Changes In Cytoskeleton Research Articles

O-GlcNAcylation regulates osteoblast differentiation through the morphological changes in mitochondria, cytoskeleton, and endoplasmic reticulum.

To explore the potential mechanisms which O-linked-N-acetylglucosaminylation (O-GlcNAcylation) regulates osteogenesis, a publicly RNA-seq dataset was re-analyzed with literature-mining and showed the primary targets of O-GlcNAcylation in osteoblasts are mitochondria/cytoskeleton. Although the O-GlcNAcylation-regulated mitochondria/cytoskeleton has been extensively studied, its specific role during osteogenesis remains unclear. To address this, we knocked out Ogt (Ogt-KO) in MC3T3-E1 osteoblastic cells. Then, significantly reduced osteoblast differentiation, motility, proliferation, mitochondria-endoplasmic reticulum (Mito-ER) coupling, volume of ER, nuclear tubulins, and oxygen metabolism were observed in Ogt-KO cells. Through artificial intelligence (AI)-predicted cellular structures, the time-lapse live cells imaging with reactive-oxygen-species/hypoxia staining showed that lower cell proliferation and altered oxygen metabolism in the Ogt-KO cells were correlated with the Mito-ER coupling. Bioinformatics analysis, combined with correlated mRNA and protein expression, suggested that Ezh2 and its downstream targets (Opa1, Gsk3a, Wnt3a, Hif1a, and Hspa9) may be involved in O-GlcNAcylation-regulated Mito-ER coupling, ultimately impacting osteoblast differentiation. In conclusion, our findings indicate that O-GlcNAcylation-regulated osteoblast differentiation is linked to morphological changes in mitochondria, cytoskeleton, and ER, with Ezh2 potentially playing a crucial role.

Quiescent cancer cells induced by high-density cultivation reveals cholesterol-mediated survival and lung metastatic traits.

The metastatic cascade, a multifaceted and highly aggressive process, is the primary cause of mortality. The survival of quiescent cancer cells in circulatory system during metastasis is crucial, yet our comprehension is constrained by the absence of universally accepted quiescent cancer models. We developed a quiescent cancer cell model using high-density cultivation. Based on the scRNA-seq analysis, IP-MS, metabolomics, mouse lung metastasis models, cholesterol assay, PLA and other molecular experiments, we explored the molecular mechanism. Immunofluorescence, atomic force microscope, FluidFM, and shear stress stimulation were used to analyze the cytoskeleton and membrane properties contributing to mechanical force resistance. We established a quiescent cancer cell model induced by high-density cultivation. Single-cell RNA sequencing (scRNA-seq) analysis reveals that CDC25A plays a crucial role in the transition to quiescence, with its expression significantly elevated in the quiescent state. Depletion of CDC25A leads to an increased proliferative capacity, and reduced metastasis under high-density conditions. Mechanistically, upregulated CDC25A in quiescent cells enhances cholesterol metabolism via endosome pathways, leading to cell cycle arrest. This increase in cholesterol reinforces the cytoskeleton, alters membrane properties, and improves resistance to mechanical forces in circulatory system. CDC25A significantly increased the cholesterol metabolism through endosome pathway in quiescent cancer cells, leading to the significant changes in cytoskeleton and membrane properties so as to enhance the resistance of mechanical force in circulatory system, facilitating lung metastasis. In high-density cultivation, quiescent cancer cells, up-regulate cholesterol metabolism by CDC25A through endosome pathway, enhancing the resistance to mechanical force in circulatory system, facilitating lung metastasis.

