Actin Accumulation Research Articles

FHOD-1 and profilin protect sarcomeres against contraction-induced deformation> in C. elegans.

Formin HOmology Domain 2-containing (FHOD) proteins are a subfamily of actin-organizing formins important for striated muscle development in many animals. We showed previously that absence of the sole FHOD protein, FHOD-1, from Caenorhabditis elegans results in thin body wall muscles with misshapen dense bodies that serve as sarcomere Z-lines. We demonstrate here that mutations predicted to specifically disrupt actin polymerization by FHOD-1 similarly disrupt muscle development, and that FHOD-1 cooperates with profilin PFN-3 for dense body morphogenesis, and with profilins PFN-2 and PFN-3 to promote body wall muscle growth. We further demonstrate that dense bodies in worms lacking FHOD-1 or PFN-2/PFN-3 are less stable than in wild-type animals, having a higher proportion of dynamic protein, and becoming distorted by prolonged muscle contraction. We also observe accumulation of actin and actin depolymerization factor/cofilin homologue UNC-60B in body wall muscle of these mutants. Such accumulations may indicate targeted disassembly of thin filaments dislodged from unstable dense bodies, possibly accounting for the abnormally slow growth and reduced body wall muscle strength in fhod-1 mutants. Overall, these results implicate FHOD protein-mediated actin assembly in forming stable sarcomere Z-lines, and identify profilin as a new contributor to FHOD activity in striated muscle development.

Multifaceted roles of NADPH oxidases in the growth and pathogenicity of Beauveria bassiana

ABSTRACT Reactive oxygen species (ROS), synthesized by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) complex, are vital molecules in biological cells, influencing various physiological processes such as fungal growth, development, and virulence. Beauveria bassiana, an entomopathogenic fungus, is a promising biopesticide for agricultural, forestry, and urban pest control. This study focuses on the characterization of NADPH oxidases (Noxs) in B. bassiana. Gene expression profiles of Noxs in B. bassiana (BbNoxs) were analysed using RT-qPCR. Knockout strains of single BbNoxA, BbNoxB, BbNoxR, and double BbNoxA and BbNoxB were constructed via homologous recombination, and their phenotypic characteristics were examined. Fungal virulence was evaluated using Galleria mellonella larvae, and infection structures formation and penetration ability were assessed on cicada wings. ROS production and actin assembly during fungal growth and infection were detected using staining and marker methods. Expression analysis revealed significant upregulation of BbNoxs during fungal growth and infection. Compared to the wild-type strain, single knockouts (ΔBbNoxA/B/R) and double knockout (ΔBbNoxAB) of BbNoxs exhibited reduced conidial yields, accelerated conidial germination rates. Deletion of BbNoxB or BbNoxR decreased fungal virulence compared to the WT strain in topical inoculation experiments. Additionally, loss of BbNoxB or BbNoxR impaired infection structures formation, penetration ability, ROS production, and actin aggregation during fungal infection. BbNoxs are crucial for fungal growth, development, and virulence in B. bassiana, playing essential roles in infection structures formation, penetration, ROS production, and actin assembly. Understanding their functions provides insights into B. bassiana’s pathogenic mechanisms.

Shionone Inhibits Glomerular Fibirosis by Suppressing NLRP3 Related Inflammasome though SESN2-NRF2/ HO-1 Pathway.

