Inhibition Of Actin Polymerization Research Articles

Epimedin C promotes mitochondrial transfer and delays thymus atrophy in 4-VCD induced mimetic-menopausal mice.

Prevention of thymus atrophy during menopause is of great significance for improving the immune function and overall health of menopausal women. Epimedin C is one of the major bioactive compounds in Herba Epimedii, a traditional herbal medicine for the treatment of menopausal syndrome in China, but the action and mechanism of Epimedin C in the treatment of menopausal thymus atrophy remains unclear. To study the effect of Epimedin C on thymus atrophy in 4-vinylcyclohexene diepoxide (4-VCD) induced mimetic-menopausal mice and explore its mechanism from new perspectives of tunneling nanotubes (TNTs) formation and mitochondrial transfer (MitoT). The effects of Epimedin C in the 4-VCD induced menopause-like phenotype in mice were observed, and the thymic output function was evaluated by the quantitative detection of T cell receptor excision circles (TRECs). The structure of the thymus was observed by H&E. The arrangement and quantity of different cell subpopulations of thymic epithelial cells (TECs) and thymocytes were detected by multiple fluorescent staining and flow cytometry. Mitochondrial morphology was observed with transmission electron microscopy. LC-MS/MS was used to analyze and identify the differential protein expression in thymus before and after Epimedin C treatment. Actin polymerization inhibitor was used to verify the possible mechanism of Epimedin C. The treadmilling-balance of actin, TNTs formation, and MitoT processes were observed by specific fluorescent probe labeling. The interaction between G-actin, Thymosin β4 (Tβ4), and Epimedin C were studied by protein cross-linking assay. Epimedin C significantly increased the thymus weight and the area of the thymus medulla, increased the grip strength and bone strength in 4-VCD induced mimetic-menopausal mice, and enhanced ovarian secretion function. It could affect the thymus output, increase CK5 and CK8 expression, maintain the reticular structure of TECs, inhibit the differentiation of thymocytes into double positive cells (CD4+CD8+) and CD4SP (CD3+TCR β+CD4+CD8-) cells. Epimedin C promoted the conversion of G-actin to F-actin and accelerated MitoT via stimulating the TNTs formation, which related with the downregulation of Tβ4 and obstruction to the formation of Tβ4-G-actin complex. Epimedin C can promote TEC activity in 4-VCD induced mimetic-menopausal mice by decreasing the expression of Tβ4, inhibiting the binding of Tβ4 to G-actin, promoting the F-actin polymerization and the TNTs-depended MitoT.

Effects of in vitro cytochalasin D and hypoxia on mitochondrial energetics and biogenesis, cell signal status and actin/tubulin/Hsp/MMP entity in air-breathing fish heart.

The cardiac actin cytoskeleton has a dynamic pattern of polymerisation. It is uncertain how far actin destabilisation impacts mitochondrial energetics and biogenesis, cell signal status, and structural entities in cardiomyocytes, particularly in hypoxic conditions. We thus tested the in vitro action of cytochalasin D (Cyt D), an inhibitor of actin polymerisation, in hypoxic ventricular explants to elucidate the role of the actin in mitochondrial energetics and biogenesis, cell signals and actin/tubulin/hsps/MMPs dynamics in hypoxic air-breathing fish hearts. The COX activity increased upon Cyt D exposure, whereas hypoxia lowered COX and SDH activities but increased LDH activity. The ROS increased, and NO decreased by Cyt D. COX and LDH activities, and NO content reversed after Cyt D exposure in hypoxic hearts. Cyt D exposure upregulated actin isoform expression (Actc1 and Actb1) but downregulated tubulin isoform (Tedc1). Hypoxia upregulated actin (Acta1a, Actb1, Actb2, Actc1a) tubulin (Tuba, Tubb5, Tedc1, Tubd1) and hsp (Hspa5, Hspa9, Hspa12a, Hspa14, Hspd1, Hsp90) isoform transcript expression and Cyt D in hypoxic hearts reversed these isoform's expression. Hypoxia upregulated Mmp2 and 9 transcript expressions but downregulated Mfn1, Fis1, Nfkb1, Prkacaa, and Aktip expressions, and Cyt D exposure reversed almost all these markers in hypoxic hearts. The data provide novel evidence for the mechanistic role of actin in integrating mitochondrial energetics and biogenesis, cell signal status and actin/tubulin/Hsp/MMP entity, indicating its critical cardioprotective role in defending against hypoxia. Besides proposing an air-breathing fish heart as a model, the study further brings the therapeutic potential of Cyt D towards hypoxia intervention.

