Stabilization Of Cytoskeleton Research Articles

Investigation of the impact of bromodomain inhibition on cytoskeleton stability and contraction

BackgroundInjury to contractile organs such as the heart, vasculature, urinary bladder and gut can stimulate a pathological response that results in loss of normal contractility. PDGF and TGFβ are among the most well studied initiators of the injury response and have been shown to induce aberrant contraction in mechanically active cells of hollow organs including smooth muscle cells (SMC) and fibroblasts. However, the mechanisms driving contractile alterations downstream of PDGF and TGFβ in SMC and fibroblasts are incompletely understood, limiting therapeutic interventions.MethodsTo identify potential molecular targets, we have leveraged the analysis of publicly available data, comparing transcriptomic changes in mechanically active cells stimulated with PDGF and TGFβ. Additional Analysis of publicly available data sets were performed on SMC and fibroblasts treated in the presence or absence of the MYC inhibitor JQ1. Validation of in silico findings were performed with qPCR, immunoblots, and collagen gel contraction assays measure the effect of JQ1 on cytoskeleton associated genes, proteins and contractility in mechanically active cells. Likelihood ratio test and FDR adjusted p-values were used to determine significant differentially expressed genes. Student ttest were used to calculate statistical significance of qPCR and contractility analyses.ResultsComparing PDGF and TGFβ stimulated SMC and fibroblasts identified a shared molecular profile regulated by MYC and members of the AP-1 transcription factor complex. Additional in silico analysis revealed a unique set of cytoskeleton-associated genes that were sensitive to MYC inhibition with JQ1. In vitro validation demonstrated JQ1 was also able to attenuate TGFβ and PDGF induced changes to the cytoskeleton and contraction of smooth muscle cells and fibroblasts in vitro.ConclusionsThese findings identify MYC as a key driver of aberrant cytoskeletal and contractile changes in fibroblasts and SMC, and suggest that JQ1 could be used to restore normal contractile function in hollow organs.

Role of BicDR in bristle shaft construction and support of BicD functions.

Cell polarization requires asymmetric localization of numerous mRNAs, proteins and organelles. The movement of cargo towards the minus end of microtubules mostly depends on cytoplasmic dynein motors. In the dynein-dynactin-Bicaudal-D transport machinery, Bicaudal-D (BicD) links the cargo to the motor. Here, we focus on the role of Drosophila BicD-related (BicDR, CG32137) in the development of the long bristles. Together with BicD, it contributes to the organization and stability of the actin cytoskeleton in the not-yet-chitinized bristle shaft. BicD and BicDR also support the stable expression and distribution of Rab6 and Spn-F in the bristle shaft, including the distal tip localization of Spn-F, pointing to the role of microtubule-dependent vesicle trafficking for bristle construction. BicDR supports the function of BicD, and we discuss the hypothesis whereby BicDR might transport cargo more locally, with BicD transporting cargo over long distances, such as to the distal tip. We also identified embryonic proteins that interact with BicDR and appear to be BicDR cargo. For one of them, EF1γ (also known as eEF1γ), we show that the encoding gene EF1γ interacts with BicD and BicDR in the construction of the bristles.

Photoresponsive peptide materials: Spatiotemporal control of self-assembly and biological functions.

Peptides work as both functional molecules to modulate various biological phenomena and self-assembling artificial materials. The introduction of photoresponsive units to peptides allows the spatiotemporal remote control of their structure and function upon light irradiation. This article overviews the photoresponsive peptide design, interaction with biomolecules, and applications in self-assembling materials over the last 30 years. Peptides modified with photochromic (photoisomerizable) molecules, such as azobenzene and spiropyran, reversibly photo-controlled the binding to biomolecules and nanostructure formation through self-assembly. Photocleavable molecular units irreversibly control the functions of peptides through cleavage of the main chain and deprotection by light. Photocrosslinking between peptides or between peptides and other biomolecules enhances the structural stability of peptide assemblies and complexes. These photoresponsive peptides spatiotemporally controlled the formation and dissociation of peptide assemblies, gene expressions, protein-drug interactions, protein-protein interactions, liposome deformation and motility, cytoskeleton structure and stability, and cell functions by appropriate light irradiation. These molecular systems can be applied to photo-control biological functions, molecular robots, artificial cells, and next-generation smart drug delivery materials.

