Actin Cytoskeletal Organization Research Articles

Protocol for the Extraction and Characterization of Trabecular Meshwork Cell Cytoskeleton Fraction.

Among various cellular attributes modulating aqueous humor (AH) outflow through the trabecular pathway and eventually intraocular pressure, the involvement of actomyosin regulated cellular contraction and relaxation, cell-extracellular matrix adhesion and cell-cell junctions, and mechanotransduction are well recognized. Although various biological and pharmacological agent-modulated AH outflows were associated with altered actin cytoskeletal organization and cell adhesive interactions of trabecular meshwork (TM) cells, these changes were analyzed largely with a biased approach to examine the specific proteins, but there were very few efforts in examining them with hypothesis-free unbiased approach in their native state. Therefore, in this chapter, we describe a protocol tailored to characterize the cytoskeleton of TM cells. This simple protocol can be applied to identifying the differentially regulated TM cell cytoskeleton proteins under any given treatment condition in conjunction with quantitative proteomic analysis.

Whole-transcriptome sequencing revealed the ceRNA regulatory network during the proliferation and differentiation of goose myoblast

The Shitou goose, the largest meat-type goose breed, is an ideal model for offering insights into enhancing meat production efficiency through understanding its genetic regulation of muscle development. Here, through whole-transcriptomic analysis of embryonic leg muscles, we identified 847 differentially expressed genes (DEG), 244 differentially expressed lncRNAs (DEL), 37 differentially expressed circRNAs (DEC), and 84 differentially expressed miRNAs (DEM). Gene ontology (GO) analysis highlighted the significant enrichment of differentially expressed RNAs in muscle structure development, actin filament-based processes, and the actin cytoskeleton pathway. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified pathways associated with the FoxO signaling pathway, AMPK signaling pathway, Wnt signaling pathway and calcium signaling pathway. Furthermore, we utilized Miranda, TargetScan, and miRDB to identify regulatory networks that involve interactions between lncRNA-mRNA, circRNA-mRNA, miRNA-mRNA, lncRNA-miRNA-mRNA, and circRNA-miRNA-mRNA, which regulated the growth and development of skeletal muscle. Notably, differentially expressed genes within the ceRNA network were most significantly enriched in the regulation of actin cytoskeletal organization. Additionally, a lncRNA/circRNA-miRNA-mRNA ceRNA network related to muscle growth and development was constructed based on protein-protein interaction (PPI) analysis and hub genes selection using Cytoscape. This further elucidated the regulatory roles of noncoding RNAs (ncRNA) in the formation of muscle fibers in Shitou goose. In summary, this study provides a valuable transcriptional regulatory network for goose muscle development laying the groundwork for further exploration of the molecular regulatory mechanisms underlying the excellent meat production performance of Shitou goose.

TAZ is involved in breast cancer cell migration via regulating actin dynamics.

Cancer metastasis is dependent on cell migration. Several mechanisms, including epithelial-to-mesenchymal transition (EMT) and actin fiber formation, could be involved in cancer cell migration. As a downstream effector of the Hippo signaling pathway, transcriptional coactivator with PDZ-binding motif (TAZ) is recognized as a key mediator of the metastatic ability of breast cancer cells. We aimed to examine whether TAZ affects the migration of breast cancer cells through the regulation of EMT or actin cytoskeleton. MCF-7 and MDA-MB-231 cells were treated with siRNA to attenuate TAZ abundance. Transwell migration assay and scratch wound healing assay were performed to study the effects of TAZ knockdown on cancer cell migration. Fluorescence microscopy was conducted to examine the vinculin and phalloidin. Semiquantitative immunoblotting and quantitative real-time PCR were performed to study the expression of small GTPases and kinases. Changes in the expression of genes associated with cell migration were examined through next-generation sequencing. TAZ-siRNA treatment reduced TAZ abundance in MCF-7 and MDA-MB-231 breast cancer cells, which was associated with a significant decrease in cell migration. TAZ knockdown increased the expression of fibronectin, but it did not exhibit the typical pattern of EMT progression. TGF-β treatment in MDA-MB-231 cells resulted in a reduction in TAZ and an increase in fibronectin levels. However, it paradoxically promoted cell migration, suggesting that EMT is unlikely to be involved in the decreased migration of breast cancer cells in response to TAZ suppression. RhoA, a small Rho GTPase protein, was significantly reduced in response to TAZ knockdown. This caused a decrease in the expression of the Rho-dependent downstream pathway, i.e., LIM kinase 1 (LIMK1), phosphorylated LIMK1/2, and phosphorylated cofilin, leading to actin depolymerization. Furthermore, myosin light chain kinase (MLCK) and phosphorylated MLC2 were significantly decreased in MDA-MB-231 cells with TAZ knockdown, inhibiting the assembly of stress fibers and focal adhesions. TAZ knockdown inhibits the migration of breast cancer cells by regulating the intracellular actin cytoskeletal organization. This is achieved, in part, by reducing the abundance of RhoA and Rho-dependent downstream kinase proteins, which results in actin depolymerization and the disassembly of stress fibers and focal adhesions.