Lead-induced actin polymerization aggravates neutrophil extracellular trap formation and contributes to vascular inflammation.

Lead (Pb) exposure is widely acknowledged as a risk factor for cardiovascular diseases. Previous studies have established neutrophil involvement in Pb-induced cardiovascular injuries; however, the underlying mechanisms remain unclear. To address this knowledge gap, the binding targets of Pb in neutrophils and their roles in regulating neutrophil extracellular trap (NET) formation were investigated. Furthermore, their impact on Pb-induced vascular inflammation and other cardiovascular injuries was studied in mice. Our findings indicate, for the first time, that Pb binds to β-actin in neutrophils, influencing NET formation. Inhibition of actin polymerization reduces the release of extracellular myeloperoxidase, neutrophil elastase, and citrullinated histone H3, indicating an impediment in NET formation. Furthermore, Pb exposure exacerbates blood pressure and vascular inflammation in vascular tissues, leading to abnormal aortic blood flow in mice. These injuries are potentially associated with NET formation, which is supported by the positive correlation between NETs and vascular inflammation. Importantly, the inhibition of actin polymerization mitigates Pb-induced vascular inflammation and regulates systolic blood pressure by reducing NET formation. Collectively, our findings provide novel insights into the mechanism underlying Pb-induced cardiovascular injury, contributing to the management of the escalating risk associated with Pb-induced cardiovascular damage.

Efficacy of natural NF-κB inhibitors in the treatment of fibrosarcoma: an in vitro model study

IntroductionNF-κB plays a pivotal role in the progression of cancers, including myosarcomas such as fibrosarcoma. Plants possess considerable potential for the provision of chemotherapeutic effects against cancer. The present study assessed, among others, the cytotoxicity, migration capacity and DNA damage induced by several natural compounds (berberine, curcumin, biochanin A, cucurbitacin E (CurE) and phenethyl caffeic acid (CAPE)) in cancer cells (WEHI-164) and normal muscle cells (L6).MethodsIC50 parameter was determined for all substances after 24-hour incubation. Molecular docking studies were performed to assess compound binding to cytoskeletal proteins. Neutral comet assay and immunocytochemical analysis were used to assess the intensity of apoptosis, and transmission electron microscopy was employed to validate these results at the ultrastructural level.Results and DiscussionThe results showed that the tested compounds had a significantly increased cytotoxic effect on cancer cells compared to normal cells. Furthermore, molecular docking studies indicated that CAPE, biochanin A, and CurE could inhibit actin polymerization, suggesting their potential role in disrupting the cytoskeleton of cancer cells. Increased expression of caspase-3 and PARP-1 in WEHI-164 cells after treatment indicated the induction of apoptosis. Transmission electron microscopy confirmed the presence of cellular stress and vacuolation in cells treated with these compounds, with more pronounced effects observed in cancer cells compared to normal cells. The results indicate that natural NF-κB inhibitors may be capable of selectively targeting cancer cells, reducing their viability and inducing apoptosis while sparing normal cells. This selectivity is of great importance for the development of safer anticancer therapies. The results of this research support the hypothesis that these natural compounds may be effective anticancer agents, particularly in the treatment of fibrosarcoma. Further, in vivo studies and clinical trials are required to gain a full understanding of their mechanisms of action and potential synergies with existing chemotherapeutic agents.

