 were identified and quantified by Western blotting. Endothelin-1 caused time-dependent phosphorylation of Smad2C which was inhibited in the presence of the endothelin B receptor antagonist, BQ788. The response to ET-1 was inhibited by the Rho/ROCK kinase antagonist, Y27632 and by cytochalasin D, an inhibitor of actin polymerization but the ET-1-mediated pSmad2C was not inhibited by the matrix metalloproteinase (MMP) inhibitor, GM6001. ET-1 increased CHSY-1 protein level, which was inhibited in the presence of BQ788, cytochalasin D and Y27632. Endothelin-1 signalling via the ETB receptor utilizes cytoskeletal rearrangement and Rho kinase but not MMPs leading to TβRI transactivation signalling and phosphorylation of Smad2C and through this pathway increased the level of CHSY-1.

Benproperine, an ARPC2 inhibitor, suppresses cancer cell migration and tumor metastasis

Metastasis is the leading cause of cancer mortality and cancer cell migration is an essential stage of metastasis. We identified benproperine (Benp, a clinically used antitussive drug) as an inhibitor of cancer cell migration and an anti-metastatic agent. Benp selectively inhibited cancer cell migration and invasion, which also suppressed metastasis of cancer cells in animal models. Actin-related protein 2/3 complex subunit 2 (ARPC2) was identified as a molecular target of Benp by affinity column chromatography with Benp-tagged Sepharose beads. Benp bound directly to ARPC2 in cells, which was validated by pull-down assay using Benp-biotin and label-free biochemical methods such as the drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA). Benp inhibited Arp2/3 function, showing disruption of lamellipodial structure and inhibition of actin polymerization. Unlike Arp2/3 inhibitors, Benp selectively inhibited the migration of cancer cells but not normal cells. ARPC2-knockdown cancer cells showed defective cell migration and suppressed metastasis in an animal model. Therefore, ARPC2 is a potential target for anti-metastatic therapy, and Benp has the clinical potential to block metastasis. Furthermore, Benp is a useful agent for studying the functions of the Arp2/3 complex in cancer cell migration and metastasis.

Regulation of gastric smooth muscle contraction via Ca2+-dependent and Ca2+-independent actin polymerization.

In gastrointestinal smooth muscle, acetylcholine induced muscle contraction is biphasic, initial peak followed by sustained contraction. Contraction is regulated by phosphorylation of 20 kDa myosin light chain (MLC) at Ser19, interaction of actin and myosin, and actin polymerization. The present study characterized the signaling mechanisms involved in actin polymerization during initial and sustained muscle contraction in response to muscarinic M3 receptor activation in gastric smooth muscle cells by targeting the effectors of initial (phospholipase C (PLC)-β/Ca2+ pathway) and sustained (RhoA/focal adhesion kinase (FAK)/Rho kinase pathway) contraction. The initial Ca2+ dependent contraction and actin polymerization is mediated by sequential activation of PLC-β1 via Gαq, IP3 formation, Ca2+ release and Ca2+ dependent phosphorylation of proline-rich-tyrosine kinase 2 (Pyk2) at Tyr402. The sustained Ca2+ independent contraction and actin polymerization is mediated by activation of RhoA, and phosphorylation of FAK at Tyr397. Both phosphorylation of Pyk2 and FAK leads to phosphorylation of paxillin at Tyr118 and association of phosphorylated paxillin with the GEF proteins p21-activated kinase (PAK) interacting exchange factor α, β (α and β PIX) and DOCK 180. These GEF proteins stimulate Cdc42 leading to the activation of nucleation promoting factor N-WASP (neuronal Wiskott-Aldrich syndrome protein), which interacts with actin related protein complex 2/3 (Arp2/3) to induce actin polymerization and muscle contraction. Acetylcholine induced muscle contraction is inhibited by actin polymerization inhibitors. Thus, our results suggest that a novel mechanism for the regulation of smooth muscle contraction is mediated by actin polymerization in gastrointestinal smooth muscle which is independent of MLC20 phosphorylation.