Diabetic nephropathy (DN) is the most common and serious complication of diabetes mellitus. Shionone (SH), an important triterpenoid compound in the root extract of Aster, might exert a protective effect in DN mice and high glucose cultivated glomerular podocytes. The current study aimed to unravel the underlying mechanism by which SH mitigates DN. We postulate that SH stimulates the expression of sestrin-2 (SESN2), a pivotal stress-inducible protein in the anti-inflammasome machinery. We utilized high-fat diet combined with streptozotocin (55 mg/kg intraperitoneal) for DN mice model, and high glucose (30 mM, 48 hours) cultured glomerular podocytes for DN cell model to evaluate the effect of SH. We also preformed experimentation on SESN2 deficiency models (SESN2 knockout mice and SESN2 siRNA in cells) to further prove our hypothesis. The results demonstrated that SH effectively suppressed glomerular fibrosis, induced adenosine monophosphate-activated protein kinase (AMPK) phosphorylation, and inhibited NLR family pyrin domain containing 3 (NLRP3) activation. Furthermore, our findings revealed that SH exerted its anti-inflammatory effect through Sesn2-dependent nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation and subsequent activation of its downstream target heme oxygenase-1 (HO-1). In summary, our findings suggest that SH serves as a promising therapeutic agent for the treatment of DN-related glomerular fibrosis. SH enhances the expression of SESN2, attenuates α-smooth muscle actin accumulation, and suppresses NLRP3-related inflammation through the Nrf2/HO-1 signaling pathway.

Expression of the locus of enterocyte effacement genes during the invasion process of the atypical enteropathogenic Escherichia coli 1711-4 strain of serotype O51:H40.

Atypical enteropathogenic Escherichia coli (aEPEC) is a significant cause of diarrhea in low- and middle-income countries. Certain aEPEC strains, including the Brazilian representative strain of serotype O51:H40 called aEPEC 1711-4, can use flagella to attach to, invade, and persist in T84 and Caco-2 intestinal cells. It can also translocate from the gut to extraintestinal sites in a rat model. Although various aspects of the virulence of this strain were studied and the requirement of a type III secretion system for the efficiency of the invasion process was demonstrated, the expression of the locus of enterocyte effacement (LEE) genes during the invasion and intracellular persistence remains unclear. To address this question, the expression of flagella and the different LEE operons was evaluated during kinetic experiments of the interaction of aEPEC 1711-4 with enterocytes in vitro. The genome of the strain was also sequenced. The results showed that flagella expression remained unchanged, but the expression of eae and escJ increased during the early interaction and invasion of aEPEC 1711-4 into Caco-2 cells, and there was no change 24 h post-infection during the persistence period. The number of actin accumulation foci formed on HeLa cells also increased during the 6-h analysis. No known gene related to the invasion process was identified in the genome of aEPEC 1711-4, which was shown to belong to the global EPEC lineage 10. These findings suggest that the LEE components and the intimate adherence promoted by intimin are necessary for the invasion and persistence of aEPEC 1711-4, but the detailed mechanism needs further study.IMPORTANCEAtypical enteropathogenic Escherichia coli (aEPEC) is a major cause of diarrhea, especially in low- and middle-income countries, like Brazil. However, due to the genome heterogeneity of each clonal group, it is difficult to comprehend the pathogenicity of this strain fully. Among aEPEC strains, 1711-4 can invade eukaryotic cells in vitro, cross the gut barrier, and reach extraintestinal sites in animal models. By studying how different known aEPEC virulence factors are expressed during the invasion process, we can gain insight into the commonalities of this phenotype among other aEPEC strains. This will help in developing preventive measures to control infections caused by invasive strains. No known virulence-encoding genes linked to the invasion process were found. Nevertheless, additional studies are still necessary to evaluate the role of other factors in this phenotype.

Nuclear actin assembly is an integral part of decidualization in human endometrial stromal cells