Single-molecule diffusivity quantification in Xenopus egg extracts elucidates physicochemical properties of the cytoplasm

The living cell creates a unique internal molecular environment that is challenging to characterize. By combining single-molecule displacement/diffusivity mapping (SMdM) with physiologically active extracts prepared from Xenopus laevis eggs, we sought to elucidate molecular properties of the cytoplasm. Quantification of the diffusion coefficients of 15 diverse proteins in extract showed that, compared to in water, negatively charged proteins diffused ~50% slower, while diffusion of positively charged proteins was reduced by ~80 to 90%. Adding increasing concentrations of salt progressively alleviated the suppressed diffusion observed for positively charged proteins, signifying electrostatic interactions within a predominately negatively charged macromolecular environment. To investigate the contribution of RNA, an abundant, negatively charged component of cytoplasm, extracts were treated with ribonuclease, which resulted in low diffusivity domains indicative of aggregation, likely due to the liberation of positively charged RNA-binding proteins such as ribosomal proteins, since this effect could be mimicked by adding positively charged polypeptides. Interestingly, in extracts prepared under typical conditions that inhibit actin polymerization, negatively charged proteins of different sizes showed similar diffusivity suppression consistent with our separately measured 2.22-fold higher viscosity of extract over water. Restoring or enhancing actin polymerization progressively suppressed the diffusion of larger proteins, recapitulating behaviors observed in cells. Together, these results indicate that molecular interactions in the crowded cell are defined by an overwhelmingly negatively charged macromolecular environment containing cytoskeletal networks.

Rosiglitazone-induced white adipocyte browning is regulated by actin and Myh9

AimsThis study investigates the role of actin polymerization and Myh9 in mediating lipid droplet (LD) fission during rosiglitazone-induced browning of white adipocytes. The aim is to understand how LD splitting might contribute to the beige conversion of white adipose tissue, providing insights into adipocyte plasticity and metabolic regulation. Materials and methodsC3H10 T1/2-differentiated adipocytes were used as a classical model to study white adipocyte browning. Rosiglitazone was applied to induce browning, and the interactions between LDs and actin, as well as the distribution of Myh9, were assessed using immunofluorescence and Western blotting. In vivo, we employed a microfilament inhibitor to block actin polymerization in cold-stimulated mice and evaluated changes in LD morphology and browning. Furthermore, dynamic live-cell imaging using confocal microscopy was conducted to observe the real-time behavior of LDs during the browning process and to determine whether they undergo fission. Main findingsOur results demonstrate that rosiglitazone significantly induces LD size reduction, a process correlated with the increased contact of LDs with microfilaments. Inhibition of actin polymerization prevented both the reduction in LD size and the browning of white adipocytes, indicating that actin plays a critical role. Myh9 was enriched at the LD fission sites, forming a structure resembling a contractile ring. Overexpression of Myh9 promoted the shrinkage of LD, suggesting that it may be involved in LD fission. SignificanceThis study identifies actin and Myh9 as key regulators of LD fission in rosiglitazone-induced browning of white adipocytes, offering new insights into the cellular mechanisms of adipocyte plasticity. The findings propose a novel pathway by which LD dynamics contribute to the beige conversion of white fat, with potential implications for metabolic disease therapies targeting adipocyte function and energy expenditure.

Vinculin-Arp2/3 interaction inhibits branched actin assembly to control migration and proliferation.

Vinculin is a mechanotransducer that reinforces links between cell adhesions and linear arrays of actin filaments upon myosin-mediated contractility. Both adhesions to the substratum and neighboring cells, however, are initiated within membrane protrusions that originate from Arp2/3-nucleated branched actin networks. Vinculin has been reported to interact with the Arp2/3 complex, but the role of this interaction remains poorly understood. Here, we compared the phenotypes of vinculin knock-out (KO) cells with those of knock-in (KI-P878A) cells, where the point mutation P878A that impairs the Arp2/3 interaction is introduced in the two vinculin alleles of MCF10A mammary epithelial cells. The interaction of vinculin with Arp2/3 inhibits actin polymerization at membrane protrusions and decreases migration persistence of single cells. In cell monolayers, vinculin recruits Arp2/3 and the vinculin-Arp2/3 interaction participates in cell-cell junction plasticity. Through this interaction, vinculin controls the decision to enter a new cell cycle as a function of cell density.