Microgravity as an Anti-Metastatic Agent in an In Vitro Glioma Model

Gravity is a primary physical force that has a profound influence on the stability of the cell cytoskeleton. In our research, we investigated the influence of microgravity on altering the cytoskeletal pathways of glioblastoma cells. The highly infiltrative behavior of glioblastoma is supported by cytoskeletal dynamics and surface proteins that allow glioblastoma cells to avoid stable connections with the tissue environment and other cells. Glioblastoma cell line C6 was exposed to a microgravity environment for 24, 48, and 72 h by 3D-RPM, a laboratory instrument recognized to reproduce the effect of microgravity in cell cultures. The immunofluorescence for GFAP, vinculin, and Connexin-43 was investigated as signals related to cytoskeleton dynamics. The polymerization of GFAP and the expression of focal contact structured by vinculin were found to be altered, especially after 48 and 72 h of microgravity. Connexin-43, involved in several intracellular pathways that critically promote cell motility and invasion of glioma cells, was found to be largely reduced following microgravity exposure. In conclusion, microgravity, by reducing the expression of Connexin-43, alters the architecture of specific cytoskeletal elements such as GFAP and increases the focal contact, which can induce a reduction in glioma cell mobility, thereby inhibiting their aggressive metastatic behavior.

Basolateral amygdala corticotropin releasing factor receptor 2 interacts with nonmuscle myosin II to destabilize memory in males

Preclinical studies show that inhibiting the actin motor ATPase nonmuscle myosin II (NMII) with blebbistatin (Blebb) in the basolateral amgydala (BLA) depolymerizes actin, resulting in an immediate, retrieval-independent disruption of methamphetamine (METH)-associated memory in male and female adult and adolescent rodents. The effect is highly selective, as NMII inhibition has no effect in other relevant brain regions (e.g., dorsal hippocampus [dPHC], nucleus accumbens [NAc]), nor does it interfere with associations for other aversive or appetitive stimuli, including cocaine (COC). To understand the mechanisms responsible for drug specific selectivity we began by investigating, in male mice, the pharmacokinetic differences in METH and COC brain exposure . Replicating METH’s longer half-life with COC did not render the COC association susceptible to disruption by NMII inhibition. Therefore, we next assessed transcriptional differences. Comparative RNA-seq profiling in the BLA, dHPC and NAc following METH or COC conditioning identified crhr2, which encodes the corticotropin releasing factor receptor 2 (CRF2), as uniquely upregulated by METH in the BLA. CRF2 antagonism with Astressin-2B (AS2B) had no effect on METH-associated memory after consolidation, allowing for determination of CRF2 influences on NMII-based susceptibility. Pretreatment with AS2B prevented the ability of Blebb to disrupt an established METH-associated memory. Alternatively, combining CRF2 overexpression and agonist treatment, urocortin 3 (UCN3), in the BLA during conditioning rendered COC-associated memory susceptible to disruption by NMII inhibition, mimicking the Blebb-induced, retrieval-independent memory disruption seen with METH. These results suggest that BLA CRF2 receptor activation during memory formation in male mice can prevent stabilization of the actin-myosin cytoskeleton supporting the memory, rendering it vulnerable to disruption by NMII inhibition. CRF2 represents an interesting target for BLA-dependent memory destabilization via downstream effects on NMII.

Levamisole Modulation of Podocytes' Actin Cytoskeleton in Nephrotic Syndrome.

Podocytes play a central role in glomerular diseases such as (idiopathic) nephrotic syndrome (iNS). Glucocorticoids are the gold standard therapy for iNS. Nevertheless, frequent relapses are common. In children with iNS, steroid-sparing agents are used to avoid prolonged steroid use and reduce steroid toxicity. Levamisole is one of these steroid-sparing drugs and although clinical effectiveness has been demonstrated, the molecular mechanisms of how levamisole exerts its beneficial effects remains poorly studied. Apart from immunomodulatory capacities, nonimmunological effects of levamisole on podocytes have also been suggested. We aimed to elaborate on the effects of levamisole on human podocytes in iNS. RNA sequencing data from a human podocyte cell line treated with levamisole showed that levamisole modulates the expression of various genes involved in actin cytoskeleton stabilization and remodeling. Functional experiments showed that podocytes exposed to puromycin aminonucleoside (PAN), lipopolysaccharides (LPS), and NS patient plasma resulted in significant actin cytoskeleton derangement, reduced cell motility, and impaired cellular adhesion when compared to controls, effects that could be restored by levamisole. Mechanistic studies revealed that levamisole exerts its beneficial effects on podocytes by signaling through the glucocorticoid receptor and by regulating the activity of Rho GTPases. In summary, our data show that levamisole exerts beneficial effects on podocytes by stabilizing the actin cytoskeleton in a glucocorticoid receptor-dependent manner.

