Alterations In Cytoskeleton Research Articles

Adseverin, an actin-binding protein, modulates hypertrophic chondrocyte differentiation and osteoarthritis progression.

In osteoarthritis (OA), a disease characterized by progressive articular cartilage degradation and calcification, the articular chondrocyte phenotype changes and this correlates with actin cytoskeleton alterations suggesting that it regulates gene expression essential for proper phenotype. This study reports that OA is associated with the loss of adseverin, an actin capping and severing protein. Adseverin deletion (Adseverin-/-) in mice compromised articular chondrocyte function, by reducing F-actin and aggrecan expression and increasing apoptosis, Indian hedgehog, Runx2, MMP13, and collagen type X expression, and cell proliferation. This led to stiffer cartilage and decreased hyaline and increased calcified cartilage thickness. Together, these changes predisposed the articular cartilage to enhanced OA severity in Adseverin-/- mice who underwent surgical induction of OA. Adseverin-/- chondrocyte RNA sequencing and in vitro studies together suggests that adseverin modulates cell viability and prevents mineralization. Thus, adseverin maintains articular chondrocyte phenotype and cartilage tissue homeostasis by preventing progression to hypertrophic differentiation in vivo. Adseverin may be chondroprotective and a potential therapeutic target.

Effects on cell cycle progression and cytoskeleton organization of five Bothrops spp. venoms in cell culture-based assays

Snake envenomation is a neglected tropical disease. In Brazil, the Bothrops genus is responsible for about 86% of snakebite accidents. Despite extensive evidence of the cytotoxicity of snake venoms, the cellular and molecular mechanisms involved are not fully understood, especially regarding the effects on cell cycle progression and cytoskeleton organization. Traditionally, the effectiveness and quality control tests of venoms and antivenoms are assessed by in vivo assays. Despite this, there is a rising effort to develop surrogate in vitro models according to the 3R principle (Replacement, Reduction, and Refinement). In this study, we treated rat liver cells (BRL-3A) with venoms from five Bothrops species (B. jararaca, B. jararacussu, B. moojeni, B. alternatus, and B. neuwiedi) and analyzed cell viability and IC50 by MTT assay, cell cycle phases distribution by flow cytometry, and morphology and cytoskeleton alterations by immunofluorescence. In addition, we evaluated the correlation between IC50 and the enzymatic and biological activities of each venom. Our results indicated that Bothrops spp. venoms decreased the cell viability of rat liver BRL-3A cells. The rank order of potency was B. jararacussu > B. moojeni > B. alternatus > B. jararaca > B. neuwiedi. The mechanisms of cytotoxicity were related to microtubules and actin network disruption, but not to cell cycle arrest. No clear correlation was found between the IC50 and retrieved literature data of in vitro enzymatic and in vivo biological activities. This work contributed to understanding cellular and molecular mechanisms underlying the Bothrops spp. venom cytotoxicity, which can help to improve envenomation treatment, as well as disclose potential therapeutic properties of snake venoms.

Loss of the extracellular matrix protein Perlecan disrupts axonal and synaptic stability during Drosophila development.

Heparan sulfate proteoglycans (HSPGs) form essential components of the extracellular matrix (ECM) and basement membrane (BM) and have both structural and signaling roles. Perlecan is a secreted ECM-localized HSPG that contributes to tissue integrity and cell-cell communication. Although a core component of the ECM, the role of Perlecan in neuronal structure and function is less understood. Here, we identify a role for Drosophila Perlecan in the maintenance of larval motoneuron axonal and synaptic stability. Loss of Perlecan causes alterations in the axonal cytoskeleton, followed by axonal breakage and synaptic retraction of neuromuscular junctions. These phenotypes are not prevented by blocking Wallerian degeneration and are independent of Perlecan's role in Wingless signaling. Expression of Perlecan solely in motoneurons cannot rescue synaptic retraction phenotypes. Similarly, removing Perlecan specifically from neurons, glia, or muscle does not cause synaptic retraction, indicating the protein is secreted from multiple cell types and functions non-cell autonomously. Within the peripheral nervous system, Perlecan predominantly localizes to the neural lamella, a specialized ECM surrounding nerve bundles. Indeed, the neural lamella is disrupted in the absence of Perlecan, with axons occasionally exiting their usual boundary in the nerve bundle. In addition, entire nerve bundles degenerate in a temporally coordinated manner across individual hemi-segments throughout larval development. These observations indicate disruption of neural lamella ECM function triggers axonal destabilization and synaptic retraction of motoneurons, revealing a role for Perlecan in axonal and synaptic integrity during nervous system development.