CNSC-40. GTP SIGNALING PATHWAY PROMOTES NHEJ REPAIR BY INDUCING NUCLEAR ACTIN POLYMERIZATION IN GLIOBLASTOMA

Abstract OBJECTIVES We recently identified that GTP promotes non-homologous end joining (NHEJ) by triggering a GTP-Rac1-PP5-Abi-1/S323 cascade and we sought to define the molecular mechanisms. METHODS Crispr/Cas9, CDNA overexpression and immunoblot were used to modulate and confirm protein levels. Cellular fractionation and immunofluorescence (IF) were used to determine the localization of proteins. Laser micro-irradiation was used to induce DNA damage and monitor the recruitment of DNA repair proteins. RESULTS We found that GTP-activated protein Rac1 and Abi-1, the two key components of the newly identified pathway, shuttled into the nucleus post-RT in glioblastoma (GBM) cells. These effects were further enhanced by the GTP precursor guanosine but blocked by GTP depletion. Furthermore, Abi-1 and Rac1 accumulated in the nucleus of the cells expressing constitutively active Rac1 but not dominant negative Rac1 after RT, which suggests that Rac1/Abi-1 nuclear translocation is Rac1 activity dependent. We previously discovered that GTP-Rac1 promotes DNA repair by facilitating the dephosphorylation of Abi-1 (S323). Here, we discovered that dephosphomimetic Abi-1 (S323A) but not phosphomimetic Abi-1 (S323D) shuttled into nucleus after RT. Because Rac1 canonically binds to Abi-1 and promotes actin polymerization in the cytosol, we next wanted to determine if RT-induced nuclear Rac1/Abi-1/S323A could enhance nuclear actin polymerization, which could enhance NHEJ repair. We found that Abi-1/S323A but not Abi-1/S323D increases nuclear filament actin formation post-RT and activating the GTP-Rac1-Abi-1/S323A axis (through guanosine supplementation, expression of constitutively active Rac1 or expression of dephosphomimetic Abi-1/S323A) enhanced the recruitment and stability of KU80 at sites of DNA damage, which could be blocked by actin polymerization inhibitor cytochalasin D. Furthermore, inhibiting actin polymerization overcame radiation resistance in GBM cells with an active GTP-Rac1-Abi-1/S323A axis. CONCLUSIONS GTP-Rac1-PP5-Abi-1/S323 cascade promotes NHEJ repair by inducing the nuclear assembly of filament actin and disrupting this regulation could improve treatment responses in GBMs.

STEM-03. TARGETING F-ACTIN DYNAMICS OF GLIOBLASTOMAS TO OVERCOME THERAPY RESISTANCE: IMPACTS ON DNA REPAIR AND GLIOMA STEM CELL PHENOTYPE

Abstract Glioblastoma (GBM) is a highly aggressive brain tumor with high recurrence and resistance to therapies. The F-actin cytoskeleton in GBM is related to cell invasion and the glioma stem cell (GSC) phenotype, affecting self-renewal and migration. Our research identifies a novel role for the F-actin pathway in DNA repair, impacting genomic stability and contributing to therapy resistance. Understanding F-actin’s role is crucial for developing targeted strategies to improve GBM treatment outcomes. This study examines the effects of pharmacological F-actin depolymerization on reversing resistance to ionizing radiation (IR) and temozolomide (TMZ) in GBM spheroids. We established resistant sublines of GBM U87-MG cells through IR and TMZ cycles, observing significant resistance increases via MTT assays. Also, resistant cells had enhanced spheroid formation capacity, with higher size and proliferation. Alterations in DNA repair and F-actin pathway were observed, with intensified and disorganized F-actin polymerization and efficient repair in resistant cells. Elevated expression of stemness markers (Nestin, CD133, and CD44) indicated a GSC-like phenotype in resistant cells, corroborated by increased tumorigenicity and de novo spheroid formation. These markers were further enhanced by DNA damage. Treating cells with actin polymerization inhibitors reduced resistance to TMZ and IR, partly due to impaired DNA repair capacity. Additionally, F-actin disassembly affected stemness marker expression, reinforcing the link between cytoskeletal dynamics and the GSC phenotype. Our findings suggest that F-actin dynamic is crucial for resistance in GBM. Targeting F-actin could resensitize recurrent GBM tumours by diminishing DNA repair capabilities and affecting the GSC-like phenotype, offering new therapeutic avenues.