Decidualization of the human endometrium is critical for establishing pregnancy and is entailed by differentiation of endometrial stromal cells (ESCs) into decidual cells. During decidualization, the actin cytoskeleton is dynamically reorganized for the ESCs’ morphological and functional changes. Although actin dynamically alters its polymerized state upon external stimuli not only in the cytoplasm, but also in the nucleus, nuclear actin dynamics during decidualization have not been elucidated. Here, we show that nuclear actin was specifically assembled during decidualization of human ESCs. This decidualization-specific formation of nuclear actin filaments was disassembled following the withdrawal of the decidualization stimulus, suggesting its reversible process. Mechanistically, RNA-seq analyses revealed that the forced disassembly of nuclear actin resulted in the suppression of decidualization, accompanied with the abnormal upregulation of cell proliferation genes, leading to incomplete cell cycle arrest. CCAAT/enhancer-binding protein beta (C/EBPβ), an important regulator for decidualization, was responsible for downregulation of the nuclear actin exporter, thus accelerating nuclear actin accumulation and its assembly for decidualization. Taken together, we demonstrate that decidualization-specific nuclear actin assembly induces cell cycle arrest for establishing the decidualized state of ESCs. We propose that not only the cytoplasmic actin, but also nuclear actin dynamics profoundly affect decidualization process in humans for ensuring pregnancy.

CHMP4B contributes to maintaining the follicular cells integrity in the panoistic ovary of the cockroach Blattella germanica.

The Endosomal Sorting Complex Required for Transport (ESCRT) is a highly conserved cellular machinery essential for many cellular functions, including transmembrane protein sorting, endosomal trafficking, and membrane scission. CHMP4B is a key component of ESCRT-III subcomplex and has been thoroughly studied in the meroistic ovaries of Drosophila melanogaster showing its relevance in maintaining this reproductive organ during the life of the fly. However, the role of the CHMP4B in the most basal panoistic ovaries remains elusive. Using RNAi, we examined the function of CHMP4B in the ovary of Blattella germanica in two different physiological stages: in last instar nymphs, with proliferative follicular cells, and in vitellogenic adults when follicular cells enter in polyploidy and endoreplication. In Chmp4b-depleted specimens, the actin fibers change their distribution, appearing accumulated in the basal pole of the follicular cells, resulting in an excess of actin bundles that surround the basal ovarian follicle and modifying their shape. Depletion of Chmp4b also determines an actin accumulation in follicular cell membranes, resulting in different cell morphologies and sizes. In the end, these changes disrupt the opening of intercellular spaces between the follicular cells (patency) impeding the incorporation of yolk proteins to the growing oocyte and resulting in female sterility. In addition, the nuclei of follicular cells appeared unusually elongated, suggesting an incomplete karyokinesis. These results proved CHMP4B essential in preserving the proper expression of cytoskeleton proteins vital for basal ovarian follicle growth and maturation and for yolk protein incorporation. Moreover, the correct distribution of actin fibers in the basal ovarian follicle emerged as a critical factor for the successful completion of ovulation and oviposition. The overall results, obtained in two different proliferative stages, suggest that the requirement of CHMP4B in B. germanica follicular epithelium is not related to the proliferative stage of the tissue.

Human mesenchymal stromal cells ameliorate cisplatin-induced acute and chronic kidney injury via TSG-6.

Cisplatin is widely used in tumor chemotherapy, but nephrotoxicity is an unavoidable side effect of cisplatin. Several studies have demonstrated that mesenchymal stromal cells (MSCs) ameliorate cisplatin-induced kidney injury, but the underlying mechanisms are unknown. In this study, the cisplatin-induced kidney injury mouse model was established by subjecting a single intraperitoneal injection with cisplatin. One hour before cisplatin injection, the mice received human bone marrow MSCs (hBM-MSCs) with or without siRNA-transfection, recombinant human tumor necrosis factor-α-stimulated gene/protein 6 (rhTSG-6), or PBS through the tail vein. In addition, cisplatin-stimulated HK-2 cells were treated with hBM-MSCs or rhTSG-6. Human BM-MSCs treatment remarkably ameliorated cisplatin-induced acute and chronic kidney injury, as evidenced by significant reductions in serum creatinine (Scr), blood urea nitrogen, tubular injury, collagen deposition, α-smooth muscle actin accumulation, as well as inflammatory responses, and by remarkable increased anti-inflammatory factor expression and Treg cells infiltration in renal tissues. Furthermore, we found that only a few hBM-MSCs engrafted into damaged kidney and that the level of human TSG-6 in the serum of mice increased significantly following hBM-MSCs administration. Moreover, hBM-MSCs significantly increased the viability of damaged HK-2 cells and decreased the levels of inflammatory cytokines in the culture supernatant. However, the knockdown of the TSG-6 gene in hBM-MSCs significantly attenuated their beneficial effects in vivo and in vitro. On the contrary, treated with rhTSG-6 achieved similar beneficial effects of hBM-MSCs. Our results indicate that systemic administration of hBM-MSCs alleviates cisplatin-induced acute and chronic kidney injury in part by paracrine TSG-6 secretion.