A Haspin-ARHGAP11A axis regulates epithelial morphogenesis through Rho-ROCK dependent modulation of LIMK1-Cofilin

Throughout mitosis, a plethora of processes must be efficiently concerted to ensure cell proliferation and tissue functionality. The mitotic spindle does not only mediate chromosome segregation, but also defines the axis of cellular division, thus determining tissue morphology. Functional spindle orientation relies on precise actin dynamics, shaped in mitosis by the LIMK1-Cofilin axis. The kinase Haspin acts as a guardian of faithful chromosome segregation that ensures amphitelic chromosome attachment and prevents unscheduled cohesin cleavage. Here, we report an unprecedented role for Haspin in the determination of spindle orientation in mitosis. We show that, during mitosis, Haspin regulates Rho-ROCK activity through ARHGAP11A, a poorly characterized GAP, and that ROCK is in turn responsible for the mitotic activation of LIMK1 and stabilization of the actin cytoskeleton, thus supporting a functional spindle orientation. By exploiting 3D cell cultures, we show that this pathway is pivotal for the establishment of a morphologically functional tissue.

Autophagy regulation and protein kinase activity of PIK3C3 controls sertoli cell polarity through its negative regulation on SCIN (scinderin)

ABSTRACT Sertoli cells are highly polarized testicular cells that provide a nurturing environment for germ cell development and maturation during spermatogenesis. The class III phosphatidylinositol 3-kinase (PtdIns3K) plays core roles in macroautophagy in various cell types; however, its role in Sertoli cells remains unclear. Here, we generated a mouse line in which the gene encoding the catalytic subunit, Pik3c3, was specifically deleted in Sertoli cells (cKO) and found that after one round of normal spermatogenesis, the cKO mice quickly became infertile and showed disruption of Sertoli cell polarity and impaired spermiogenesis. Subsequent proteomics and phosphoproteomics analyses enriched the F-actin cytoskeleton network involved in the disorganized Sertoli-cell structure in cKO testis which we identified a significant increase of the F-actin negative regulator SCIN (scinderin) and the reduced phosphorylation of HDAC6, an α-tubulin deacetylase. Our results further demonstrated that the accumulation of SCIN in cKO Sertoli cells caused the disorder and disassembly of the F-actin cytoskeleton, which was related to the failure of SCIN degradation through the autophagy-lysosome pathway. Additionally, we found that the phosphorylation of HDAC6 at site S59 by PIK3C3 was essential for its degradation through the ubiquitin-proteasome pathway. As a result, the HDAC6 that accumulated in cKO Sertoli cells deacetylated SCIN at site K189 and led to a disorganized F-actin cytoskeleton. Taken together, our findings elucidate a new mechanism for PIK3C3 in maintaining the polarity of Sertoli cells, in which both its autophagy regulation or protein kinase activities are required for the stabilization of the actin cytoskeleton. Abbreviations: ACTB: actin, beta; AR: androgen receptor; ATG14: autophagy related 14; BafA1: bafilomycin A1; BECN1: beclin 1, autophagy related; BTB: blood-testis barrier; CASP3: caspase 3; CDC42: cell division cycle 42; CDH2: cadherin 2; CHX: cycloheximide; CTNNA1: catenin (cadherin associated protein), alpha 1; CYP11A1: cytochrome P450, family 11, subfamily A, polypeptide 1; EBSS: Earle’s balanced salt solution; ES: ectoplasmic specialization; FITC: fluorescein isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCNA: germ cell nuclear acidic protein; GJA1: gap junction protein, alpha 1; H2AX: H2A.X variant histone; HDAC6: histone deacetylase 6; KIT: KIT proto-oncogene, receptor tyrosine kinase; LAMP1: lysosomal associated membrane protein 1; MAP3K5: mitogen-activated protein kinase kinase kinase 5; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; OCLN: occludin; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4: phosphoinositide-3-kinase regulatory subunit 4; PNA: arachis hypogaea lectin; RAC1: Rac family small GTPase 1; SCIN: scinderin; SQSTM1/p62: sequestosome 1; SSC: spermatogonia stem cell; STK11: serine/threonine kinase 11; TJP1: tight junction protein 1; TubA: tubastatin A; TUBB3: tubulin beta 3 class III; TUNEL: TdT-mediated dUTP nick-end labeling; UB: ubiquitin; UVRAG: UV radiation resistance associated gene; VIM: vimentin; WT1: WT1 transcription factor; ZBTB16: zinc finger and BTB domain containing 16.