Formation of a giant unilocular vacuole via macropinocytosis-like process confers anoikis resistance

Cell survival in metazoans depends on cell attachment to the extracellular matrix (ECM) or to neighboring cells. Loss of such attachment triggers a type of programmed cell death known as anoikis, the acquisition of resistance to which is a key step in cancer development. The mechanisms underlying anoikis resistance remain unclear, however. The intracellular F-actin cytoskeleton plays a key role in sensing the loss of cell–ECM attachment, but how its disruption affects cell fate during such stress is not well understood. Here, we reveal a cell survival strategy characterized by the formation of a giant unilocular vacuole (GUVac) in the cytoplasm of the cells whose actin cytoskeleton is disrupted during loss of matrix attachment. Time-lapse imaging and electron microscopy showed that large vacuoles with a diameter of >500 nm accumulated early after inhibition of actin polymerization in cells in suspension culture, and that these vacuoles subsequently coalesced to form a GUVac. GUVac formation was found to result from a variation of a macropinocytosis-like process, characterized by the presence of inwardly curved membrane invaginations. This phenomenon relies on both F-actin depolymerization and the recruitment of septin proteins for micron-sized plasma membrane invagination. The vacuole fusion step during GUVac formation requires PI(3)P produced by VPS34 and PI3K-C2α on the surface of vacuoles. Furthermore, its induction after loss of matrix attachment conferred anoikis resistance. Our results thus show that the formation of a previously unrecognized organelle promotes cell survival in the face of altered actin and matrix environments.

Formation of a giant unilocular vacuole via macropinocytosis-like process confers anoikis resistance.

Cell survival in metazoans depends on cell attachment to the extracellular matrix (ECM) or to neighboring cells. Loss of such attachment triggers a type of programmed cell death known as anoikis, the acquisition of resistance to which is a key step in cancer development. The mechanisms underlying anoikis resistance remain unclear, however. The intracellular F-actin cytoskeleton plays a key role in sensing the loss of cell-ECM attachment, but how its disruption affects cell fate during such stress is not well understood. Here, we reveal a cell survival strategy characterized by the formation of a giant unilocular vacuole (GUVac) in the cytoplasm of the cells whose actin cytoskeleton is disrupted during loss of matrix attachment. Time-lapse imaging and electron microscopy showed that large vacuoles with a diameter of >500 nm accumulated early after inhibition of actin polymerization in cells in suspension culture, and that these vacuoles subsequently coalesced to form a GUVac. GUVac formation was found to result from a variation of a macropinocytosis-like process, characterized by the presence of inwardly curved membrane invaginations. This phenomenon relies on both F-actin depolymerization and the recruitment of septin proteins for micron-sized plasma membrane invagination. The vacuole fusion step during GUVac formation requires PI(3)P produced by VPS34 and PI3K-C2α on the surface of vacuoles. Furthermore, its induction after loss of matrix attachment conferred anoikis resistance. Our results thus show that the formation of a previously unrecognized organelle promotes cell survival in the face of altered actin and matrix environments.

The completing of the second meiotic division by MII mouse oocytes correlates with the positioning of F-actin and mitochondria in the ooplasm

Actin filaments play an essential role in the process of oocyte maturation and completion of meiosis. However, whether the localization of F-actin in the ooplasm is associated with normal completion of the second meiotic division remains unclear. Mitochondrial distribution is another important parameter correlating directly with MII oocyte capacity to finalize meiosis. Our objective was to examine the role of actin microfilaments in the distribution of mitochondria and, respectively, Metaphase II (MII) oocytes meiotic potential. We show monoclonal antibody-mediated inhibition of actin polymerization in young mouse oocytes, reduction of the amount of F-actin, and induction of mitochondrial clustering induced by antibody treatment. Similar phenotype, even in untreated eggs, was observed in in vitro oocyte aging experiments. Observed changes correlate with reduced ability of MII oocytes to extrude the second polar body and form the pronuclei. Changes in colocalization of F-actin and mitochondria likely resulted from disturbed cytoskeleton architecture. The perturbations in the amount of F-actin and its distribution largely coincide with mitochondrial redistribution. Based on these data, we suggest actin microfilament’s participation in redistribution of mitochondria during MII oocyte aging in vitro. Accordingly, patterning of F-actin is indicative of high rate of the completed second meiotic division. These results help evaluating oocyte’s quality and choosing optimal time between placement into culture and in vitro fertilization.