Focal adhesions are controlled by microtubules through local contractility regulation

Microtubules regulate cell polarity and migration via local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with talin, the major component of focal adhesions. Here, local optogenetic activation of KANK1-mediated microtubule/talin linkage promoted microtubule targeting to an individual focal adhesion and subsequent withdrawal, resulting in focal adhesion centripetal sliding and rapid disassembly. This sliding is preceded by a local increase of traction force due to accumulation of myosin-II and actin in the proximity of the focal adhesion. Knockdown of the Rho activator GEF-H1 prevented development of traction force and abolished sliding and disassembly of focal adhesions upon KANK1 activation. Other players participating in microtubule-driven, KANK-dependent focal adhesion disassembly include kinases ROCK, PAK, and FAK, as well as microtubules/focal adhesion-associated proteins kinesin-1, APC, and αTAT. Based on these data, we develop a mathematical model for a microtubule-driven focal adhesion disruption involving local GEF-H1/RhoA/ROCK-dependent activation of contractility, which is consistent with experimental data.

IQGAP1 Regulates Actin Polymerization and Contributes to Bleomycin-Induced Lung Fibrosis.

We previously found IQ motif containing GTPase activating protein (IQGAP1) to be consistently elevated in lung fibroblasts (LF) isolated from patients with scleroderma (systemic sclerosis, SSc)-associated interstitial lung disease (ILD) and reported that IQGAP1 contributed to SSc by regulating expression and organization of α-smooth muscle actin (SMA) in LF. The aim of this study was to compare the development of ILD in the presence and absence of IQGAP1. Pulmonary fibrosis was induced in IQGAP1 knockout (KO) and wild-type (WT) mice by a single-intratracheal instillation of bleomycin. Two and three weeks later, mice were euthanized and investigated. We observed that the IQGAP1 KO mouse was characterized by a reduced rate of actin polymerization with reduced accumulation of actin in the lung compared to the WT mouse. After exposure to bleomycin, the IQGAP1 KO mouse demonstrated decreased contractile activity of LF, reduced expression of SMA, TGFβ, and collagen, and lowered overall fibrosis scores compared to the WT mouse. The numbers of inflammatory cells and expression of pro-inflammatory cytokines in lung tissue were not significantly different between IQGAP1 KO and WT mice. We conclude that IQGAP1 plays an important role in the development of lung fibrosis induced by bleomycin, and the absence of IQGAP1 reduces the contractile activity of lung fibroblast and bleomycin-induced pulmonary fibrosis. Thus, IQGAP1 may be a potential target for novel anti-fibrotic therapies for lung fibrosis.