Loss of the extracellular matrix protein Perlecan disrupts axonal and synaptic stability during Drosophila development

Heparan sulfate proteoglycans (HSPGs) form essential components of the extracellular matrix (ECM) and basement membrane (BM) and have both structural and signaling roles. Perlecan is a secreted ECM-localized HSPG that contributes to tissue integrity and cell-cell communication. Although a core component of the ECM, the role of Perlecan in neuronal structure and function is less understood. Here, we identify a role for Drosophila Perlecan in the maintenance of larval motoneuron axonal and synaptic stability. Loss of Perlecan causes alterations in the axonal cytoskeleton, followed by axonal breakage and synaptic retraction of neuromuscular junctions. These phenotypes are not prevented by blocking Wallerian degeneration and are independent of Perlecan’s role in Wingless signaling. Expression of Perlecan solely in motoneurons cannot rescue synaptic retraction phenotypes. Similarly, removing Perlecan specifically from neurons, glia, or muscle does not cause synaptic retraction, indicating the protein is secreted from multiple cell types and functions non-cell autonomously. Within the peripheral nervous system, Perlecan predominantly localizes to the neural lamella, a specialized ECM surrounding nerve bundles. Indeed, the neural lamella is disrupted in the absence of Perlecan, with axons occasionally exiting their usual boundary in the nerve bundle. In addition, entire nerve bundles degenerate in a temporally coordinated manner across individual hemi-segments throughout larval development. These observations indicate disruption of neural lamella ECM function triggers axonal destabilization and synaptic retraction of motoneurons, revealing a role for Perlecan in axonal and synaptic integrity during nervous system development.

New Ruthenium-Cyclopentadienyl Complexes Affect Colorectal Cancer Hallmarks Showing High Therapeutic Potential.

Colorectal cancer (CRC) is among the most deadly cancers worldwide. Current therapeutic strategies have low success rates and several side effects. This relevant clinical problem requires the discovery of new and more effective therapeutic alternatives. Ruthenium drugs have arisen as one of the most promising metallodrugs, due to their high selectivity to cancer cells. In this work we studied, for the first time, the anticancer properties and mechanisms of action of four lead Ru-cyclopentadienyl compounds, namely PMC79, PMC78, LCR134 and LCR220, in two CRC-derived cell lines (SW480 and RKO). Biological assays were performed on these CRC cell lines to evaluate cellular distribution, colony formation, cell cycle, proliferation, apoptosis, and motility, as well as cytoskeleton and mitochondrial alterations. Our results show that all the compounds displayed high bioactivity and selectivity, as shown by low half-maximal inhibitory concentrations (IC50) against CRC cells. We observed that all the Ru compounds have different intracellular distributions. In addition, they inhibit to a high extent the proliferation of CRC cells by decreasing clonogenic ability and inducing cell cycle arrest. PMC79, LCR134, and LCR220 also induce apoptosis, increase the levels of reactive oxygen species, lead to mitochondrial dysfunction, induce actin cytoskeleton alterations, and inhibit cellular motility. A proteomic study revealed that these compounds cause modifications in several cellular proteins associated with the phenotypic alterations observed. Overall, we demonstrate that Ru compounds, especially PMC79 and LCR220, display promising anticancer activity in CRC cells with a high potential to be used as new metallodrugs for CRC therapy.

Loss of PRDX6 Aborts Proliferative and Migratory Signaling in Hepatocarcinoma Cell Lines.