Dynamic microtubules drive fibroblast spreading

ABSTRACTWhen cells with a mesenchymal type of motility come into contact with an adhesive substrate they adhere and start spreading by the formation of lamellipodia. Using a label-free approach and virtual synchronization approach we analyzed spreading in fibroblasts and cancer cells. In all cell lines spreading is a non-linear process undergoing isotropic or anisotropic modes with first fast (5–20 min) and then slow (30–120 min) phases. In the first 10 min cell area increases 2–4 times, while the absolute rate of initial spreading decreases 2–8 times. Fast spreading depends on actin polymerization and dynamic microtubules. Inhibition of microtubule growth was sufficient for a slowdown of initial spreading. Inhibition of myosin II in the presence of stable microtubules restored fast spreading. Inhibition of actin polymerization or complete depolymerization of microtubules slowed down fast spreading. However, in these cases inhibition of myosin II only partially restored spreading kinetics. We conclude that rapid growth of microtubules towards cell margins at the first stage of cell spreading temporarily inhibits phosphorylation of myosin II and is essential for the fast isotropic spreading. Comparison of the fibroblasts with cancer cells shows that fast spreading in different cell types shares similar kinetics and mechanisms, and strongly depends on dynamic microtubules.

.Clostridioides difficile.

Clostridioides difficile is a spore-forming, anaerobic, intestinal pathogen that causes severe diarrhea that can lead to death. In 2011, C. difficile infected ∼500000 people in the USA and killed ∼29000 people. C. difficile infection (CDI) is the most common healthcare-related infection in the USA, leading to increased healthcare costs of $4.8 billion. This pathogen transmits via the oral-fecal route as a highly contagious and resilient spore. Upon exposure to primary bile acids in the intestine, C. difficile germinates, and in the absence of colonization resistance from the normal microbiota, the bacterium colonizes the colon and produces toxins. These toxins inhibit actin polymerization in host cells, leading to cell death. C. difficile cells can then sporulate in the intestine and exit the body via diarrheal shedding. In culture, sporulation is induced at stationary phase in a nutrient-limiting environment, but the intestinal triggers of sporulation are still unknown.

Heme Induces Significant Neutrophil Adhesion in Vitro Via an Nfκb-Dependent Pathway

Background: Intravascular hemolysis, a major complication of sickle cell anemia and malaria among other diseases, incurs the release of excessive quantities of hemoglobin and heme from red blood cells. If not adequately sequestered by hemoglobin- and heme-binding proteins, these molecules may incur oxidative stress, thrombosis and cellular activation. Heme is now recognized as a red cell DAMP (damage-associated molecular pattern) that stimulates inflammasome formation in macrophages and can induce neutrophil extracellular trap release under certain circumstances. Aim: This study evaluated the in vitro effects of heme on the adhesive properties of human neutrophils. Methods: Neutrophils were separated from the peripheral blood of healthy individuals and their adhesion in the presence/absence of heme was compared by static adhesion assays using myeloperoxidase, for quantification of cell adhesion (30 min, 37oC, 5% CO2). Results: Heme (50 µM) significantly increased the adhesion of neutrophils to fibronectin (FN) and to recombinant ICAM-1 (an endothelial ligand), when compared to non-treated neutrophils (43.7±4.6%; 10.9±21.4 %, respectively, n=5, P<0.001 for FN and 38.1±3.6%; 6.9±0.8%, respectively, n=6, P<0.001 for ICAM-1). Interestingly, heme induced neutrophil adhesion even more efficiently than the potent pro-inflammatory cytokine, TNF-α (200 ng/ml) (35.5±5.2% adhesion to FN, n=6, P<0.05). Furthermore, inhibition of the NFκB transcription factor with the pharmacological inhibitor, BAY 11-7082 (20 μM), abolished heme-stimulated neutrophils adhesion to FN (reduced from 41.8±6.7% to 7.4±0.5%. n=6; P<0.001). Flow cytometry demonstrated that while TNF-α significantly increases the expression of the Mac-1 integrin subunit, CD11b (data not shown, P<0.05), but not the LFA-1 integrin subunit CD11a (data not shown, P>0.05), on the surface of neutrophils, heme did not augment CD11b or CD11a expression (P>0.05). In contrast, heme significantly augmented the active conformations of these two β2 integrin subunits, as demonstrated by epitope-specific antibodies (CD11b; heme: 520.5±51.8; basal 123.7±16.7 MFI, n=10, P<0.001 and CD11a; heme: 69.8±3.9; basal: 43.8±2.0 MFI, n=6, P>0.001). Inhibition of NFκB translocation with BAY 11-7082 (20 μM) significantly decreased the activity of the LFA-1 integrin on the surface of neutrophils after heme stimulation (reduced to 47.6±4.2 MIF, n=6; P<0.05). To assess whether heme-induced neutrophil adhesive properties are mediated by cytoskeletal rearrangements, we evaluated the effects of cytochalasin D (0.5 μg/ml), an inhibitor of actin polymerization. While cytochalasin D inhibited TNF-α-induced neutrophils adhesion (data not shown, P<0.05), this compound did not significantly alter the adhesive properties of heme-stimulated neutrophils to FN (heme: 22.8±2.3%, p<0.001; cytochalasin D: 22.5±2.0%, p=0.4; n=6). Pre-incubation of heme-stimulated neutrophils with the antioxidants, ascorbic acid (120 µM; 3.5 hours) and α-tocopherol (1mM; 15 min), reduced their adhesion to FN by 14.4±9.2% and 46.5±5.5% respect. n=6, p<0.05). Conclusion: We therefore demonstrate, herein, that heme is a potent activator of neutrophil adhesive properties, increasing the ligand affinity of the β2 integrins via a mechanism that is apparently mediated by an NFkB-dependent pathway. The mechanism of neutrophil activation appears to differ from that stimulated by TNF-α and may involve, in part, the generation of reactive oxygen species. Given the fundamental role that the adhesion of neutrophils to the vascular wall plays in the vaso-occlusive process in sickle cell disease and other vascular inflammatory processes, our findings further support the idea that cell-free heme represents a major therapeutic target in the hemolytic diseases. DisclosuresNo relevant conflicts of interest to declare.