Evaluating Physiological and Organizational Properties of the Tight Junction ZO-1 ZU-5 Domain in MDCK Cells

Zonula occludens-1 (ZO-1) is an essential TJ component contributing to junctional formation by mediating interactions between junctional proteins and the actomyosin cytoskeleton. MDCK II cells, a model epithelial cell line, with ZO-1 knocked-out (ZO-1 KO) have observable phenotypic changes such as alterations in the actin cytoskeletal organization and physiological barrier properties. This study focuses on the ZO-1-ZU5 domain by rescuing ZO-1 with full-length and a panel of ZO-1-ZU5 mutants. ZO-1-ZU-5 is known to bind cingulin and through this interaction ZO-1 adopts an extended conformation. We examined actomyosin cytoskeleton organization, ZO-1 mobility, gene expression and barrier function to determine whether the ZO-1-ZU5 mutants rescue full-length activity. We found that the ZO-1- ZU5 truncation mutant cell line rescued transepithelial electrical resistance (TER) but did not rescue tight junction distribution or the amount of total gamma-actin. We will present evaluation of additional ZU5 mutants. This study expands our understanding of how extended ZO-1 regulates the mechanical, genetic, and phenotypic properties of epithelial cells. Trinity University Biology Summer Undergraduate Research Fellowship. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dopamine receptor D2 confers colonization resistance via microbial metabolites.

The gut microbiome has major roles in modulating host physiology. One such function is colonization resistance, or the ability of the microbial collective to protect the host against enteric pathogens1-3, including enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7, an attaching and effacing (AE) food-borne pathogen that causes severe gastroenteritis, enterocolitis, bloody diarrhea and acute renal failure4,5 (haemolytic uremic syndrome). Although gut microorganisms can provide colonization resistance by outcompeting some pathogens or modulating host defence provided by the gut barrier and intestinal immune cells6,7, this phenomenon remains poorly understood. Here, we show that activation of the neurotransmitter receptor dopamine receptor D2 (DRD2) in the intestinal epithelium by gut microbial metabolites produced upon dietary supplementation with the essential amino acid L-tryptophan protects the host against Citrobacter rodentium, a mouse AE pathogen that is widely used as a model for EHEC infection8,9. We further find that DRD2 activation by these tryptophan-derived metabolites decreases expression of a host actin regulatory protein involved in C. rodentium and EHEC attachment to the gut epithelium via formation of actin pedestals. Our results reveal a noncanonical colonization resistance pathway against AE pathogens that features an unconventional role for DRD2 outside the nervous system in controlling actin cytoskeletal organization in the gut epithelium. Our findings may inspire prophylactic and therapeutic approaches targeting DRD2 with dietary or pharmacological interventions to improve gut health and treat gastrointestinal infections, which afflict millions globally.

Influence of the calcium voltage-gated channel auxiliary subunit (CACNA2D1) absence on intraocular pressure in mice