Abstract 2067: Myeloid Cell Lgr4 Contributes To Atherosclerotic Lesion Formation

Background: R-Spondin 2 (RSPO2), a matricellular protein, primarily functions via binding to cell surface receptors including leucine-rich repeat-containing G protein-coupled receptor (LGR) 4, 5, and 6. We previously identified elevated RSPO2 levels in both human and mouse atherosclerotic arteries. Our preliminary experiments demonstrated increased expression of LGR4 in human atherosclerotic aortic segments, identified Lgr4 as the major RSPO2’s receptor in primary murine macrophages, and atherogenic molecule oxLDL stimulated LGR4 levels in macrophages. However, the precise role of macrophage LGR4 in atherosclerosis development is unknown. Methods and Results: We generated novel myeloid cell-specific Lgr4 knockout mice ( Lgr4 fl/fl LysM Cre +/- , Lgr4 ΔM ) by breeding Lgr4 fl/fl mice with LysM Cre +/- mice. AAV8-h PCSK9 -injected Lgr4 ΔM and littermate control Lgr4 fl/fl ( Lgr4 WT ) were fed a Western diet (16 weeks) to investigate for atherosclerosis. Oil red O (ORO) staining of whole aortas (en face) and aortic root sections showed reduced atherosclerosis in male Lgr4 ΔM mice compared with sex-matched Lgr4 WT mice. Further histochemical staining performed on aortic root sections revealed smaller necrotic cores in Lgr4 ΔM mice. However, there were no significant differences in plasma total cholesterol and body weight. Interestingly, male Lgr4 ΔM mice had decreased fat mass percentage, blood glucose, and epididymal adipose tissue weight. Initial studies with female mice ( n = 4/group) demonstrated no differences in atherosclerosis and metabolic parameters. Additional in vitro studies exhibited that RSPO2 treatment induces oxLDL uptake, reduces efferocytic capacity, and promotes activation of VASP protein (inducer of actin aggregates) in macrophages. Further, the deletion of Lgr4 reduces lipid accumulation in macrophages. Conclusions: Taken together, these findings suggest that deletion of myeloid cell Lgr4 suppresses atherosclerotic lesion formation and identify Lgr4 as a novel therapeutic target for atherosclerosis.

The effect of resolvin D1n-3 DPA on primary oral epithelial cell migration in vitro.

Specialized pro-resolving lipid mediators (SPMs) are known for their anti-inflammatory and pro-resolving actions. The aim of the present study was to find new functions of the SPM resolvin D1n-3 DPA (RvD1n-3 DPA) on oral epithelial cells. As a starting point, we used a dataset obtained by RNA high-throughput sequencing of oral epithelial cells exposed to TNF-α and RvD1n-3 DPA versus TNF-α alone. GOrilla enrichment analysis showed that the actin cytoskeleton was significantly overrepresented after adjustment for multiple hypothesis testing. As actin, amongst others, is closely related to cell migration, we then explored whether RvD1n-3 DPA can modulate oral epithelial cell migration. To this end, we used an in vitro cell migration model, including TNF-α treatment, to mimic an inflammatory cell state. The analysis revealed that RvD1n-3 DPA increased oral epithelial cell migration in the presence but not in the absence of TNF-α. Addition of RvD1n-3 DPA also induced F actin accumulation around the cell nucleus, indicating that RvD1n-3 DPA potentially can mediate processes of intracellular transport. This indicates that this lipid mediator may be a promising therapeutic candidate in oral mucosal wound healing.

Amyloid β interferes with wound healing of brain microvascular endothelial cells by disorganizing the actin cytoskeleton

Cerebral amyloid angiopathy (CAA) is a disease in which amyloid β (Aβ) is deposited in the cerebral blood vessels, reducing compliance, tearing and weakening of vessel walls, leading to cerebral hemorrhage. The mechanisms by which Aβ leads to focal wall fragmentation and intimal damage are not well understood. We analyzed the motility of human brain microvascular endothelial cells (hBMECs) in real-time using a wound-healing assay. We observed the suppression of cell migration by visualizing Aβ aggregation using quantum dot (QD) nanoprobes. In addition, using QD nanoprobes and a SiR-actin probe, we simultaneously observed Aβ aggregation and F-actin organization in real-time for the first time. Aβ began to aggregate at the edge of endothelial cells, reducing cell motility. In addition, Aβ aggregation disorganized the actin cytoskeleton and induced abnormal actin aggregation. Aβ aggregated actively in the anterior group, where cell motility was active. Our findings may be a first step toward explaining the mechanism by which Aβ causes vascular wall fragility, bleeding, and rebleeding in CAA.

Bioenergetic and excitotoxic determinants of cofilactin rod formation.