In vitro rejuvenation of brain mitochondria by the inhibition of actin polymerization

The oxygen consumption rate (OCR) and cytochrome c oxidase (CcO) activity of respiratory complex IV (CIV) in brain mitochondria significantly decline in middle-aged male mice compared to younger male mice. To explore the mechanisms underlying the regulation of brain mitochondrial function, we examined CIV-associated proteins, and identified actin inside the isolated brain mitochondria. Inhibiting actin polymerization using cytochalasin B (CB) significantly enhanced the OCR and CcO activity of CIV in the mitochondria. These changes were accompanied by a significant reduction in the amount of CIV-bound cytochrome c (cyt c). Actin was also associated with respiratory complex III (CIII); however, the amount of CIII-bound cyt c increased significantly after treatment of the mitochondria with CB. In contrast, no significant alteration in the assembly or the CcO activity of CIV in CIV-containing supercomplexes or CIV monomers was induced by CB. These results suggest that mitochondrial actin plays a crucial role in the regulation of the CcO activity and OCR of CIV with modification of the retention of cyt c between CIV and CIII.

Investigating HCMV entry into host cells by STEM tomography

Human cytomegalovirus (HCMV) entry into susceptible cells is a fast intricate process that is not fully understood. Although, previous studies explored different aspects of this process by means of biochemical and inhibitors assays, a clear morphological characterization of its steps at the ultrastructural level is still lacking. We attempted to characterize those intermediates involved during HCMV entry by developing a methodological approach that resulted in optimal ultrastructure preservation and allowed for 3D imaging. It involves rapid freezing and cryosubstitution which ensure a clear visibility of membranous leaflets as well as retained membranous continuity. Likewise, it delivered a reproducible optimization of the growth and infection conditions that are pivotal towards maintaining biologically active enriched input virus particles. Data acquisition was achieved through STEM tomography in a 3D context. Indeed, several intermediates that characterize HCMV entry-related events were observed both extra- and intracellularly. Some of the cell-membrane associated viral particles that we referred to as “Pinocchio particles” were morphologically altered in comparison to the cell-free virions. We were also able to characterize intracellular fusion intermediates taking place between the viral envelope and the vesicular membranes. Furthermore, inhibiting actin polymerization by Latrunculin-A enabled us to spot fusion-like intermediates of the viral envelope with the host cell plasma membrane that we did not observe in the untreated infected cells. Our data also suggests that Dyngo-4a; a dynamin-2 inhibitor, does not interfere with the internalization of the HCMV into the host cells as previously deduced.

Direct Observation of Tunneling Nanotubes within Human Mesenchymal Stem Cell Spheroids.

Tunneling nanotubes (TNTs) play an important role in cell-cell communication. TNTs have been predominantly reported among cells in monolayer culture. Using various imaging modalities, including scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM), this work reports the finding of TNTs between cells within human mesenchymal stem cell (MSC) spheroids. TNTs visualized by SEM are consistent in size and geometry with those observed in cellular monolayer culture. LSCM imaging of living spheroids confirms the presence of F-actin filaments within the TNTs, which are known to maintain nanotube integrity. In addition, LSCM revealed the distribution of F-actin fibers across the entire spheroid body instead of being confined within individual cells. Intracellular material transport by TNTs was tested in MSC spheroids treated with cytochalasin D (CytoD), a known actin polymerization inhibitor for disrupting TNT formation. CytoD treatment decreased the transport of cytosolic material by at least four-fold compared to untreated spheroids. To the best of our knowledge, this work represents the first direct observation of TNTs within MSC spheroids. These findings offer new physical insight into cellular interactions within spheroids, providing structural information for increasing interests in spheroid-based cell therapy.