The etiology of elevated intraocular pressure (IOP), a major risk factor for glaucoma (optic nerve atrophy), is poorly understood despite continued efforts. Although the gene variant of CACNA2D1 (encoding α2δ1), a calcium voltage-gated channel auxiliary subunit, has been reported to be associated with primary open-angle glaucoma, and the pharmacological mitigation of α2δ1 activity by pregabalin lowers IOP, the cellular basis for α2δ1 role in the modulation of IOP remains unclear. Our recent findings reveled readily detectable levels of α2δ1 and its ligand thrombospondin in the cytoskeletome fraction of human trabecular meshwork (TM) cells. To understand the direct role of α2δ1 in the modulation of IOP, we evaluated α2δ1 null mice for changes in IOP and found a moderate (∼10%) but significant decrease in IOP compared to littermate wild type control mice. Additionally, to gain cellular insights into α2δ1 antagonist (pregabalin) induced IOP changes, we assessed pregabalin's effects on human TM cell actin cytoskeletal organization and cell adhesive interactions in comparison with a Rho kinase inhibitor (Y27632), a known ocular hypotensive agent. Unlike Y27632, pregabalin did not have overt effects on cell morphology, actin cytoskeletal organization, or cell adhesion in human TM cells. These results reveal a modest but significant decrease in IOP in α2δ1 deficient mice, and this response appears to be not associated with the contractile and cell adhesive characteristics of TM cells based on the findings of pregabalin effects on isolated TM cells. Therefore, the mechanism by which pregabalin lowers IOP remains elusive.

Alteration of actin cytoskeletal organisation in fetal akinesia deformation sequence

Fetal akinesia deformation sequence (FADS) represents the severest form of congenital myasthenic syndrome (CMS), a diverse group of inherited disorders characterised by impaired neuromuscular transmission. Most CMS originate from defects in the muscle nicotinic acetylcholine receptor, but the underlying molecular pathogenesis is only poorly understood. Here we show that RNAi-mediated silencing of FADS-related proteins rapsyn and NUP88 in foetal fibroblasts alters organisation of the actin cytoskeleton. We show that fibroblasts from two independent FADS individuals have enhanced and shorter actin stress fibre bundles, alongside with an increased number and size of focal adhesions, with an otherwise normal overall connectivity and integrity of the actin-myosin cytoskeleton network. By proximity ligation assays and bimolecular fluorescence complementation, we show that rapsyn and NUP88 localise nearby adhesion plaques and that they interact with the focal adhesion protein paxillin. Based on these findings we propose that a respective deficiency in rapsyn and NUP88 in FADS alters the regulation of actin dynamics at focal adhesions, and thereby may also plausibly dictate myofibril contraction in skeletal muscle of FADS individuals.

Investigating the influence of titanium particle size and concentration on osteogenic response of human osteoblasts - in vitro study.

The purpose of this study was to investigate the correlation between the size and concentration of titanium particles and the osteogenic response of human osteoblasts (HOB). Different concentrations of titanium dioxide nano- and micro-particles were prepared and their biocompatibility on HOBs was analyzed using XTT assay. The changes in the actin cytoskeletal organization were studied by confocal laser scanning microscopy. The generation of intracellular reactive oxygen species (ROS) by HOBs after exposure to titanium dioxide particles was analyzed using ROS assay. Besides, the osteogenic potential represented by alkaline phosphatase activity, osteoprotegerin, macrophage colony stimulating factor levels, and biomineralization were analyzed. Short-term interaction of titanium dioxide nano- and micro-particles did not induce toxicity to HOBs. However, cells treated with 100 μg/mL titanium dioxide nano- and micro-particles demonstrated higher ROS generation compared to control. Besides, cells treated with 100 μg/mL titanium dioxide nanoparticles showed higher alkaline phosphatase activity, osteoprotegerin, macrophage colony stimulating factor levels and biomineralization compared to titanium dioxide microparticles. Collectively, the study found titanium dioxide nanoparticles to be more biocompatible than microparticles providing an insight into the capability of nanostructures in supporting osteoblast differentiation and its plausibility in biomedical applications.

Recombinant human secretory leukocyte protease inhibitor (rhSLPI) coated titanium enhanced human osteoblast adhesion and differentiation