Peroxiredoxin 6 (PRDX6), the only mammalian 1-Cys member of the peroxiredoxin family, has peroxidase, phospholipase A2 (PLA2), and lysophosphatidylcholine (LPC) acyltransferase (LPCAT) activities. It has been associated with tumor progression and cancer metastasis, but the mechanisms involved are not clear. We constructed an SNU475 hepatocarcinoma cell line knockout for PRDX6 to study the processes of migration and invasiveness in these mesenchymal cells. They showed lipid peroxidation but inhibition of the NRF2 transcriptional regulator, mitochondrial dysfunction, metabolic reprogramming, an altered cytoskeleton, down-regulation of PCNA, and a diminished growth rate. LPC regulatory action was inhibited, indicating that loss of both the peroxidase and PLA2 activities of PRDX6 are involved. Upstream regulators MYC, ATF4, HNF4A, and HNF4G were activated. Despite AKT activation and GSK3β inhibition, the prosurvival pathway and the SNAI1-induced EMT program were aborted in the absence of PRDX6, as indicated by diminished migration and invasiveness, down-regulation of bottom-line markers of the EMT program, MMP2, cytoskeletal proteins, and triggering of the "cadherin switch". These changes point to a role for PRDX6 in tumor development and metastasis, so it can be considered a candidate for antitumoral therapies.

SYP-3343 drives abnormal vascularization in zebrafish through regulating endothelial cell behavior.

SYP-3343 is a novel strobilurin fungicide with excellent and broad-spectrum antifungal activity, and its potential toxicity raises public health concerns. However, the vascular toxicity of SYP-3343 to zebrafish embryos is still not well understood. In the present study, we investigated the effects of SYP-3343 on vascular growth and its potential mechanism of action. SYP-3343 inhibited zebrafish endothelial cell (zEC) migration, altered nuclear morphology, and triggered abnormal vasculogenesis and zEC sprouting angiogenesis, resulting in angiodysplasia. RNA sequencing showed that SYP-3343 exposure altered the transcriptional levels of vascular development-related biological processes in zebrafish embryos including angiogenesis, sprouting angiogenesis, blood vessel morphogenesis, blood vessel development, and vasculature development. Whereas, the addition of NAC exerted an improvement effect on zebrafish vascular defects owing to SYP-3343 exposure. Additionally, SYP-3343 altered cell cytoskeleton and morphology, obstructed migration and viability, disrupted cell cycle progression, and depolarized mitochondrial membrane potential, as well as promoted apoptosis and reactive oxygen species (ROS) in HUVEC. SYP-3343 also caused an imbalance of the oxidation and antioxidant systems and irritated the alterations in the cell cycle- and apoptosis-related genes in HUVECs. Collectively, SYP-3343 has high cytotoxicity, possibly by up-regulating p53 and caspase3 expressions and bax/bcl-2 ratio via ROS, leading to malformed vascular development.

Eukaryotic Initiation Factor 5A2 localizes to actively translating ribosomes to promote cancer cell protrusions and invasive capacity