Fluconazole-induced actin cytoskeleton remodeling requires phosphatidylinositol 3-phosphate 5-kinase in the pathogenic yeast Candida glabrata.

SummaryKnown azole antifungal resistance mechanisms include mitochondrial dysfunction and overexpression of the sterol biosynthetic target enzyme and multidrug efflux pumps. Here, we identify, through a genetic screen, the vacuolar membrane‐resident phosphatidylinositol 3‐phosphate 5‐kinase (CgFab1) to be a novel determinant of azole tolerance. We demonstrate for the first time that fluconazole promotes actin cytoskeleton reorganization in the emerging, inherently less azole‐susceptible fungal pathogen Candida glabrata, and genetic or chemical perturbation of actin structures results in intracellular sterol accumulation and azole susceptibility. Further, CgFAB1 disruption impaired vacuole homeostasis and actin organization, and the F‐actin‐stabilizing compound jasplakinolide rescued azole toxicity in cytoskeleton defective‐mutants including the Cgfab1Δ mutant. In vitro assays revealed that the actin depolymerization factor CgCof1 binds to multiple lipids including phosphatidylinositol 3,5‐bisphosphate. Consistently, CgCof1 distribution along with the actin filament‐capping protein CgCap2 was altered upon both CgFAB1 disruption and fluconazole exposure. Altogether, these data implicate CgFab1 in azole tolerance through actin network remodeling. Finally, we also show that actin polymerization inhibition rendered fluconazole fully and partially fungicidal in azole‐susceptible and azole‐resistant C. glabrata clinical isolates, respectively, thereby, underscoring the role of fluconazole‐effectuated actin remodeling in azole resistance.

Characterization of the glucosyltransferase activity of Legionella pneumophila effector SetA.

Legionella pneumophila glucosyltransferase SetA, which is introduced into target cells by a type IV secretion system, affects the intracellular traffic of host cells. Here, we characterized the enzyme activity of the Legionella effector. We report that Asp118 and Arg121 of SetA are essential for glucohydrolase and glucotransferase activities. Exchange of Trp36 to alanine reduced the enzyme activity of SetA. All three amino acids were crucial for the cytotoxic effects of SetA in yeast. We observed that phosphatidylinositol-3-phosphate (PI3P) increased the glucosyltransferase activity of SetA severalfold, while the glucohydrolase activity was not affected. In the presence of PI3P, we observed the glucosylation of actin, vimentin and the chaperonin CCT5 in the cytosolic fraction of target cells. Studies on the functional consequences of glucosylation of skeletal muscle α-actin in vitro revealed inhibition of actin polymerization by glucosylation.

Fasudil inhibits actin polymerization and collagen synthesis and induces apoptosis in human urethral scar fibroblasts via the Rho/ROCK pathway.

PurposeTo examine the effects and mechanism of action of fasudil on cytoskeletal polymerization, collagen synthesis, and apoptosis in fibroblasts derived from human urethral scar tissue.Materials and methodsFibroblasts treated with or without transforming growth factor β1 (TGF-β1, 10 ng/mL) were incubated with fasudil (12.5, 25, 50 μmol/L) for 24 hours. Quantitative real-time polymerase chain reaction and Western blotting were used to determine the expression of Arp2, Arp3, WASP, and WAVE2. Collagen I and III protein levels were also evaluated by Western blotting. The filamentous actin cytoskeleton was examined by immunofluorescence and epifluorescence microscopy. An Annexin V-FITC/PI staining assay was used to investigate apoptosis.ResultsTGF-β1-dependent induction of actin polymerization and collagen synthesis and promotion of apoptosis were dose dependent. When compared with untreated controls, fasudil significantly decreased the expression of Arp2, Arp3, WASP, WAVE2, Collagen I, and Collagen III in cells treated with or without TGF-β1. Fasudil also promoted apoptosis in cells, irrespective of TGF-β1 treatment.ConclusionIrrespective of TGF-β1 activation status, fasudil suppressed actin polymerization and collagen synthesis and induced apoptosis in human urethral scar fibroblasts via the Rho/ROCK signaling pathway.

Hydrogen Sulfide Disturbs Actin Polymerization via S-Sulfhydration Resulting in Stunted Root Hair Growth.