Osseointegration is vital to success in orthopedic and dental reconstructions with implanted materials. The bone matrix or cells—particularly osteoblasts—are required to achieve functional contact on the implant surface. Osteoblast induction is therefore essential for osteogenesis to occur. Enhancement of osteoblast adhesion, proliferation, and differentiation, particularly by implant surface modifications, have been found challenging to develop. Secretory Leukocyte Protease Inhibitor (SLPI), a cation ionic protein with anti-inflammatory and anti-bacterial activities, showed activation in osteoblast proliferation and differentiation. However, the effects of coating recombinant human (rh) SLPI on a titanium alloy surface on human osteoblast adhesion, proliferation, and differentiation has never been investigated. In this study, titanium alloys (Ti–6Al–4V) were coated with rhSLPI, while human osteoblast adhesion, proliferation, differentiation, actin cytoskeletal organization, and gene expressions involved in cell adhesion and differentiation were investigated. The results indicate that coating titanium with 10–100 µg/ml rhSLPI enhanced the physical properties of the Ti surface and enhanced human osteoblast (hFOB 1.19) cell adhesion, activated actin dynamic, enhanced adhesive forces, upregulated integrins α1, α2, and α5, enhanced cell proliferation, mineralization, alkaline phosphatase activity, and upregulated ALP, OCN, and Runx2. This is the first study to demonstrate that coating SLPI on titanium surfaces enhances osseointegration and could be a candidate molecule for surface modification in medical implants.

Segregated localization of two calponin-related proteins within sarcomeric thin filaments in Caenorhabditis elegans striated muscle.

The calponin family proteins are expressed in both muscle and non-muscle cells and involved in the regulation of cytoskeletal dynamics and cell contractility. In the nematode Caenorhabditis elegans, UNC-87 and CLIK-1 are calponin-related proteins with 42% identical amino acid sequences containing seven calponin-like motifs. Genetic studies demonstrated that UNC-87 and CLIK-1 have partially redundant function in regulating actin cytoskeletal organization in striated and non-striated muscle cells. However, biochemical studies showed that UNC-87 and CLIK-1 are different in their ability to bundle actin filaments. In this study, I extended comparison between UNC-87 and CLIK-1 and found additional differences in vitro and in vivo. Although UNC-87 and CLIK-1 bound to actin filaments similarly, UNC-87, but not CLIK-1, bound to myosin and inhibited actomyosin ATPase in vitro. In striated muscle, UNC-87 and CLIK-1 were segregated into different subregions within sarcomeric actin filaments. CLIK-1 was concentrated near the actin pointed ends, whereas UNC-87 was enriched toward the actin barbed ends. Restricted localization of UNC-87 was not altered in a clik-1-null mutant, suggesting that their segregated localization is not due to competition between the two related proteins. These results suggest that the two calponin-related proteins have both common and distinct roles in regulating actin filaments.

A genome‑wide approach to the systematic and comprehensive analysis of LIM gene family in sorghum (Sorghum bicolor L.).

The LIM domain-containing proteins are dominantly found in plants and play a significant role in various biological processes such as gene transcription as well as actin cytoskeletal organization. Nevertheless, genome-wide identification as well as functional analysis of the LIM gene family have not yet been reported in the economically important plant sorghum (Sorghum bicolor L.). Therefore, we conducted an in silico identification and characterization of LIM genes in S. bicolor genome using integrated bioinformatics approaches. Based on phylogenetic tree analysis and conserved domain, we identified five LIM genes in S. bicolor (SbLIM) genome corresponding to Arabidopsis LIM (AtLIM) genes. The conserved domain, motif as well as gene structure analyses of the SbLIM gene family showed the similarity within the SbLIM and AtLIM members. The gene ontology (GO) enrichment study revealed that the candidate LIM genes are directly involved in cytoskeletal organization and various other important biological as well as molecular pathways. Some important families of regulating transcription factors such as ERF, MYB, WRKY, NAC, bZIP, C2H2, Dof, and G2-like were detected by analyzing their interaction network with identified SbLIM genes. The cis-acting regulatory elements related to predicted SbLIM genes were identified as responsive to light, hormones, stress, and other functions. The present study will provide valuable useful information about LIM genes in sorghum which would pave the way for the future study of functional pathways of candidate SbLIM genes as well as their regulatory factors in wet-lab experiments.

Indoloquinazoline alkaloids suppress angiogenesis and inhibit metastasis of melanoma cells