7449 A Genome-wide CRISPR Screen Identifies Drivers of Impaired Glucose Uptake in Human Insulin Resistance

Abstract Disclosure: N. Haider: None. Y. Lee: None. P. Yi: None. C.R. Kahn: None. Insulin resistance is a major risk factor in the development of type 2 diabetes (T2D), and metabolic syndrome. Although 25% of people within the general non-diabetic population are insulin resistant, the primary underlying cause of insulin resistance remains elusive. In this study, we have used induced pluripotent stem cells (iPSC) derived from non-diabetic humans at both ends of the insulin sensitivity spectrum, i.e., the top 20% of insulin resistance vs. the top 20% of insulin sensitive, as well as individuals with T2D to model human insulin resistance. We find that iPSCs differentiated into myoblasts (iMyos) from T2D and non-diabetic individuals in the highest quintile of insulin resistance (I-Res) show impaired insulin signaling and defective insulin-stimulated glucose uptake, as compared to iMyos from the insulin sensitive individuals (I-Sen), indicating that these cells in vitro mirror the alterations seen in vivo in a cell autonomous manner. To uncover the primary upstream drivers that contribute to the impaired glucose uptake associated with insulin resistance, we conducted a genome-wide loss-of-function screen using a CRISPR-based approach to identify genes that positively or negatively regulated glucose uptake in I-Res and T2D iMyos. Knockdown of ∼525 genes show effect to rescue the impaired glucose uptake in both I-Res and T2D iMyos, i.e., were negative regulators of glucose uptake. We hypothesized that important negative regulators of glucose uptake might be over-expressed at the protein level in I-Res and T2D iMyos and crossing the iMyos proteomics data with the CRISPR screen revealed 8 top candidates that are increased at the protein level and whose knockdown rescued glucose uptake in I-Res and T2D iMyos. These included candidates associated with cytoskeleton changes, protein kinases, transmembrane transport, mitochondrial and nuclear proteins. In summary, iPSC derived myoblasts from patients with insulin resistance and T2D mirror defects in glucose uptake observed in vivo. Combining CRISPR screening and global proteomics, we have identified important upstream regulators of insulin resistance in humans. Presentation: 6/2/2024

Poly(ethylene glycol) dimethacrylate (PEGDMA) multi-functional pillar-patterned surface for osteogenic differentiation of pre-osteoblast and anti-bacterial effects to Escherichia coli and Staphylococcus aureus

Cells are sensitive to the surface topographical environment, which can subsequently induce changes in cell morphology, cytoskeleton, and differentiation. Therefore, the topological environment in bone implant devices is known to influence the cell behavior, adhesion, and differentiation of osteoblasts. Furthermore, bone implant devices with anti-bacterial properties could be necessary to prevent surgical site infections (SSIs). Consequently, there is a demand for research into multi-functional surfaces that can facilitate osteoblast differentiation while also possessing an anti-bacterial effect. In this study, we fabricated and assessed the poly(ethylene glycol) dimethacrylate (PEGDMA) nano/micro pillar-patterned surface to find the optimized dimensional characteristics of the pillar pattern for multi-functional effect. Results observed that the pillar pattern enhanced differentiation to osteoblast on the pillar-patterned surface (D1S0.5) having pillars with 0.5 μm height, 1 μm diameter, and 0.5 μm spacing between pillars and pillar-patterned surface (D1S1) having pillars with 0.5 μm height, 1 μm diameter, and 1 μm spacing between pillars. Subsequently, results of evaluating anti-bacterial effect revealed that the optimized pillar-patterned surface exhibited remarkable antibacterial effect, with the D1S0.5 pillar-patterned surface showing the highest antibacterial effect. Consequently, this study proposed and verified a multi-functional pillar-patterned surface capable of promoting osteoblast differentiation with anti-bacterial effects.

Comparative analysis of transcriptomics and metabolomics provides insights into the mechanisms of VPAHPND invasion and hepatopancreatic damage in Litopenaeus vannamei

Acute hepatopancreatic necrosis disease (AHPND) poses significant threats to the global shrimp farming industry; however, its molecular mechanisms remain largely unknown. Previous research has primarily focused on comparisons between infected and non-infected states, limiting our understanding of VPAHPND mechanisms. We integrated transcriptomic and metabolomic analyses to investigate the pathogenic mechanism underpinning AHPND in highly vulnerable post-larvae (PL) stage shrimp. By comparing shrimp infected with VPAHPND, those infected with non-VPAHPND, and uninfected shrimp (controls), we identified different VPAHPND infection responses, including significant cytoskeleton and metabolic reprogramming changes. Specifically, VPAHPND infection disturbed lipid, glutathione, and bile acid metabolism, while a key regulatory factor Farnesoid X Receptor (FXR) in these pathways was down-regulated. These findings suggest that VPAHPND manipulates host metabolism to enhance infectivity, leading to severe and irreparable hepatopancreas damage. Our study highlights the molecular interactions between VPAHPND and shrimp, and provides potential targets to mitigate the impact of AHPND in aquaculture.

Klebsiella pneumoniae increases invasion inintestinal epithelial cells by disrupting thecytoskeleton.