BackgroundEukaryotic Initiation Factor 5A (eIF-5A), an essential translation factor, is post-translationally activated by the polyamine spermidine. Two human genes encode eIF-5A, being eIF5-A1 constitutively expressed whereas eIF5-A2 is frequently found overexpressed in human tumours. The contribution of both isoforms with regard to cellular proliferation and invasion in non-small cell lung cancer remains to be characterized.MethodsWe have evaluated the use of eIF-5A2 gene as prognosis marker in lung adenocarcinoma (LUAD) patients and validated in immunocompromised mice. We have used cell migration and cell proliferation assays in LUAD lines after silencing each eIF-5A isoform to monitor their contribution to both phenotypes. Cytoskeleton alterations were analysed in the same cells by rhodamine-phalloidin staining and fluorescence microscopy. Polysome profiles were used to monitor the effect of eIF-5A2 overexpression on translation. Western blotting was used to study the levels of eIF-5A2 client proteins involved in migration upon TGFB1 stimulation. Finally, we have co-localized eIF-5A2 with puromycin to visualize the subcellular pattern of actively translating ribosomes.ResultsWe describe the differential functions of both eIF-5A isoforms, to show that eIF5-A2 properties on cell proliferation and migration are coincident with its features as a poor prognosis marker. Silencing of eIF-5A2 leads to more dramatic consequences of cellular proliferation and migration compared to eIF-5A1. Overexpression of eIF-5A2 leads to enhanced global translation. We also show that TGFβ signalling enhances the expression and activity of eIF-5A2 which promotes the translation of polyproline rich proteins involved in cytoskeleton and motility features as it is the case of Fibronectin, SNAI1, Ezrin and FHOD1. With the use of puromycin labelling we have co-localized active ribosomes with eIF-5A2 not only in cytosol but also in areas of cellular protrusion. We have shown the bulk invasive capacity of cells overexpressing eIF-5A2 in mice.ConclusionsWe propose the existence of a coordinated temporal and positional interaction between TFGB and eIF-5A2 pathways to promote cell migration in NSCLC. We suggest that the co-localization of actively translating ribosomes with hypusinated eIF-5A2 facilitates the translation of key proteins not only in the cytosol but also in areas of cellular protrusion.5zFBY1s4Yn-gzgeYzNdWy3Video

Abstract P6-14-09: Non-canonical Wnt/Ror2 signaling regulates tumor cell invasion and dissemination in breast cancer through cell-matrix crosstalk

Abstract BACKGROUND: Metastasis is the main cause of mortality for breast cancer patients. The early steps of cancer metastasis require that tumor cells actively invade and disseminate from the primary tumor to distant organs. Cell-extracellular matrix (ECM) interactions represent fundamental interactions during tumor invasion and metastasis, yet how particular signal-transduction factors prompt the conversion of tumor cells into migratory populations capable of systemic dissemination remains elusive. OBJECTIVES: Wnt signaling is a known regulator of cell fate, migration, and polarity during various key biological processes. We previously discovered an inverse correlation between the canonical Wnt signature and a non-canonical Wnt receptor, Ror2, across breast cancers in TCGA. The objective of this study is to investigate how canonical and non-canonical Wnt signaling orchestrate tumor cell behavior during cancer invasion and metastasis. METHODS: We used clinically relevant syngeneic TP53-null Genetically Engineered Mouse Model (GEMM) tumors that molecularly reflect the different breast cancer intrinsic subtypes. In vivo transplantation of GEMM models and in vitro 3D tumor organoids were employed to elucidate the molecular mechanism underlying the migration, invasion, and metastasis of tumor cells upon genetic perturbation of Wnt/Ror2 signaling. RESULTS/DISCUSSION: From 3-dimensional (3D) tumor organoid models, we identified novel transcriptional alterations encompassing cell-cell adhesion, cytoskeletal remodeling, and ECM organization upon Ror2 loss. At protein levels, we discovered a significant increase in collagen fibril organization and integrin-α5 and integrin-β3 expression following Ror2 depletion. In addition, the matrisomal protein fibronectin (FN) was concomitantly upregulated and assembled in Ror2-deficient tumor cells at sites of invasion. Consequently, we observed FAK activation and actin cytoskeleton alterations in Ror2-deficient tumor cells, leading to a promigratory tumor cell behavior. The altered cytoskeleton and cell-ECM interaction enhanced the rigidity of the Ror2-depleted cells. Furthermore, Inhibition of either integrin or FAK activation abrogated the increased invasion driven by Ror2-loss. Unexpectedly, these changes were distinct from processes regulated by Wnt/ß-catenin activation. From both spontaneous metastasis and experimental metastasis models, we discovered a shift from canonical to non-canonical Wnt signaling throughout the progression of lung metastasis, indicating that the balance of Wnt signaling may serve as a switch to dictate cell functions at different stages of metastatic development. Consistently, we found enhanced initial colonization in the lungs when Ror2 is depleted in these breast cancer cells. Together, these studies provide new insight into how canonical and alternative Wnt pathways coordinate cell-cell and cell-ECM exchanges during breast cancer progression and metastasis. Supported by grant NIH-CA016303 Citation Format: Hongjiang Si, Na Zhao, Andrea Pedroza, Chad Creighton, Jeffrey Rosen, Kevin Roarty. Non-canonical Wnt/Ror2 signaling regulates tumor cell invasion and dissemination in breast cancer through cell-matrix crosstalk [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-09.