Metastasis is the leading cause of cancer-related mortality, targeting angiogenesis emerges as a therapeutic strategy for the treatment of melanoma metastasis. Discovery of new antiangiogenic compounds with specific mechanism of action is still desired. In present study, a bioassay-guidance uncovers the EtOAc extract of a marine-derived fungus Aspergillus clavutus LZD32-24 with significant inhibitory activity against the angiogenesis in Tg (fli1a: EGFP) zebrafish model. Extensive chromatographic fractionation led to the isolation of 48 indoloquinazoline alkaloids, including 21 new analogues namely clavutoines A-U (1–21). Their structures were determined by the spectroscopic data, including the ECD, single crystal X-ray diffraction and quantum chemical calculation for the configurational assignments. Among the bioactive analogues, quinadoline B (QB) showed the most efficacy to suppress the zebrafish vascular outgrowth in zebrafish embryos. QB markedly inhibited the migration, invasion and tube formation with weak cytotoxicity in human umbilical vein endothelial cells (HUVECs). Investigation of the mode of action revealed QB suppressed the ROCK/MYPT1/MLC2/coffin and FAK /Src signaling pathways, and subsequently disrupted actin cytoskeletal organization. In addition, QB reduced the number of new vessels sprouting from the ex vivo chick chorioallantoic membrane (CAM), and inhibited the metastasis of B16F10 melanoma cells in lung of C57BL/6 mice through suppressing angiogenesis. These findings suggest that QB is a potential lead for the development of new antiangiogenic agent to inhibit melanoma metastasis.

The myosin and RhoGAP MYO9B influences osteocyte dendrite growth and responses to mechanical stimuli.

Introduction: Myosin IXB (MYO9B) is an unconventional myosin with RhoGAP activity and thus is a regulator of actin cytoskeletal organization. MYO9B was previously shown to be necessary for skeletal growth and health and to play a role in actin-based functions of both osteoblasts and osteoclasts. However, its role in responses to mechanical stimulation of bone cells has not yet been described. Therefore, experiments were undertaken to determine the role of MYO9B in bone cell responses to mechanical stress both in vitro and in vivo. Methods: MYO9B expression was knocked down in osteoblast and osteocyte cell lines using RNA interference and the resulting cells were subjected to mechanical stresses including cyclic tensile strain, fluid shear stress, and plating on different substrates (no substrate vs. monomeric or polymerized collagen type I). Osteocytic cells were also subjected to MYO9B regulation through Slit-Robo signaling. Further, wild-type or Myo9b -/- mice were subjected to a regimen of whole-body vibration (WBV) and changes in bone quality were assessed by micro-CT. Results: Unlike control cells, MYO9B-deficient osteoblastic cells subjected to uniaxial cyclic tensile strain were unable to orient their actin stress fibers perpendicular to the strain. Osteocytic cells in which MYO9B was knocked down exhibited elongated dendrites but were unable to respond normally to treatments that increase dendrite length such as fluid shear stress and Slit-Robo signaling. Osteocytic responses to mechanical stimuli were also found to be dependent on the polymerization state of collagen type I substrates. Wild-type mice responded to WBV with increased bone tissue mineral density values while Myo9b -/- mice responded with bone loss. Discussion: These results demonstrate that MYO9B plays a key role in mechanical stress-induced responses of bone cells in vitro and in vivo.

Diagnostic yield and novel candidate genes for neurodevelopmental disorders by exome sequencing in an unselected cohort with microcephaly

ObjectivesMicrocephaly is caused by reduced brain volume and most usually associated with a variety of neurodevelopmental disorders (NDDs). To provide an overview of the diagnostic yield of whole exome sequencing (WES) and promote novel candidates in genetically unsolved families, we studied the clinical and genetic landscape of an unselected Chinese cohort of patients with microcephaly.MethodsWe performed WES in an unselected cohort of 103 NDDs patients with microcephaly as one of the features. Full evaluation of potential novel candidate genes was applied in genetically undiagnosed families. Functional validations of selected variants were conducted in cultured cells. To augment the discovery of novel candidates, we queried our genomic sequencing data repository for additional likely disease-causing variants in the identified candidate genes.ResultsIn 65 families (63.1%), causative sequence variants (SVs) and clinically relevant copy number variants (CNVs) with a pathogenic or likely pathogenic (P/LP) level were identified. By incorporating coverage analysis to WES, a pathogenic or likely pathogenic CNV was detected in 15 families (16/103, 15.5%). In another eight families (8/103, 7.8%), we identified variants in newly reported gene (CCND2) and potential novel neurodevelopmental disorders /microcephaly candidate genes, which involved in cell cycle and division (PWP2, CCND2), CDC42/RAC signaling related actin cytoskeletal organization (DOCK9, RHOF), neurogenesis (ELAVL3, PPP1R9B, KCNH3) and transcription regulation (IRF2BP1). By looking into our data repository of 5066 families with NDDs, we identified additional two cases with variants in DOCK9 and PPP1R9B, respectively.ConclusionOur results expand the morbid genome of monogenic neurodevelopmental disorders and support the adoption of WES as a first-tier test for individuals with microcephaly.