Dennd2c Negatively Controls Multinucleation and Differentiation in Osteoclasts by Regulating Actin Polymerization and Protrusion Formation.

Osteoclasts are bone-resorbing multinucleated giant cells formed by the fusion of monocyte/macrophage lineages. Various small GTPases are involved in the multinucleation and differentiation of osteoclasts. However, the roles of small GTPases regulatory molecules in osteoclast differentiation remain unclear. In the present study, we examined the role of Dennd2c, a putative guanine nucleotide exchange factor for Rab GTPases, in osteoclast differentiation. Knockdown of Dennd2c promoted osteoclast differentiation, resorption, and expression of osteoclast markers. Morphologically, Dennd2c knockdown induced the formation of larger osteoclasts with several protrusions. In contrast, overexpression of Dennd2c inhibited the multinucleation and differentiation of osteoclasts, bone resorption, and the expression of osteoclast markers. Dennd2c-overexpressing macrophages exhibited spindle-shaped mononuclear cells and long thin protrusions. Treatment of Dennd2c-overexpressing cells with the Cdc42 inhibitor ML-141 or the Rac1 inhibitor 6-thio-GTP prevented protrusion formation. Moreover, treatment of Dennd2c-overexpressing cells with the actin polymerization inhibitor latrunculin B restored multinucleated and TRAP-positive osteoclast formation. These results indicate that Dennd2c negatively regulates osteoclast differentiation and multinucleation by modulating protrusion formation in macrophages.

Comprehensive Cell Biological Investigation of Cytochalasin B Derivatives with Distinct Activities on the Actin Network.

In search of a more comprehensive structure-activity relationship (SAR) regarding the inhibitory effect of cytochalasin B (2) on actin polymerization, a virtual docking of 2 onto monomeric actin was conducted. This led to the identification of potentially important functional groups of 2 (i.e., the NH group of the isoindolone core (N-2) and the hydroxy groups at C-7 and C-20) involved in interactions with the residual amino acids of the binding pocket of actin. Chemical modifications of 2 at positions C-7, N-2, and C-20 led to derivatives 3-6, which were analyzed for their bioactivities. Compounds 3-5 exhibited reduced or no cytotoxicity in murine L929 fibroblasts compared to that of 2. Moreover, short- and long-term treatments of human osteosarcoma cells (U-2OS) with 3-6 affected the actin network to a variable extent, partially accompanied by the induction of multinucleation. Derivatives displaying acetylation at C-20 and N-2 were subjected to slow intracellular conversion to highly cytotoxic 2. Together, this study highlights the importance of the hydroxy group at C-7 and the NH function at N-2 for the potency of 2 on the inhibition of actin polymerization.

Long-term zinc treatment alters the mechanical properties and metabolism of prostate cancer cells

The failure of intracellular zinc accumulation is a key process in prostate carcinogenesis. Although prostate cancer cells can accumulate zinc after long-term exposure, chronic zinc oversupply may accelerate prostate carcinogenesis or chemoresistance. Because cancer progression is associated with energetically demanding cytoskeletal rearrangements, we investigated the effect of long-term zinc presence on biophysical parameters, ATP production, and EMT characteristics of two prostate cancer cell lines (PC-3, 22Rv1). Prolonged exposure to zinc increased ATP production, spare respiratory capacity, and induced a response in PC-3 cells, characterized by remodeling of vimentin and a shift of cell dry mass density and caveolin-1 to the perinuclear region. This zinc-induced remodeling correlated with a greater tendency to maintain actin architecture despite inhibition of actin polymerization by cytochalasin. Zinc partially restored epithelial characteristics in PC-3 cells by decreasing vimentin expression and increasing E-cadherin. Nevertheless, the expression of E-cadherin remained lower than that observed in predominantly oxidative, low-invasive 22Rv1 cells. Following long-term zinc exposure, we observed an increase in cell stiffness associated with an increased refractive index in the perinuclear region and an increased mitochondrial content. The findings of the computational simulations indicate that the mechanical response cannot be attributed exclusively to alterations in cytoskeletal composition. This observation suggests the potential involvement of an additional, as yet unidentified, mechanical contributor. These findings indicate that long-term zinc exposure alters a group of cellular parameters towards an invasive phenotype, including an increase in mitochondrial number, ATP production, and cytochalasin resistance. Ultimately, these alterations are manifested in the biomechanical properties of the cells.