The oxygen dissociation curve of blood in COVID-19-An update.

An impressive effect of the infection with SARS-Co-19 is the impairment of oxygen uptake due to lung injury. The reduced oxygen diffusion may potentially be counteracted by an increase in oxygen affinity of hemoglobin. However, hypoxia and anemia associated with COVID-19 usually decrease oxygen affinity due to a rise in [2,3-bisphosphoglycerate]. As such, COVID-19 related changes in the oxygen dissociation curve may be critical for oxygen uptake and supply, but are hard to predict. A Pubmed search lists 14 publications on oxygen affinity in COVID-19. While some investigations show no changes, three large studies found an increased affinity that was related to a good prognosis. Exact causes remain unknown. The cause of the associated anemia in COVID-19 is under discussion. Erythrocytes with structural alterations of membrane and cytoskeleton have been observed, and virus binding to Band 3 and also to ACE2 receptors in erythroblasts has been proposed. COVID-19 presentation is moderate in many subjects suffering from sickle cell disease. A possible explanation is that COVID-19 counteracts the unfavorable large right shift of the oxygen dissociation curve in these patients. Under discussion for therapy are mainly affinity-increasing drugs.

The Effect of Acidic and Alkaline Seawater on the F-Actin-Dependent Ca2+ Signals Following Insemination of Immature Starfish Oocytes and Mature Eggs.

In starfish, the addition of the hormone 1-methyladenine (1-MA) to immature oocytes (germinal vesicle, GV-stage) arrested at the prophase of the first meiotic division induces meiosis resumption (maturation), which makes the mature eggs able to respond to the sperm with a normal fertilization response. The optimal fertilizability achieved during the maturation process results from the exquisite structural reorganization of the actin cytoskeleton in the cortex and cytoplasm induced by the maturing hormone. In this report, we have investigated the influence of acidic and alkaline seawater on the structure of the cortical F-actin network of immature oocytes of the starfish (Astropecten aranciacus) and its dynamic changes upon insemination. The results have shown that the altered seawater pH strongly affected the sperm-induced Ca2+ response and the polyspermy rate. When immature starfish oocytes were stimulated with 1-MA in acidic or alkaline seawater, the maturation process displayed a strong dependency on pH in terms of the dynamic structural changes of the cortical F-actin. The resulting alteration of the actin cytoskeleton, in turn, affected the pattern of Ca2+ signals at fertilization and sperm penetration.

Glucocorticoids increase tissue cell protection against pore-forming toxins from pathogenic bacteria

Many species of pathogenic bacteria damage tissue cells by secreting toxins that form pores in plasma membranes. Here we show that glucocorticoids increase the intrinsic protection of tissue cells against pore-forming toxins. Dexamethasone protected several cell types against the cholesterol-dependent cytolysin, pyolysin, from Trueperella pyogenes. Dexamethasone treatment reduced pyolysin-induced leakage of potassium and lactate dehydrogenase, limited actin cytoskeleton alterations, reduced plasma membrane blebbing, and prevented cytolysis. Hydrocortisone and fluticasone also protected against pyolysin-induced cell damage. Furthermore, dexamethasone protected HeLa and A549 cells against the pore-forming toxins streptolysin O from Streptococcus pyogenes, and alpha-hemolysin from Staphylococcus aureus. Dexamethasone cytoprotection was not associated with changes in cellular cholesterol or activating mitogen-activated protein kinase (MAPK) cell stress responses. However, cytoprotection was dependent on the glucocorticoid receptor and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR). Collectively, our findings imply that glucocorticoids could be exploited to limit tissue damage caused by pathogens secreting pore-forming toxins.