Targeting Rho kinase to restore endothelial barrier function following vascular scaffold implantation

Vascular scaffold implantation induces injury to the intimal layer and causes discontinuity of the regenerated endothelial monolayer, compromising barrier integrity, increasing permeability, and allowing the transmigration of leukocytes and lipoproteins into the subendothelial space. Mechanical vascular wall stretching triggers Ras homolog family member A (RhoA)/Rho kinase-mediated actomyosin contractility and destabilization of adherens junctions, leading to endothelial barrier dysfunction. Assembly of intercellular adhesion and actin cytoskeletal organization of interendothelial junctions are controlled by downregulation of RhoA guanosine triphosphatase (GTPase)-mediated barrier-disruptive activity and upregulation of repressor-activator protein 1 (Rap1) and Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase-mediated cytoskeletal reorganization, leading to endothelial barrier stabilization. This review highlights the involvement of Rho GTPases in the disruption of endothelial barrier integrity following vascular scaffold implantation and the targeting of downstream Rho-associated protein kinases, which signal the network to restore endothelial barrier integrity and stability.

Simvastatin Attenuates Glucocorticoid-Induced Human Trabecular Meshwork Cell Dysfunction via YAP/TAZ Inactivation

Purpose Impairment of the trabecular meshwork (TM) is the principal cause of increased outflow resistance in the glaucomatous eye. Yes-associated protein (YAP) and transcriptional coactivator with PDZ binding motif (TAZ) are emerging as potential mediators of TM cell/tissue dysfunction. Furthermore, YAP/TAZ activity was recently found to be controlled by the mevalonate pathway in non-ocular cells. Clinically used statins block the mevalonate cascade and were shown to improve TM cell pathobiology; yet, the link to YAP/TAZ signaling was not investigated. In this study, we hypothesized that simvastatin attenuates glucocorticoid-induced human TM (HTM) cell dysfunction via YAP/TAZ inactivation. Methods Primary HTM cells were seeded atop or encapsulated within bioengineered extracellular matrix (ECM) hydrogels. Dexamethasone was used to induce a pathologic phenotype in HTM cells in the absence or presence of simvastatin. Changes in YAP/TAZ activity, actin cytoskeletal organization, phospho-myosin light chain levels, hydrogel contraction/stiffness, and fibronectin deposition were assessed. Results Simvastatin potently blocked pathologic YAP/TAZ nuclear localization/activity, actin stress fiber formation, and myosin light chain phosphorylation in HTM cells. Importantly, simvastatin co-treatment significantly attenuated dexamethasone-induced ECM contraction/stiffening and fibronectin mRNA and protein levels. Sequential treatment was similarly effective but did not match clinically-used Rho kinase inhibition. Conclusions YAP/TAZ inactivation with simvastatin attenuates HTM cell pathobiology in a tissue-mimetic ECM microenvironment. Our data may help explain the association of statin use with a reduced risk of developing glaucoma via indirect YAP/TAZ inhibition as a proposed regulatory mechanism.

Abstract 454: The Effect Of High-fat Diet On Vascular Smooth Muscle Cell Mechanics And Migration In Apoe -/- Mice

Atherosclerosis remains a leading cause of cardiovascular diseases, which cause causes about 30% of global death. Vascular smooth muscle cells (VSMCs) are the major cellular components of arterial wall. VMSCs play critical role in the atherosclerosis development by phenotypic switching from a contractile phenotype to a synthetic phenotype, migration from media to intima of arterial wall, and deposition as foam cells in atherosclerotic plaque. In this study, we tested the effect of high-fat diet on the VSMC mechanics by comparing the mechanics and migration of VSMCs isolated from apolipoprotein-E knockout (ApoE -/- ) and wild type (WT) mice. Atomic force microscope (AFM) was employed to test cell adhesion and stiffness of VSMCs. AFM was also used to examine live VSMC submembranous cytoskeleton organization. A data-driven mathematical models-based image and signal processing software was used to analyze AFM data. Two-way ANOVA was used to test statistical significance for all experiments. The results demonstrated significant differences in the actin cytoskeletal organization and cell mechanics between ApoE -/- VSMCs and WT cells upon high-fat diet. However, no significant difference in cell stiffness was observed between normal diet-fed ApoE -/- and WT VSMCs. These interesting findings on the effect of high-fat diet on VSMC mechanics will improve our knowledge of the mechanism underlying the contribution of VSMC in the development of atherosclerosis.