Klebsiella pneumoniae is an opportunistic pathogen and it can cause human mucosal lesions through the intestine, leading to bacteremia and abscess formation in liver and spleen. Previous studies have shown that K. pneumoniae can enter or cross cells through the intestinal epithelium, but the mechanism is unknown. In this study, we treated the intestinal epithelial cell line Caco-2 with KP1195, a clinically isolated strain with high adhesion and invasion of intestinal epithelial cells. The results showed that thetreatment of K. pneumoniae could increase the expression of integrin gene and further disrupt the changes of cytoskeleton. Treating Caco-2 with cytoskeletal inhibitor cytorelaxin D can significantly increase the efficiency of K. pneumoniae invading Caco-2 cells. These data suggest that disruption of the cytoskeleton through integrins may be one of the mechanisms by which K. pneumoniae increases intracellular invasion. This study provides a theoretical basis for further understanding of the mechanism of K. pneumoniae entering intestinal epithelial cells.

CYTOSKELETON DYNAMICS AND SPATIAL ORGANIZATION DURING EPITHELIAL-TO-MESENCHYMAL TRANSITION

Epithelial-to-mesenchymal transition (EMT) is a crucial process that occurs during normal development, such as embryogenesis and organ formation. If it is inappropriately activated, it can lead to pathological processes like metastasis. Although EMT is well studied at the morphological and transcriptome level, cytoskeleton changes during this process are less understood. In our work we aimed to describe the morphological changes that occurred in MCF-7, A-549 and HaCaT cells after EMT, analyze microtubule dynamics, spatial distribution and its contribution to cell motility, identify changes in actin filament organization and study focal adhesion turnover in the cells after EMT. We hypothesized that the dynamics of microtubules in cells undergoing EMT might change. Cells undergoing EMT were expected to have more dynamic microtubules. Also, cells undergoing EMT are expected to adhere more efficiently to diverse substrates, and therefore spread and move more easily. Focal contacts in cells undergoing EMT were expected to be more pronounced and dynamic than in cells that not undergoing EMT. The research methods used in the study included inducing EMT in modified MCF-7 cells through an inserted inducible Tet-on system and stimulation of EMT in A-549 and HaCaT cells using TGF-β. The cells were observed using bright field microscopy, and immunofluorescence analysis was conducted to visualize microtubules and actin filaments. Transfection with EB-3–RFP protein was done to describe and measure microtubule dynamics, while transduction with Talin-RFP and transient transfection with Ptag-RFP-vinculin was done to visualize focal adhesions. Time-lapse fluorescent microscopy was used to record films, and the Fiji Image J program was used to analyze the data. All statistical analysis was performed using GraphPad Prism (Dotmatics, USA), and a nonparametric Mann-Whitney U test or parametric t-test with Welch correction. The actin filament measurements were completed using Matlab scripts. The major research findings: In all three-cell models after EMT, cell morphology changed. Cells increased in size. MCF-7 and HaCaT became spread out, while A-549 became elongated. Some of the cells lost cell-cell contacts. All three cell models after EMT had alterations in microtubule organization and dynamics. MCF-7 and HaCaT cells showed more frequently individual MTs at cell edges, while A-549 had less covered nucleus by MTs after EMT. Microtubule dynamics (growth velocity) increased, and the length of microtubule growth tracks became longer. The average angle of MT growth trajectories to cell radius decreased. Actin fibers rearranged into more pronounced stress fibers after EMT. Alterations in the focal adhesion behaviors were different in three models used. These results indicate that cytoskeletal changes during EMT mainly include increased microtubule dynamics.