Rab27a GTPase and its effector Myosin Va are host factors required for efficient Oropouche virus cell egress.

Oropouche fever, a debilitating illness common in South America, is caused by Oropouche virus (OROV), an arbovirus. OROV belongs to the Peribunyaviridae family, a large group of RNA viruses. Little is known about the biology of Peribunyaviridae in host cells, especially assembly and egress processes. Our research reveals that the small GTPase Rab27a mediates intracellular transport of OROV induced compartments and viral release from infected cells. We show that Rab27a interacts with OROV glycoproteins and colocalizes with OROV during late phases of the infection cycle. Moreover, Rab27a activity is required for OROV trafficking to the cell periphery and efficient release of infectious particles. Consistently, depleting Rab27a's downstream effector, Myosin Va, or inhibiting actin polymerization also hinders OROV compartments targeting to the cell periphery and infectious viral particle egress. These data indicate that OROV hijacks Rab27a activity for intracellular transport and cell externalization. Understanding these crucial mechanisms of OROV's replication cycle may offer potential targets for therapeutic interventions and aid in controlling the spread of Oropouche fever.

Excess microtubule and F-actin formation mediates shortening and loss of primary cilia in response to a hyperosmotic milieu.

The primary cilium is a small organelle protruding from the cell surface that receives signals from the extracellular milieu. Although dozens of studies have reported that several genetic factors can impair the structure of primary cilia, evidence for environmental stimuli affecting primary cilia structures is limited. Here, we investigated an extracellular stress that affected primary cilia morphology and its underlying mechanisms. Hyperosmotic shock induced reversible shortening and disassembly of the primary cilia of murine intramedullary collecting duct cells. The shortening of primary cilia caused by hyperosmotic shock followed delocalization of the pericentriolar material (PCM). Excessive microtubule and F-actin formation in the cytoplasm coincided with the hyperosmotic shock-induced changes to primary cilia and the PCM. Treatment with a microtubule-disrupting agent, nocodazole, partially prevented the hyperosmotic shock-induced disassembly of primary cilia and almost completely prevented delocalization of the PCM. An actin polymerization inhibitor, latrunculin A, also partially prevented the hyperosmotic shock-induced shortening and disassembly of primary cilia and almost completely prevented delocalization of the PCM. We demonstrate that hyperosmotic shock induces reversible morphological changes in primary cilia and the PCM in a manner dependent on excessive formation of microtubule and F-actin.

Fat Phagocytosis Promotes Anti-Inflammatory Responses of Macrophages in a Mouse Model of Osteonecrosis.

Osteonecrosis (ON) of the femoral head (ONFH) is a devastating bone disease affecting over 20 million people worldwide. ONFH is caused by a disruption of the blood supply, leading to necrotic cell death and increased inflammation. Macrophages are the key cells mediating the inflammatory responses in ON. It is unclear what the dynamic phenotypes of macrophages are and what mechanisms may affect macrophage polarization and, therefore, the healing process. In our preliminary study, we found that there is an invasion of macrophages into the repair tissue during ON healing. Interestingly, in both ONFH patients and a mouse ON model, fat was co-labeled within macrophages using immunofluorescence staining, indicating the phagocytosis of fat by macrophages. To study the effects of fat phagocytosis on the macrophage phenotype, we set up an in vitro macrophage and fat co-culture system. We found that fat phagocytosis significantly decreased M1 marker expression, such as IL1β and iNOS, in macrophages, whereas the expression of the M2 marker Arg1 was significantly increased with fat phagocytosis. To investigate whether the polarization change is indeed mediated by phagocytosis, we treated the cells with Latrunculin A (LA, which inhibits actin polymerization and phagocytosis). LA supplementation significantly reversed the polarization marker gene changes induced by fat phagocytosis. To provide an unbiased transcriptional gene analysis, we submitted the RNA for bulk RNA sequencing. Differential gene expression (DGE) analysis revealed that the top upregulated genes were related to anti-inflammatory responses, while proinflammatory genes were significantly downregulated. Additionally, using pathway enrichment and network analyses (Metascape), we confirmed that gene-enriched categories related to proinflammatory responses were significantly downregulated in macrophages with fat phagocytosis. Finally, we validated the similar macrophage phenotype changes in vivo. To summarize, we discovered that fat phagocytosis occurs in both ONFH patients and an ON mouse model, which inhibits proinflammatory responses with increased anabolic gene expression in macrophages. This fat-phagocytosis-induced macrophage phenotype is consistent with the in vivo changes shown in the ON mouse model. Our study reveals a novel phagocytosis-mediated macrophage polarization mechanism in ON, which fills in our knowledge gaps of macrophage functions and provides new concepts in macrophage immunomodulation as a promising treatment for ON.