Cofilactin rods (CARs), which are 1:1 aggregates of cofilin-1 and actin, lead to neurite loss in ischemic stroke and other disorders. The biochemical pathways driving CAR formation are well-established, but how these pathways are engaged under ischemic conditions is less clear. Brain ischemia produces both ATP depletion and glutamate excitotoxicity, both of which have been shown to drive CAR formation in other settings. Here, we show that CARs are formed in cultured neurons exposed to ischemia-like conditions: oxygen-glucose deprivation (OGD), glutamate, or oxidative stress. Of these conditions, only OGD produced significant ATP depletion, showing that ATP depletion is not required for CAR formation. Moreover, the OGD-induced CAR formation was blocked by the glutamate receptor antagonists MK-801 and kynurenic acid; the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors GSK2795039 and apocynin; as well as an ROS scavenger. The findings identify a biochemical pathway leading from OGD to CAR formation in which the glutamate release induced by energy failure leads to activation of neuronal glutamate receptors, which in turn activates NADPH oxidase to generate oxidative stress and CARs.

Peripheral thickening of the sarcomeres and pointed end elongation of the thin filaments are both promoted by SALS and its formin interaction partners.

During striated muscle development the first periodically repeated units appear in the premyofibrils, consisting of immature sarcomeres that must undergo a substantial growth both in length and width, to reach their final size. Here we report that, beyond its well established role in sarcomere elongation, the Sarcomere length short (SALS) protein is involved in Z-disc formation and peripheral growth of the sarcomeres. Our protein localization data and loss-of-function studies in the Drosophila indirect flight muscle strongly suggest that radial growth of the sarcomeres is initiated at the Z-disc. As to thin filament elongation, we used a powerful nanoscopy approach to reveal that SALS is subject to a major conformational change during sarcomere development, which might be critical to stop pointed end elongation in the adult muscles. In addition, we demonstrate that the roles of SALS in sarcomere elongation and radial growth are both dependent on formin type of actin assembly factors. Unexpectedly, when SALS is present in excess amounts, it promotes the formation of actin aggregates highly resembling the ones described in nemaline myopathy patients. Collectively, these findings helped to shed light on the complex mechanisms of SALS during the coordinated elongation and thickening of the sarcomeres, and resulted in the discovery of a potential nemaline myopathy model, suitable for the identification of genetic and small molecule inhibitors.

Friction forces determine cytoplasmic reorganization and shape changes of ascidian oocytes upon fertilization

Contraction and flow of the actin cell cortex have emerged as a common principle by which cells reorganize their cytoplasm and take shape. However, how these cortical flows interact with adjacent cytoplasmic components, changing their form and localization, and how this affects cytoplasmic organization and cell shape remains unclear. Here we show that in ascidian oocytes, the cooperative activities of cortical actomyosin flows and deformation of the adjacent mitochondria-rich myoplasm drive oocyte cytoplasmic reorganization and shape changes following fertilization. We show that vegetal-directed cortical actomyosin flows, established upon oocyte fertilization, lead to both the accumulation of cortical actin at the vegetal pole of the zygote and compression and local buckling of the adjacent elastic solid-like myoplasm layer due to friction forces generated at their interface. Once cortical flows have ceased, the multiple myoplasm buckles resolve into one larger buckle, which again drives the formation of the contraction pole—a protuberance of the zygote’s vegetal pole where maternal mRNAs accumulate. Thus, our findings reveal a mechanism where cortical actomyosin network flows determine cytoplasmic reorganization and cell shape by deforming adjacent cytoplasmic components through friction forces.

WAVE2 Is a Vital Regulator in Myogenic Differentiation of Progenitor Cells through the Mechanosensitive MRTFA-SRF Axis.