Platelet transcriptome analysis in patients with germline RUNX1 mutations

BackgroundGermline mutations in RUNX1 can cause a familial platelet disorder that may lead to acute myeloid leukemia, an autosomal dominant disorder characterized by moderate thrombocytopenia, platelet dysfunction, and a high risk of developing acute myeloid leukemia or myelodysplastic syndrome. Discerning the pathogenicity of novel RUNX1 variants is critical for patient management. ObjectivesTo extend the characterization of RUNX1 variants and evaluate their effects by transcriptome analysis. MethodsThree unrelated patients with long-standing thrombocytopenia carrying heterozygous RUNX1 variants were included: P1, who is a subject with recent development of myelodysplastic syndrome, with c.802 C>T[p.Gln268∗] de novo; P2 with c.586A>G[p.Thr196Ala], a variant that segregates with thrombocytopenia and myeloid neoplasia in the family; and P3 with c.476A>G[p.Asn159Ser], which did not segregate with thrombocytopenia or neoplasia. Baseline platelet evaluations were performed. Ultrapure platelets were prepared for platelet transcriptome analysis. ResultsIn P1 and P2, but not in P3, transcriptome analysis confirmed aberrant expression of genes recognized as RUNX1 targets. Data allowed grouping patients by distinct gene expression profiles, which were partitioned with clinical parameters. Functional studies and platelet mRNA expression identified alterations in the actin cytoskeleton, downregulation of GFI1B, defective GPVI downstream signaling, and reduction of alpha granule proteins, such as thrombospondin-1, as features likely implicated in thrombocytopenia and platelet dysfunction. ConclusionPlatelet phenotype, familial segregation, and platelet transcriptomics support the pathogenicity of RUNX1 variants p.Gln268∗ and p.Thr196Ala, but not p.Asn159Ser. This study is an additional proof of concept that platelet RNA analysis could be a tool to help classify pathogenic RUNX1 variants and identify novel RUNX1 targets.

Y-27632 Impairs Angiogenesis on Extra-Embryonic Vasculature in Post-Gastrulation Chick Embryos

Y-27632 inhibits Rho-associated coiled-coil-containing protein kinase (ROCK) signaling, which is involved in various embryonic developmental processes, including angiogenesis, by controlling actin cytoskeleton assembly and cell contractility. Administration of Y-27632 impairs cytoskeletal arrangements in post-gastrulation chick embryos, leading to ventral body wall defects (VBWDs). Impaired angiogenesis has been hypothesized to contribute to VBWDs. ROCK is essential in transmitting signals downstream of vascular endothelial growth factor (VEGF). VEGF-mediated angiogenesis induces gene expressions and alterations of the actin cytoskeleton upon binding to VEGF receptors (VEGFRs). The aim of this study was to investigate effects of Y-27632 on angiogenesis in post-gastrulation chick embryos during early embryogenesis. After 60 h incubation, embryos in shell-less culture were treated with Y-27632 or vehicle for controls. Y-27632-treated embryos showed reduced extra-embryonic blood vessel formation with impaired circulation of the yolk sac, confirmed by fractal analysis. Western blot confirmed impaired ROCK downstream signaling by decreased expression of phosphorylated myosin light chain. Interestingly, RT-PCR demonstrated increased gene expression of VEGF and VEGFR-2 1 h post-treatment. Protein levels of VEGF were higher in Y-27632-treated embryos at 8 h following treatment, whereas no difference was seen in membranes. We hypothesize that administration of Y-27632 impairs vessel formation during angiogenesis, which may contribute to failure of VWB closure, causing VBWDs.