MP03-14 INHIBITION OF HUMAN DETRUSOR SMOOTH MUSCLE CELL GROWTH AND MODULATION OF CYTOSKELETAL ORGANIZATION BY IMMUNO-MODULATORY IMIDE DRUGS (IMIDS)

You have accessJournal of UrologyCME1 Apr 2023MP03-14 INHIBITION OF HUMAN DETRUSOR SMOOTH MUSCLE CELL GROWTH AND MODULATION OF CYTOSKELETAL ORGANIZATION BY IMMUNO-MODULATORY IMIDE DRUGS (IMIDS) Alexander Tamalunas, Amin Wendt, Florian Springer, Ru Huang, Ruixiao Wang, Yuhan Liu, Beata Rutz, Anna Ciotkowska, Giuseppe Magistro, Christian Stief, and Martin Hennenberg Alexander TamalunasAlexander Tamalunas More articles by this author , Amin WendtAmin Wendt More articles by this author , Florian SpringerFlorian Springer More articles by this author , Ru HuangRu Huang More articles by this author , Ruixiao WangRuixiao Wang More articles by this author , Yuhan LiuYuhan Liu More articles by this author , Beata RutzBeata Rutz More articles by this author , Anna CiotkowskaAnna Ciotkowska More articles by this author , Giuseppe MagistroGiuseppe Magistro More articles by this author , Christian StiefChristian Stief More articles by this author , and Martin HennenbergMartin Hennenberg More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003214.14AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: While lower urinary tract symptoms (LUTS) consist of voiding (BPH) and storage disorders (OAB), new AUA and EAU guidelines take into account the mounting number of patients suffering from both, so called “mixed LUTS”. Recently, we could show that IMiDs (thalidomide, lenalidomide and pomalidomide) inhibit prostate smooth muscle contraction, modulate cytoskeletal actin organization, and reduce prostate stromal cell growth at the same time, without showing cytotoxic effects. Based on these promising data, we now investigated the effects of IMiDs on cellular functions, including cytoskeletal organization, and growth in bladder cells. METHODS: Experiments were carried out in an immortalized line of cultured human bladder detrusor smooth muscle cells (HBdSMC). Cytoskeletal organization was visualized by phalloidin staining, while cell growth was assessed using an EdU and cell colony assay. Cell viability was quantified in CCK8 assay, and FACS. RESULTS: IMiDs ([A] thalidomide 10-100 µM, [B] lenalidomide 5-30 µM, and [C] pomalidomide 2.5-10 µM) significantly reduced the number of viable WPMY-1 cells in a concentration- and time-dependent manner (Figure 1). Correspondingly, proliferation of WPMY-1 cells was significantly reduced in a concentration-dependent manner (Figure 2 and 3), without showing pro-apoptotic effects (Figure 4). In parallel, IMiDs induced cytoskeletal disorganization: while the cellular shape of control cells was characterized by many long and thin protrusions containing bundles of actin filaments, this structure collapsed after treatment with IMiDs (Figure 5). CONCLUSIONS: IMiDs impair human detrusor smooth muscle growth, which is paralleled by a breakdown of the cytoskeleton, which may inhibit exaggerated bladder smooth muscle contraction in OAB. Urodynamic effects in vivo and a possible application in LUTS appear possible. Together with our previous data, this may suggest a possible novel drug class in LUTS treatment. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e27 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alexander Tamalunas More articles by this author Amin Wendt More articles by this author Florian Springer More articles by this author Ru Huang More articles by this author Ruixiao Wang More articles by this author Yuhan Liu More articles by this author Beata Rutz More articles by this author Anna Ciotkowska More articles by this author Giuseppe Magistro More articles by this author Christian Stief More articles by this author Martin Hennenberg More articles by this author Expand All Advertisement PDF downloadLoading ...