DATA DRIVEN AND CELL SPECIFIC DETERMINATION OF NUCLEI-ASSOCIATED ACTIN STRUCTURE

In the specialized field of cellular mechanosignaling, the intricate relationship between the cytoskeleton and nucleus is crucial for determining the behavior and fate of mesenchymal stem cells (MSCs). Existing analytical methods for filamentous actin fibers (F-actin) suffer from issues of accuracy and reproducibility. To address this, our research introduces a significant advancement in image analysis by utilizing two deep-learning segmentation Convolutional Neural Networks (CNNs) based on U-Net architecture. This algorithm precisely quantifies F-actin and offers reproducible data from 3D confocal microscopy images. Our CNNs automatically extract the 3D shape of both the nucleus and individual actin fibers, thereby enabling the geometric reconstruction of these critical cellular components. We applied this methodology to MSCs exposed to low-intensity vibration, and hypothesized that mechanical stimuli will induce perinuclear actin remodeling. Confocal Z-stack images of MSCs were captured using a 40x oil lens and a Zeiss LSM 900 microscope. Nucleus and F-actin structures were stained with Hoechst 33342 and Phalloidin, respectively. Nucleus-based images were employed to mask and isolate actin fibers within the nuclear vicinity. A specialized deep-learning segmentation model was engineered within the PyTorch framework. For model training, a selective 2% of nuclei and F-actin images were annotated. Specifically, three layers from each of six representative nuclei, among approximately 500 cross-sectional layers, were meticulously selected; a similar approach was employed for actin. The model underwent 200 epochs of training. Post-training, the deep-learning model was employed to process all collected cross-sectional images of F-actin and nuclei. A subsequent analytical step involved the scrutiny of intersecting segmented fibers between consecutive layers for geometrically accurate actin fiber reconstruction. We analyzed 102 control and 110 vibration-treated mesenchymal stem cells (MSCs) by utilizing our deep-learning-based reconstruction algorithm on confocal Z-stack images. Our data revealed an 8% decrease in nuclear height from 5.73 µm to 5.28 µm (p < 0.001) upon application of low-intensity vibration. In contrast, the two groups observed no significant difference in nuclear volume. Accompanying these changes at the nuclear level, the following changes in the F-actin cytoskeleton were observed upon LIV application. The total number of actin filaments per cell increased from 117 to 150. The average volume of individual actin fibers rose from 0.40 µm³ to 0.43 µm³. Notably, the volume of fibers at the apical nuclear surface increased from 0.35 µm³ to 0.46 µm³. While the average length of actin fibers in the apical nuclear surface remained largely unchanged due to the addition of smaller fibers in LIV groups, the LIV group had a larger number of fibers that were larger than 15µm.

Signal flow in the NMDA receptor-dependent phosphoproteome regulates postsynaptic plasticity for aversive learning.

Structural plasticity of dendritic spines in the nucleus accumbens (NAc) is crucial for learning from aversive experiences. Activation of NMDA receptors (NMDARs) stimulates Ca2+-dependent signaling that leads to changes in the actin cytoskeleton, mediated by the Rho family of GTPases, resulting in postsynaptic remodeling essential for learning. We investigated how phosphorylation events downstream of NMDAR activation drive the changes in synaptic morphology that underlie aversive learning. Large-scale phosphoproteomic analyses of protein kinase targets in mouse striatal/accumbal slices revealed that NMDAR activation resulted in the phosphorylation of 194 proteins, including RhoA regulators such as ARHGEF2 and ARHGAP21. Phosphorylation of ARHGEF2 by the Ca2+-dependent protein kinase CaMKII enhanced its RhoGEF activity, thereby activating RhoA and its downstream effector Rho-associated kinase (ROCK/Rho-kinase). Further phosphoproteomic analysis identified 221 ROCK targets, including the postsynaptic scaffolding protein SHANK3, which is crucial for its interaction with NMDARs and other postsynaptic scaffolding proteins. ROCK-mediated phosphorylation of SHANK3 in the NAc was essential for spine growth and aversive learning. These findings demonstrate that NMDAR activation initiates a phosphorylation cascade crucial for learning and memory.

Abstract P213: Mechanisms of Renin-Angiotensin System-Mediated Nitric Oxide Signaling in Podocytes

Introduction: The renin-angiotensin system (RAS) is a crucial cardiovascular and renal function regulator. Podocytes, glomerular epithelial cells that play a pivotal role in maintaining the renal filtration barrier, are key targets for RAS peptides. Our findings showed that RAS can promote calcium (Ca 2+ ) and nitric oxide (NO) signaling in podocytes. Moreover, a decrease in NO bioavailability directly correlates with glomerular filtration barrier damage, Ca 2+ overload, and the development of salt-sensitive hypertension. We hypothesized that RAS-mediated NO production is crucial for Ca 2+ control and cytoskeletal rearrangements in podocytes, contributing to glomerular filtration regulation. Methods: We performed live confocal imaging on human podocytes and freshly isolated rat glomeruli to detect the production of NO (DAF-FM) and Ca 2+ (Fluo-8). RAS peptides (Ang II, III, IV, 1-7, 1-9, and TRV120027) were used to stimulate cells, and NO and Ca 2+ production were measured. Blockers for Ang II Type 1 (AT1R), Ang II Type 2 (AT2R), and MAS receptors were used to identify the pathways involved in NO production. The dynamic changes in podocyte cytoskeleton mediated by RAS-NO intracellular signaling were detected using rhodamine-phalloidin staining and scanning ion microscopy. The NOS isoform-specific blockers were used to determine the source of NO in podocytes. ANOVA with post-hoc was used for comparisons. Results: Angiotensins II and III generate the most profound increases in NO levels in podocytes. Ang II, Ang III, and Ang 1-7 promote NO production by activating AT2R (n ≥ 20, p < 0.01). Interestingly, the increase in NO modulates AT1R- and β-arrestin-mediated Ca 2+ signaling (n ≥ 20, p < 0.05), suggesting the role of the NO/AT2R axis in Ca 2+ control in podocytes. NO production correlates with podocyte volume increase, and scanning ion microscopy experiments further confirmed that AT2R activation and corresponding NO elevation are responsible for the protrusion of podocyte's lamellipodia. Finally, we found that in podocytes, NOS1 is responsible for NO bioavailability, accounting for 69±5% of the total NO response, while the remaining NO levels are generated by NOS2 (39±5%). Conclusion: Our data show that RAS activation promotes NO production in podocytes, mainly via the AT2R pathway. NO is crucial for Ca 2+ control in podocytes and lamellipodia formation, suggesting the essential role of NO in glomerular filtration barrier function.