Chronic MAP4343 reverses escalated alcohol drinking in a mouse model of alcohol use disorder

Alcohol use disorders can be driven by negative reinforcement. Alterations of the microtubule cytoskeleton have been associated with mood regulation in the context of depression. Notably, MAP4343, a pregnenolone derivative known to promote tubulin assembly, has antidepressant properties. In the present study, we tested the hypothesis that MAP4343 may reduce excessive alcohol drinking in a mouse model of alcohol dependence by normalizing affect during withdrawal. Adult male C57BL/6J mice were given limited access to voluntary alcohol drinking and ethanol intake escalation was induced by chronic intermittent ethanol (CIE) vapor inhalation. Chronic, but not acute, administration of MAP4343 reduced ethanol intake and this effect was more pronounced in CIE-exposed mice. There was a complex interaction between the effects of MAP4343 and alcohol on affective behaviors. In the elevated plus maze, chronic MAP4343 tended to increase open-arm exploration in alcohol-naive mice but reduced it in alcohol-withdrawn mice. In the tail suspension test, chronic MAP4343 reduced immobility selectively in Air-exposed alcohol-drinking mice. Finally, chronic MAP4343 countered the plasma corticosterone reduction induced by CIE. Parallel analysis of tubulin post-translational modifications revealed lower α-tubulin acetylation in the medial prefrontal cortex of CIE-withdrawn mice. Altogether, these data support the relevance of microtubules as a therapeutic target for the treatment of AUD.

Alterations in Cytoskeleton and Mitochondria in the Development and Reversal of Steatosis in Human Hepatocytes

Alterations in mitochondrial morphology and function and increased oxidative stresses in hepatocytes are well-established in non-alcoholic fatty liver disease (NAFLD). Patients can undergo lifestyle changes, especially in earlier NAFLD stages, to reverse disease-induced phenotypes on a gross level. Yet, little is known about whether mitochondrial function and injuries recover upon reversal. We, thus, elucidated this question and interplays between cytoskeletal network and mitochondria in the development and reversal of steatosis. We cultured primary human hepatocytes stably for 2 weeks and used free-fatty-acid (FAA) supplementation to induce steatosis over 7 days and reversed steatosis by FAA withdrawal over the next 7 days. We assessed cytoskeletal and mitochondrial morphologies using immunocytochemistry and confocal microscopy. We evaluated mitochondrial respiration and function via the Seahorse analyzer where we fully optimized reagent dosing specifically for human hepatocytes. During early steatosis, intracellular lipid droplets displaced microtubules altering mitochondrial distribution, and disrupted the F-actin network leading to loss of bile canaliculi in steatotic hepatocytes. Basal mitochondrial respiration, maximum respiratory capacity, and resistance to H2O2-induced cell death also increased as an adaptative response. Upon reversal of steatosis, F-actin and bile canaliculi were restored in hepatocytes. Nevertheless, we observed an increase in elongated mitochondrial branches accompanied by decreases in α-tubulin expression, mitochondrial proton leak, and susceptibility to H2O2-induced cell death. Despite the restoration of cytoskeletons morphologically upon reversal of steatosis, mitochondria, in hepatocytes, were impaired due to early adaptative respiratory increase. Hepatocytes were thus highly predisposed to H2O2-induced cell death. These results indicate the persistence of potential health risks for recovering NAFLD patients.

Microenvironment factors promoting the quality of vitrified cat oocytes

In oocyte cryopreservation programs, vitrification has overthrown conventional slow freezing both in veterinary and human medicine. In animals, its feasibility in field conditions makes it the preferred technique for the safeguard of genetic resources from zoo or wild animals, including threatened felids, for which the domestic cat is an excellent model. However, many cellular injuries, such as cytoskeleton, mitochondria and meiotic spindle alterations, DNA damage, zona pellucida hardening and cumulus cell loss, might occur following vitrification. After warming, although the exact mechanisms are still unclear, degeneration is a frequent outcome for cat vitrified oocytes. For immature (germinal vesicle) gametes, in vitro maturation after warming is a challenge, and cleavage after fertilization barely reaches 15–30%, while for mature (metaphase II) cryopreserved gametes it can get to 30–50%. Anyway, the progression to late embryos stages is often impaired, and improvements are needed. Standard cryopreservation protocol and the use of conventional in vitro culture systems after warming may not be enough for vitrified oocytes to recover and demonstrate their full developmental potential. Physical or chemical factors applied to oocytes undergoing vitrification, as an enrichment to the vitrification step, or to the culture microenvironment, could create more favorable conditions and promote vitrified oocyte survival and development. From the use of three-dimensional culture systems to the regulation of metabolic activities and cellular pathways, this review aims to explore all the possibilities employed so far, including the studies performed by our own lab, and the future perspectives, to present the most effective strategies for cat oocyte vitrification and the best time for their application (i.e., before, during, or after vitrification-warming).