Abstract PR007: Physical and metabolic aspects of therapy induced senescence and polyploidy in an evolving tumor microenvironment

Abstract Radiation and chemotherapy are highly effective at killing cancer cells but cells that survive treatment often develop therapy-induced senescence (TIS). Since growth is arrested in TIS, this has been considered a positive treatment outcome; however, senescent cells that remain metabolically active and develop a senescence associated secretory phenotype (SASP) can also promote cancer progression. In addition, a small number of cancer cells are able to escape this dormant state and contribute to cancer progression. Polyploid giant cancer cells (PGCCs) represent a small subpopulation of non-mitotic cancer cells that evades treatment through periods of transient dormancy and then relapses into full-blown disease through amitotic budding into chemoresistant progenitor cells. The PGCC phenotype mimics senescent cells in multiple ways, including enlarged size, increased beta-galactosidase expression, increased metabolic activity, and pro-inflammatory SASP; yet, there is a significant gap in our understanding of how PGCCs contribute to TIS escape and subsequent chemoresistance. In addition, increased numbers of large PGCCs are seen in late stage and metastatic cancers; yet, their role in tumor recurrence and metastasis has not been established. We previously showed PGCCs have a unique actin cytoskeletal organization, giving rise to elevated stiffness and migratory persistence. These findings prompted us to further examine vimentin intermediate filaments (VIFs) that act as shock absorbers in the cell, protecting cells from compressive loads. Based on this, we postulated that PGCCs have unique adaptations in their vimentin structure to help support their enlarged morphology and drive their persistent migration. Indeed, we showed that PGCCs have increased levels of cytoplasmic vimentin and evenly distributed VIFs compared to non-PGCCs. This dispersed network of VIFs often polarized at the leading-edge during migration, and was necessary for PGCCs phenotype, as disruption of VIFs decreased PGCC volume and blocked migratory persistence. The VIF network also scaffolds lysosomes and autophagosomes in the cytosol to regulate their fusion during autophagy. Our new data clearly shows that inhibiting autophagy with Bafilomycin limits PGCC migration. In addition, targeting VIF structure also blocks autophagy. Thus, the structure of VIFs and autophagic flux are both critical in directing PGCCs migratory persistence. We have developed a novel 3D tumor microenvironment model that allows us to monitor cancer cell behavior over 4-6 weeks, which allows us to monitor cell recovery from TIS and dormancy. Using this model, we show that PGCC’s unique biophysical properties are directly linked to their dysregulated metabolism and altered cell structure. These studies provide critical information about how aging, TIS, and polyploidy affect evolving tumor microenvironments. Citation Format: Michelle R. Dawson, Deepraj Ghosh. Physical and metabolic aspects of therapy induced senescence and polyploidy in an evolving tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr PR007.

Sec14-like-Lipid-Binding-4 Enables Hemoglobin Clearance By Liver Sinusoidal Endothelial Cells

Hemolysis of banked red blood cells (RBC storage lesion) contributes to lack of therapeutic benefit and higher mortality following transfusion therapy in patients. However, the molecular mechanism underlying storage lesion remains poorly understood. Using a multiple-ancestry genome-wide association analysis among blood donors, our group has recently identified Sec14-like-lipid-binding-4 (Sec14L4) protein to be associated with increased rate of hemolysis following blood storage. Although Sec14L4 is highly similar to yeast phosphatidylinositol transfer protein Sec14, which is known to be involved in the synthesis of golgi-derived transport vesicles, the exact function of Sec14L4 and how it promotes RBC storage lesions remains unknown. Our initial studies using fluorescence microscopy in our newly generated Sec14L4 null mice revealed significant enrichment of Sec14L4 in the live sinusoidal endothelial cells (LSECs), which positively correlated with elevated levels of iron in the liver. Scanning and transmission electron micrographs revealed impaired actin cytoskeleton organization in LSECs of Sec14L4 null mice, which was associated with upregulation of major GTPases RhoA, RhoB and Rac1 activity. The impairment of actin cytoskeletal organization in Sec14L4 null mice lead to impaired vesicle trafficking in LSECs and higher retention of RBCs in the liver sinusoids compared to control mice. These data are the first to demonstrate a previously unrecognized role for Sec14L4 protein in LSEC mediated endocytic trafficking by promoting actin polymerization. These findings also suggest that Sec14L4 may regulate RBC storage lesion through impaired hemoglobin clearance by LSECs secondary to cytoskeletal impairment.