Renin-angiotensin system-mediated nitric oxide signaling in podocytes.

Nitric oxide (NO) is widely recognized for its role in regulating renal function and blood pressure. However, the precise mechanisms by which NO affects renal epithelial cells remain understudied. Our previous research has shown that NO signaling in glomerular podocytes can be initiated by Angiotensin II (ANG II) but not by ATP. This study aims to elucidate the crucial interplay between the renin-angiotensin system (RAS) and NO production in podocytes. To conduct our research, we used cultured human podocytes and freshly isolated rat glomeruli. A variety of RAS peptides were used, alongside confocal microscopy, to detect NO production and NO/Ca2+ cross talk. Dynamic changes in the podocyte cytoskeleton, mediated by RAS-NO intracellular signaling, were observed using fluorescent labeling for F-actin and scanning probe microscopy. The experiments demonstrated that ANG II and ANG III generated high levels of NO by activating the angiotensin II type 2 receptor (AT2R). We did not detect functional MAS receptor presence in podocytes, and the moderate NO response to ANG 1-7 was also mediated through AT2R. Furthermore, NO production impacted intracellular Ca2+ signaling and correlated with an increase in podocyte volume and growth. Scanning probe experiments revealed that AT2R activation and the corresponding NO generation are responsible for the protrusion of podocyte lamellipodia. Taken together, our data indicate that AT2R activation enhances NO production in podocytes and subsequently mediates changes in Ca2+ signaling and podocyte volume dynamics. These mechanisms may play a significant role in both physiological and pathophysiological interactions between the RAS and podocytes.NEW & NOTEWORTHY The renin-angiotensin system plays a crucial role in the production of intracellular nitric oxide within podocytes. This mechanism operates through the activation of the angiotensin II type 2 receptor, leading to dynamic modifications in intracellular calcium levels and the actin filament network. This intricate process is vital for linking the activity of angiotensin receptors to podocyte function.

The Immunosuppressive Drug LF15-0195 Acts Also on Glomerular Lesions, by a Change in Cytoskeleton Distribution in Podocyte

Introduction: Buffalo/Mna rats spontaneously develop nephrotic syndrome (NS) which recurs after renal transplantation. The immunosuppressive drug LF15-0195 can promote regression of the initial and post-transplantation nephropathy via induction of regulatory T cells. We investigate if this drug has an additional effect on the expression and localization of podocyte specific proteins. Methods: Buffalo/Mna kidney samples were collected before and after the occurrence of proteinuria, and after the remission of proteinuria induced by LF15-0195 treatment and compared by quantitative RT-PCR, Western blot, electron, and confocal microscopy to kidney samples of age-matched healthy rats. Cytoskeleton changes were assessed in culture by stress fibers induction by TNFα. Results: We observed, by electron microscopy, a restoration of foot process architecture in the LF15-0195-treated Buff/Mna kidneys, consistent with proteinuria remission. Nephrin, podocin, CD2AP, and α-actinin-4 mRNA levels remained low during the active disease in the Buff/Mna, in comparison with healthy rats which increase, while podocalyxin and synaptopodin transcripts were elevated before the occurrence of the disease but did not differ from healthy animals after. No difference in the mRNA and protein expression between the untreated and the LF15-0195-treated proteinuric Buff/Mna were seen for these 6 proteins. No changes were observed by confocal microscopy in the protein distribution at a cellular level, but a more homogenous distribution similar to healthy rats, was observed within the glomeruli of LF15-0195-treated rats. In addition, LF15-0195 could partially restore actin cytoskeleton of endothelial cells in TNFα-induced-cell stress experiment. Conclusion: The effect of LF15-0195 treatment appears to be mediated by 2 mechanisms: an immunomodulatory effect via regulatory T cells induction, described in our previous work and which can act on immune cell involved in the disease pathogenesis, and an effect on the restoration of podocyte cytoskeleton, independent of expression levels of the proteins involved in the slit diaphragm and podocyte function, showed in this article.