Overstretching alveolar epithelial type II cells decreases surfactant secretion via actin polymerization and intracellular trafficking alteration

Pulmonary surfactant is essential for maintaining proper lung function. Alveolar epithelial type II (AE2) cells secrete surfactants via lamellar bodies (LBs). In tidal loading during each breath, the physiological cyclic stretching of AE2 cells promotes surfactant secretion. Excessive stretching inhibits surfactant secretion, which is considered to contribute to the development of lung damage. However, its precise mechanism remains unknown. This study tested whether actin polymerization and intracellular transport are required for pulmonary surfactant secretion and the association of actin polymerization and transport in identical human AE2-derived A549 cells using live-cell imaging, not in the bulk cells population. We found that overstretching approximately doubled actin polymerization into filaments (F-actin) and suppressed LB secretion by half in the fluorescent area ratio, compared with physiological stretching (F-actin: 1.495 vs 0.643 (P < 0.01); LB: 0.739 vs 0.332 (P < 0.01)). An inhibitor of actin polymerization increased LB secretion. Intracellular tracking using fluorescent particles revealed that cyclic stretching shifted the particle motion perpendicularly to the direction of stretching according to the orientation of the F-actin (proportion of perpendicular axis motion prior particle: 0h 40.12 % vs 2h 63.13 % (P < 0.01)), and particle motion was restricted over time in the cells subjected to overstretching, indicating that overstretching regulates intracellular transport dynamics (proportion of stop motion particle: 0h 1.01 % vs 2h 11.04 % (P < 0.01)). These findings suggest that overstretching changes secretion through the cytoskeleton: overstretching AE2 cells inhibits pulmonary surfactant secretion, at least through accelerating actin polymerization and decreasing intracellular trafficking, and the change in actin orientation would modulate intracellular trafficking.

PKC-α is involved in the signaling of phagocytosis induced by two snake venom secretory PLA2S in macrophages

Phagocytosis, an essential process for host defense, requires the coordination of a variety of signaling reactions. MT-II, an enzymatically inactive Lys49 phospholipase A2 (PLA2) homolog, and MT-III, a catalytically-active Asp49 PLA2, are known to activate phagocytosis in macrophages. In this study, the signaling pathways mediating phagocytosis, focusing on protein kinases, were investigated. Macrophages from male Swiss mice peritoneum were obtained 96 h after intraperitoneal thioglycolate injection. Phagocytosis was evaluated using non-opsonized zymosan particles in the presence or absence of specific inhibitors, as well as PKC and PKC-α localization by confocal microscopy. Moreover, protein kinase C (PKC) activity was assessed by γP32 ATP in macrophages stimulated by both PLA2s. Data showed that both sPLA2s increased phagocytosis. Cytochalasin D, staurosporine/H7, wortmannin, and herbimycin, inhibitors of actin polymerization, PKC, phosphoinositide 3-kinase (PI3K), and protein tyrosine kinase (PTK), respectively, significantly reduced phagocytosis induced by both PLA2s. PKC activity was increased in macrophages stimulated by both PLA2s. Actin polymerization and talin were evidenced by immunofluorescence and talin was recruited 5 min after both PLA2s stimulation. PKC and PKC-α localization within the cell were increased after 60 min of MT-II and MT-III stimulation. These data suggest that the effect of both PLA2s depends on actin cytoskeleton rearrangements and the activation of PKC, PI3K, and PTK signaling events required for phagocytosis.