Hydrogen sulfide (H2S) is an important signaling molecule in plants. Our previous report suggested that H2S signaling affects the actin cytoskeleton and root hair growth. However, the underlying mechanisms of its effects are not understood. S-Sulfhydration of proteins is regulated directly by H2S, which converts the thiol groups of cysteine (Cys) residues to persulfides and alters protein function. In this work, we studied the effects of S-sulfhydration on actin dynamics in Arabidopsis (Arabidopsis thaliana). We generated transgenic plants overexpressing the H2S biosynthesis-related genes l-CYSTEINE DESULFHYDRASE (LCD) and d-CYSTEINE DESULFHYDRASE in the O-acetylserine(thiol)lyase isoform a1 (oasa1) mutant and Columbia-0 backgrounds. The H2S content increased significantly in overexpressing LCD/oasa1 plants. The density of filamentous actin (F-actin) bundles and the F-actin/globular actin ratio decreased in overexpressing LCD/oasa1 plants. S-Sulfhydration also was enhanced in overexpressing LCD/oasa1 plants. An analysis of actin dynamics suggested that S-sulfhydration inhibited actin polymerization. We also found that ACTIN2 (ACT2) was S-sulfhydrated at Cys-287. Cys-287 is adjacent to the D-loop, which acts as a central region for hydrophobic and electrostatic interactions and stabilizes F-actin filaments. Overaccumulation of H2S caused the depolymerization of F-actin bundles and inhibited root hair growth. Introduction of ACT2 carrying a Cys-287-to-Ser mutation into an act2-1 mutant partially suppressed H2S-dependent inhibition of root hair growth. We conclude that H2S regulates actin dynamics and affects root hair growth.

Super-Resolution Correlative Light and Electron Microscopy (SR-CLEM) Reveals Novel Ultrastructural Insights Into Dendritic Cell Podosomes.

Podosomes are multimolecular cytoskeletal structures that coordinate the migration of tissue-resident dendritic cells (DCs). They consist of a protrusive actin-rich core and an adhesive integrin-rich ring that contains adaptor proteins such as vinculin and zyxin. Individual podosomes are typically interconnected by a dense network of actin filaments giving rise to large podosome clusters. The actin density in podosome clusters complicates the analysis of podosomes by light microscopy alone. Here, we present an optimized procedure for performing super-resolution correlative light and electron microscopy (SR-CLEM) to study the organization of multiple proteins with respect to actin in podosome clusters at the ventral plasma membrane of DCs. We demonstrate that our procedure is suited to correlate at least three colors in super-resolution Airyscan microscopy with scanning electron microscopy (SEM). Using this procedure, we first reveal an intriguing complexity in the organization of ventral and radiating actin filaments in clusters formed by DCs which was not properly detected before by light microscopy alone. Next, we demonstrate a differential organization of vinculin and zyxin with respect to the actin filaments at podosomes. While vinculin mostly resides at sites where the actin filaments connect to the cell membrane, zyxin is primarily associated with filaments close to and on top of the core. Finally, we reveal a novel actin-based structure with SEM that connects closely associated podosome cores and which may be important for podosome topography sensing. Interestingly, these interpodosomal connections, in contrast to the radiating and ventral actin filaments appear to be insensitive to inhibition of actin polymerization suggesting that these pools of actin are not dynamically coupled. Together, our work demonstrates the power of correlating different imaging modalities for studying multimolecular cellular structures and could potentially be further exploited to study processes at the ventral plasma membrane of immune cells such as clathrin-mediated endocytosis or immune synapse formation.

Cell injury triggers actin polymerization to initiate epithelial restitution.

The role of the actin cytoskeleton in the sequence of physiological epithelial repair in the intact epithelium has yet to be elucidated. Here, we explore the role of actin in gastric repair in vivo and in vitro gastric organoids (gastroids). In response to two-photon-induced cellular damage of either an in vivo gastric or in vitro gastroid epithelium, actin redistribution specifically occurred in the lateral membranes of cells neighboring the damaged cell. This was followed by their migration inward to close the gap at the basal pole of the dead cell, in parallel with exfoliation of the dead cell into the lumen. The repair and focal increase of actin was significantly blocked by treatment with EDTA or the inhibition of actin polymerization. Treatment with inhibitors of myosin light chain kinase, myosin II, trefoil factor 2 signaling or phospholipase C slowed both the initial actin redistribution and the repair. While Rac1 inhibition facilitated repair, inhibition of RhoA/Rho-associated protein kinase inhibited it. Inhibitors of focal adhesion kinase and Cdc42 had negligible effects. Hence, initial actin polymerization occurs in the lateral membrane, and is primarily important to initiate dead cell exfoliation and cell migration to close the gap.