Chemotherapeutic Potential of Chlorambucil-Platinum(IV) Prodrugs against Cisplatin-Resistant Colorectal Cancer Cells.

Chlorambucil-platinum(IV) prodrugs exhibit multi-mechanistic chemotherapeutic activity with promising anticancer potential. The platinum(II) precursors of the prodrugs have been previously found to induce changes in the microtubule cytoskeleton, specifically actin and tubulin of HT29 colon cells, while chlorambucil alkylates the DNA. These prodrugs demonstrate significant anticancer activity in 2D cell and 3D spheroid viability assays. A notable production of reactive oxygen species has been observed in HT29 cells 72 h post treatment with prodrugs of this type, while the mitochondrial membrane potential was substantially reduced. The cellular uptake of the chlorambucil-platinum(IV) prodrugs, assessed by ICP-MS, confirmed that active transport was the primary uptake mechanism, with platinum localisation identified primarily in the cytoskeletal fraction. Apoptosis and necrosis were observed at 72 h of treatment as demonstrated by Annexin V-FITC/PI assay using flow cytometry. Immunofluorescence measured via confocal microscopy showed significant changes in actin and tubulin intensity and in architecture. Western blot analysis of intrinsic and extrinsic pathway apoptotic markers, microtubule cytoskeleton markers, cell proliferation markers, as well as autophagy markers were studied post 72 h of treatment. The proteomic profile was also studied with a total of 1859 HT29 proteins quantified by mass spectroscopy, with several dysregulated proteins. Network analysis revealed dysregulation in transcription, MAPK markers, microtubule-associated proteins and mitochondrial transport dysfunction. This study confirms that chlorambucil-platinum(IV) prodrugs are candidates with promising anticancer potential that act as multi-mechanistic chemotherapeutics.

A Novel Strategy for Glioblastoma Treatment by Natural Bioactive Molecules Showed a Highly Effective Anti-Cancer Potential.

Glioblastoma (GBM) is a severe form of brain tumor that has a high fatality rate. It grows aggressively and most of the time results in resistance to traditional treatments like chemo- and radiotherapy and surgery. Biodiversity, beyond representing a big resource for human well-being, provides several natural compounds that have shown great potential as anticancer drugs. Many of them are being extensively researched and significantly slow GBM progression by reducing the proliferation rate, migration, and inflammation and also by modulating oxidative stress. Here, the use of some natural compounds, such as Allium lusitanicum, Succisa pratensis, and Dianthus superbus, was explored to tackle GBM; they showed their impact on cell number reduction, which was partially given by cell cycle quiescence. Furthermore, a reduced cell migration ability was reported, accomplished by morphological cytoskeleton changes, which even highlighted a mesenchymal-epithelial transition. Furthermore, metabolic studies showed an induced cell oxidative stress modulation and a massive metabolic rearrangement. Therefore, a new therapeutic option was suggested to overcome the limitations of conventional treatments and thereby improve patient outcomes.

Communicating pain: emerging axonal signaling in peripheral neuropathic pain.

Peripheral nerve damage often leads to the onset of neuropathic pain (NeuP). This condition afflicts millions of people, significantly burdening healthcare systems and putting strain on families' financial well-being. Here, we will focus on the role of peripheral sensory neurons, specifically the Dorsal Root Ganglia neurons (DRG neurons) in the development of NeuP. After axotomy, DRG neurons activate regenerative signals of axons-soma communication to promote a gene program that activates an axonal branching and elongation processes. The results of a neuronal morphological cytoskeleton change are not always associated with functional recovery. Moreover, any axonal miss-targeting may contribute to NeuP development. In this review, we will explore the epidemiology of NeuP and its molecular causes at the level of the peripheral nervous system and the target organs, with major focus on the neuronal cross-talk between intrinsic and extrinsic factors. Specifically, we will describe how failures in the neuronal regenerative program can exacerbate NeuP.