Cancer cells can be killed mechanically or with combinations of cytoskeletal inhibitors.

For over two centuries, clinicians have hypothesized that cancer developed preferentially at the sites of repeated damage, indicating that cancer is basically “continued healing.” Tumor cells can develop over time into other more malignant types in different environments. Interestingly, indefinite growth correlates with the depletion of a modular, early rigidity sensor, whereas restoring these sensors in tumor cells blocks tumor growth on soft surfaces and metastases. Importantly, normal and tumor cells from many different tissues exhibit transformed growth without the early rigidity sensor. When sensors are restored in tumor cells by replenishing depleted mechanosensory proteins that are often cytoskeletal, cells revert to normal rigidity-dependent growth. Surprisingly, transformed growth cells are sensitive to mechanical stretching or ultrasound which will cause apoptosis of transformed growth cells (Mechanoptosis). Mechanoptosis is driven by calcium entry through mechanosensitive Piezo1 channels that activate a calcium-induced calpain response commonly found in tumor cells. Since tumor cells from many different tissues are in a transformed growth state that is, characterized by increased growth, an altered cytoskeleton and mechanoptosis, it is possible to inhibit growth of many different tumors by mechanical activity and potentially by cytoskeletal inhibitors.

Rho/SRF Inhibitor Modulates Mitochondrial Functions.

CCG-1423 is a Rho A pathway inhibitor that has been reported to inhibit Rho/SRF-mediated transcriptional regulation. Serum response factor and its cofactors, which include ternary complex factors and myocardin-related transcription factors, regulate various cellular functions. In this study, we observed that CCG-1423 modulates the mitochondrial functions. The effect of this small molecule drug was determined by measuring mitochondrial function using an XFe96 Analyzer and an Oxygraph 2k (O2k) high-resolution respirometer. CCG-1423 treatment significantly reduced oxidative phosphorylation in a dose-dependent manner. However, CCG-1423 increased the glycolytic rate. We also observed that histone 4 at lysine-16 underwent hyperacetylation with the treatment of this drug. Immunolabeling with F-actin and MitoTracker revealed the alteration in the actin cytoskeleton and mitochondria. Taken together, our findings highlight a critical role of CCG-1423 in inhibiting the transcription of SRF/p49 and PGC-1α, β, resulting in the downregulation of mitochondrial genes, leading to the repression of mitochondrial oxidative phosphorylation and overall ATP reduction. This study provides a better understanding of the effects of CCG-1423 on mitochondria, which may be useful for the assessment of the potential clinical application of CCG-1423 and its derivatives.

Biomechanics of cancer stem cells.

Cancer stem cells (CSCs) have been believed to be one driving force for tumor progression and drug resistance. Despite the significance of biochemical signaling in malignancy, highly malignant tumor cells or CSCs exhibit lower cellular stiffness than weakly malignant cells or non-CSCs, which are softer than their healthy counterparts, suggesting the inverse correlation between cell stiffness and malignancy. Recent years have witnessed the rapid accumulation of evidence illustrating the reciprocity between cell cytoskeleton/mechanics and CSC functions and the potential of cellular stiffness for specific targeting of CSCs. However, a systematic understanding of tumor cell mechanics and their role in CSCs and tumor progression is still lacking. The present review summarizes the recent progress in the alterations of tumor cell cytoskeleton and stiffness at different stages of tumor progression and recapitulates the relationship between cellular stiffness and CSC functions. The altered cell mechanics may mediate the mechanoadaptive responses that possibly empower CSCs to survive and thrive during metastasis. Furthermore, we highlight the possible impact of tumor cell mechanics on CSC malignancy, which may potentiate low cell stiffness as a mechanical marker for CSC targeting.