Skeletal myogenesis is an intricate process involving the differentiation of progenitor cells into myofibers, which is regulated by actin cytoskeletal dynamics and myogenic transcription factors. Although recent studies have demonstrated the pivotal roles of actin-binding proteins (ABPs) as mechanosensors and signal transducers, the biological significance of WAVE2 (Wiskott-Aldrich syndrome protein family member 2), an ABP essential for actin polymerization, in myogenic differentiation of progenitor cells has not been investigated. Our study provides important insights into the regulatory roles played by WAVE2 in the myocardin-related transcription factor A (MRTFA)-serum response factor (SRF) signaling axis and differentiation of myoblasts. We demonstrate that WAVE2 expression is induced during myogenic differentiation and plays a pivotal role in actin cytoskeletal remodeling in C2C12 myoblasts. Knockdown of WAVE2 in C2C12 cells reduced filamentous actin levels, increased globular actin accumulation, and impaired the nuclear translocation of MRTFA. Furthermore, WAVE2 depletion in myoblasts inhibited the expression and transcriptional activity of SRF and suppressed cell proliferation in myoblasts. Consequently, WAVE2 knockdown suppressed myogenic regulatory factors (i.e., MyoD, MyoG, and SMYD1) expressions, thereby hindering the differentiation of myoblasts. Thus, this study suggests that WAVE2 is essential for myogenic differentiation of progenitor cells by modulating the mechanosensitive MRTFA-SRF axis.

TrCla4 promotes actin polymerization at the hyphal tip and mycelial growth in Trichophyton rubrum.

Dermatophytes invade and colonize host superficial tissues via hyphal growth. Although cytoskeletal reorganization and its regulation are essential for hyphal growth, the molecular mechanisms in dermatophytes and their applicability as antifungal drug targets remain poorly understood. The p21-activated kinase (PAK) is a downstream effector of the small GTPases Rac and CDC42, also known as p21, and is involved in various molecular and cellular functions, including actin polymerization and cell morphogenesis. In this study, we investigated the contribution of the PAK protein TrCla4 to morphogenesis and mycelial growth in Trichophyton rubrum, the most frequently isolated fungus in dermatophytosis (athlete's foot). The actin polymerization inhibitor, cytochalasin A inhibited actin accumulation at the hyphal tip and mycelial growth of T. rubrum, suggesting the involvement of the actin cytoskeleton in mycelial growth. In the Trcla4 knockout strain (ΔTrcla4), we observed defects in mycelial growth, hyphal branching, and the accumulation of polymerized actin at the hyphal tip. Chemical inhibitors of TrRac-dependent TrCla4 kinase activity, FRAX486 and IPA-3, also inhibited spore germination and mycelial growth. Interestingly, ΔTrcla4 showed no additional inhibition of mycelial growth when treated with these inhibitors, indicating that their inhibitory effects are primarily mediated through TrCla4. In an invertebrate dermatophyte infection model, animals infected with ΔTrcla4 had higher survival rates than those infected with the wild-type, and IPA-3 and FRAX486 treatments both significantly improved animal survival rates. These results suggest that the dermatophyte PAK promotes mycelial growth by facilitating actin polymerization at the hyphal tip, making it a potential therapeutic target for dermatophytosis. IMPORTANCE Superficial fungal infections, such as athlete's foot, affect more than 10% of the world's population and have a significant impact on quality of life. Despite the fact that treatment-resistant fungi are a concern, there are just a few antifungal drug targets accessible, as opposed to the wide range of therapeutic targets found in bacterial infections. As a result, additional alternatives are sought. In this study, we generated a PAK TrCla4 deletion strain (∆Trcla4) of Trichophyton rubrum. The ∆Trcla4 strain exhibited deficiencies in mycelial growth, hyphal morphology, and polarized actin localization at the hyphal tip. IPA-3 and FRAX486, small chemical inhibitors of mammalian PAK, were discovered to limit fungal mycelial proliferation. According to our findings, fungal PAKs are interesting therapeutic targets for the development of new antifungal medicines.

Dynamics of actomyosin filaments in the contractile ring revealed by ultrastructural analysis.