Comparative cytotoxicity of Acanthamoeba castellanii-derived conditioned medium on human corneal epithelial and stromal cells

Soluble factors in the secretome of Acanthamoeba castellanii play crucial roles in the pathogenesis of Acanthamoeba keratitis (AK). Investigating the pathological effects of A. castellanii-derived conditioned medium (ACCM) on ocular cells can provide insights into the damage inflicted during AK. This study examined ACCM-induced cytotoxicity in primary human corneal stromal cells (CSCs) and a human SV40 immortalized corneal epithelial cell line (ihCECs) at varying ACCM concentrations (25 %, 50 %, 75 %, and 100 %). MTT, AlamarBlue, Sulforhodamine B, lactate dehydrogenase, and Caspase-3/7 activation assays were used to assess the impact of ACCM on the cell viability, proliferation and apoptosis. Additionally, fluorescent staining was used to reveal actin cytoskeleton changes. ACCM exposure significantly decreased cell viability, increased apoptosis, and disrupted the actin cytoskeleton, particularly at higher concentrations and longer exposures. Proteases were found to mediate these cytopathogenic effects, highlighting the need for characterization of A. castellanii proteases as key virulence factors in AK pathogenesis.

First person – David Velásquez-Carvajal

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. David Velásquez-Carvajal is first author on ‘ Microtubule reorganization during mitotic cell division in the dinoflagellate Ostreospis cf. ovata’, published in JCS. David conducted the research described in this article while a PhD student in Stefania Castagnetti's lab at Sorbonne Universités, CNRS, Villefranche-sur-Mer, France. He is now an Assistant Professor at Corporación Universitaria Remington, Medellín, Colombia. He has an interest in two related topics: understanding cytoskeleton changes and its modulation during neuroplasticity, and understanding how cells react to ‘expanded states of consciousness’ induced by psychedelics, and how these states impact changes in the cytoskeleton.

Slow Sulfide Donor GYY4137 Increased the Sensitivity of Two Breast Cancer Cell Lines to Paclitaxel by Different Mechanisms.

Paclitaxel (PTX) is a chemotherapeutic agent affecting microtubule polymerization. The efficacy of PTX depends on the type of tumor, and its improvement would be beneficial in patients' treatment. Therefore, we tested the effect of slow sulfide donor GYY4137 on paclitaxel sensitivity in two different breast cancer cell lines, MDA-MB-231, derived from a triple negative cell line, and JIMT1, which overexpresses HER2 and is resistant to trastuzumab. In JIMT1 and MDA-MB-231 cells, we compared IC50 and some metabolic (apoptosis induction, lactate/pyruvate conversion, production of reactive oxygen species, etc.), morphologic (changes in cytoskeleton), and functional (migration, angiogenesis) parameters for PTX and PTX/GYY4137, aiming to determine the mechanism of the sensitization of PTX. We observed improved sensitivity to paclitaxel in the presence of GYY4137 in both cell lines, but also some differences in apoptosis induction and pyruvate/lactate conversion between these cells. In MDA-MB-231 cells, GYY4137 increased apoptosis without affecting the IP3R1 protein, changing the morphology of the cytoskeleton. A mechanism of PTX sensitization by GYY4137 in JIMT1 cells is distinct from MDA-MB-231, and remains to be further elucidated. We suggest different mechanisms of action for H2S on the paclitaxel treatment of MDA-MB-231 and JIMT1 breast cancer cell lines.

Microtubule-induced restriction of nuclear deformability in hypertrophic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Leducq foundation Background Hypertrophic cardiomyopathy (HCM) is characterized by abnormal thickening of the left ventricular (LV) wall in conjunction with diastolic dysfunction with a prevalence of ~1:200. HCM pathophysiology is incompletely understood. We hypothesized that misregulated mechanotransduction plays an important role in the disease process. Extensive cytoskeletal remodeling has been observed in HCM patients, with a robust increase in tubulin and desmin levels, with a concomitant increase in stabilizing post-translational modifications, which we previously showed contributes to diastolic dysfunction. Microtubules are connected to the nucleus by the LINC complex. Little is known about the interaction of the non-sarcomeric cytoskeleton with the nucleus and the role this may have in the development of HCM. Purpose Relate cytoskeleton changes to nuclear and chromatin morphology in HCM patient myocardium and assess nuclear deformability in contracting cardiomyocytes with and without microtubule destabilizing drugs. Methods Nuclear morphology and chromatin organization was assessed using electron microscopy in cardiac septal tissue from 19 patients with obstructive HCM and compared to samples from 4 non-failing donors (NF). Further nuclear changes were studied using wild type (WT) and homozygous Mybpc3 c.2373InsG mice (Mybpc3c.2373InsG, KI), which have a HCM phenotype. Nuclear morphology was assessed in slices of 6 WT and 6 Mybpc3c.2373InsG paraffin-embedded mouse hearts. To study nuclear deformability during contractions, adult cardiomyocytes were isolated, stained with live cell dyes and imaged live while electrically paced. Lastly, DNA damaged was assessed in 6 WT and 6 Mybpc3c.2373InsG paraffin-embedded mouse hearts. Results Cardiomyocyte nuclei of HCM patients had a significantly higher number of invaginations (0.32±0.09 invaginations/µm in HCM versus 0.18±0.03 invaginations/µm in NF hearts), were more irregular of shape and had altered chromatin organization compared to those from NF hearts. Nuclei of Mybpc3c.2373InsG mice were considerably larger (322±75 µm³ versus 221±24 µm³). While sarcomere shortening is similar (7.0±1.4% in Mybpc3c.2373InsG versus 6.5±1.7% in WT), nuclei from Mybpc3c.2373InsG mice are less deformable than WT nuclei (figure). Addition of microtubule destabilizer nocodazole or detyrosination inhibitor EPO-Y restored nuclear deformability to WT levels, indicating a role of detyrosinated microtubules in nuclear deformability. In addition, nuclei from Mybpc3c.2373InsG have more extensive DNA damage (19.7±3.4 foci in Mybpc3c.2373InsG versus 9.1±3.2 foci in WT). Conclusion Extensive nuclear changes are observed in HCM patients and a HCM mouse model. Cytoskeletal remodeling results in less deformable nuclei that might contribute to chromatin alterations and increased DNA damage. Nuclear deformability can be restored by microtubule modifying drugs, specifically those that decrease microtubule detyrosination.

Endothelial cells in chronic thromboembolic pulmonary hypertension exhibit cytoskeletal alteration

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Health Research Fund (Fondo de Investigación Sanitaria [FIS]) from the Instituto de Salud Carlos III Introduction Endothelial dysfunction (ED) is pivotal in Chronic Thromboembolic Pulmonary Hypertension (CTEPH) due to structural and functional alterations in endothelial. Previous studies have shown the critical function of cytoskeletal elements, particularly actin microfilaments and microtubules, along with associated cell adhesion complexes, in the endothelial integrity. Nevertheless, precise alterations in the cytoskeletal elements of endothelial cells (ECs) in CTEPH patients remain unclear. Purpose This study aims to investigate the cytoskeleton changes in CTEPH-ECs compared to healthy ECs, characterizing morphological and structural differences. Objectives involve assessing microtubules and microfilaments expression, and their influence on ECs migratory capacity. Finally, our study aims to gain novel insights into the spatial organization of cytoskeleton components in CTEPH patients. Methods CTEPH-ECs were isolated from freshly resected pulmonary endarterectomy specimens of CTEPH patients, while human pulmonary artery ECs (HPAE) were used as healthy controls. Fluorescence immunocytochemistry assessed phenotypic and compositional characteristics of the cytoskeleton. Morphometric analysis described morphology and filament organization of cytoskeletal proteins using a custom-built pipeline. X-ray microscopy examined the phenotype and cytoskeletal components distribution. The migration assay evaluated how these structural differences affected ECs migratory capacity and the cytoskeletal elements expression. Results CTEPH-ECs exhibited larger surface area (1352 vs 662µm2, p<0.01) and a more stellate morphology (0.365 vs 0.289, p<0.01) than HPAE-ECs. Immunostaining indicated altered cytoskeletal composition in CTEPH-ECs with significantly higher tubulin expression, expressed in microtubules, (3327 vs 2238AU, p<0.01) and F-actin microfilaments (2068 vs 1300AU, p<0.01) compared to HPAE. Similarly, CTEPH-ECs showed reduced G-actin expression (3002 vs 7461AU, p<0.01). Morphometric analysis demonstrated that microfilaments were thinner (0.623 vs 0.831AU, p<0.01), more straight (0.135 vs. 0.172, p<0.01) and more aligned (0.132 vs. 0.156, p<0.01) in CTEPH-ECs. Microtubule thickness decreased compared to HPAE-ECs (0.341 vs 1.371AU, p<0.01). CTEPH-ECs showed impaired wound healing (60 vs 89% closure, p<0.05). CTEPH group showed an increase in microtubules and microfilaments expression during wound healing, but with irregular patterns, unlike the constant levels in HPAE. TXM-technology revealed spatial organization differences, especially expansive microtubules distribution in CTEPH-ECs (4.2*10^7 vs. 8.7*10^6 nm³, p<0.05). Conclusion These preliminary findings highlight significant structural and compositional differences in CTEPH-ECs, along with altered wound healing capacity, suggesting dynamic cytoskeletal and cellular changes in CTEPH. Understanding these alterations is crucial for advancing CTEPH knowledge and identifying potential therapeutic targets.