Cytokinesis, the final process of cell division, involves the accumulation of actin and myosin II filaments at the cell's equator, forming a contractile ring that facilitates the division into two daughter cells. While light microscopy has provided valuable insights into the molecular mechanism of this process, it has limitations in examining individual filaments in vivo. In this study, we utilized transmission electron microscopy to observe actin and myosin II filaments in the contractile rings of dividing Dictyostelium cells. To synchronize cytokinesis, we developed a novel method that allowed us to visualize dividing cells undergoing cytokinesis with a frequency as high as 18%. This improvement enabled us to examine the lengths and alignments of individual filaments within the contractile rings. As the furrow constricted, the length of actin filaments gradually decreased. Moreover, both actin and myosin II filaments reoriented perpendicularly to the long axis during furrow constriction. Through experiments involving myosin II null cells, we discovered that myosin II plays a role in regulating both the lengths and alignments of actin filaments. Additionally, dynamin-like protein A was found to contribute to regulating the length of actin filaments, while cortexillins were involved in regulating their alignment.

RACK1 promotes Shigella flexneri actin-mediated invasion, motility, and cell-to-cell spreading

Shigella flexneri is an intracellular bacterium that hijacks the host actin cytoskeleton to invade and disseminate within the colonic epithelium. Shigella's virulence factors induce actin polymerization, leading to bacterial uptake, actin tail formation, actin-mediated motility, and cell-to-cell spreading. Many host factors involved in the Shigella-prompted actin rearrangements remain elusive. Here, we studied the role of a host protein receptor for activated C kinase 1 (RACK1) in actin cytoskeleton dynamics and Shigella infection. We used time-lapse imaging to demonstrate that RACK1 facilitates Shigella-induced actin cytoskeleton remodeling at multiple levels during infection of epithelial cells. Silencing RACK1 expression impaired Shigella-induced rapid polymerizing structures, reducing host cell invasion, bacterial motility, and cell-to-cell spreading. In uninfected cells, RACK1 silencing reduced jasplakinolide-mediated filamentous actin aggregate formation and negatively affected actin turnover in fast polymerizing structures, such as membrane ruffles. Our findings provide a role of RACK1 in actin cytoskeleton dynamics and Shigella infection.

Occidiofungin inhibition of Candida biofilm formation on silicone elastomer surface.

Biofilms are the leading cause of clinically acquired fungal infections and contribute to significantly high morbidity and mortality in immunocompromised and hospitalized patients. Candida biofilms exhibit increased resistance to currently available antifungal agents that contribute to persistent reoccurring infections and are driving efforts to identify novel fungicidal compounds. The natural product, occidiofungin, is an antifungal compound with demonstrated activity against hyphal morphogenesis in polymorphic Candida species. In this study, we use an in vitro static biofilm model to demonstrate the efficacy of occidiofungin against C. albicans and C. tropicalis at all stages of biofilm development, including inhibiting cell dispersal. Consistent with prior findings, we demonstrate that actin organization is altered following occidiofungin exposure to include loss of F-actin cables and accumulation of actin aggregates. Altogether, our results provide strong evidence of the antibiofilm activity of occidiofungin toward Candida biofilms and support its potential as a therapeutic for Candida infections. IMPORTANCE Candida are opportunistic fungal pathogens with medical relevance given their association with superficial to life-threatening infections. An important component of Candida virulence is the ability to form a biofilm. These structures are highly resistant to antifungal therapies and are often the cause of treatment failure. In this work, we evaluated the efficacy of the antifungal compound, occidiofungin, against Candida biofilms developed on a silicone surface. We demonstrate that occidiofungin eliminated cells at all stages of biofilm formation in a dose-dependent manner. Consistent with our understanding of occidiofungin bioactivity, we noted alterations to actin organization and cell morphology following antifungal exposure. Given the challenges associated with the treatment of biofilm-associated infections, occidiofungin exhibits potential as a therapeutic antifungal agent in the future.