Galactosylation of xyloglucan is essential for the stabilization of the actin cytoskeleton and endomembrane system through the proper assembly of cell walls.

Xyloglucan, a major hemicellulose, interacts with cellulose and pectin to assemble primary cell walls in plants. Loss of the xyloglucan galactosyltransferase MURUS3 (MUR3) leads to the deficiency of galactosylated xyloglucan and perturbs plant growth. However, it is unclear whether defects in xyloglucan galactosylation influence the synthesis of other wall polysaccharides, cell wall integrity, cytoskeleton behaviour, and endomembrane homeostasis. Here, we found that in mur3-7 etiolated seedlings cellulose was reduced, CELLULOSE SYNTHASE (CESA) genes were down-regulated, the density and mobility of cellulose synthase complexes (CSCs) were decreased, and cellulose microfibrils become discontinuous. Pectin, rhamnogalacturonan II (RGII), and boron contents were reduced in mur3-7 plants, and B-RGII cross-linking was abnormal. Wall porosity and thickness were significantly increased in mur3-7 seedlings. Endomembrane aggregation was also apparent in the mur3-7 mutant. Furthermore, mutant seedlings and their actin filaments were more sensitive to Latrunculin A (LatA) treatment. However, all defects in mur3-7 mutants were substantially restored by exogenous boric acid application. Our study reveals the importance of MUR3-mediated xyloglucan galactosylation for cell wall structural assembly and homeostasis, which is required for the stabilization of the actin cytoskeleton and the endomembrane system.

404-P: Single-Cell RNA Profiling Changes in Glomerular Cells in Diabetic Nephropathy (DN)

Hyperglycemia, the main risk factor for DN, causes complex pathological damages in glomeruli. To characterize detail changes in cell differentiation and gene expressions induced by diabetes, we performed unbiased single-cell RNA sequencing (scRNA-seq) on glomerular cells from diabetic and nondiabetic mice of long duration. Glomerular cells were isolated from C57 mice after 7-months of STZ-induced diabetes, when clear glomerular pathologies have been documented. Total number of 48,337 high-qualitied glomerular cells were isolated and sequenced from WT and STZ-treated mice. Based on clustering analysis and cell-type specific markers, 12 different cell types were identified, including endothelial cells (EC), podocytes, mesangial cells (MC), proximal tubule cells, parietal epithelial cells (PEC), collecting duct cells, ascending loop of Henle, macrophages, B cells, T cells, NK cells and neutrophils. Over 90% of the cells were from glomeruli (41% EC, 34 % podocytes, 14% MC, 2% of PEC), and 6% immune cells. Interestingly, gene profiling identified 9 clusters of EC, 5 clusters of MC and 7 clusters of podocytes. Diabetes did not change the total cell numbers for EC, MC and podocytes, although macrophage and B cell numbers were increased from 2.2% to 4.8% and 0.35% to 1%, respectively. Cluster subset analysis of EC, MC and podocytes showed that diabetes induced substantial changes. For podocytes, diabetes increased those clusters (5% to 37%) with high expressions of stressed proteins such as Hspa1a, Hspa1b and Hsp90aa1, while decreased those clusters with high expressions of ECM production and CDC42 GTPase cycle, that were involved in cytoskeleton stabilization and foot process formation. Findings from scRNA-seq of glomeruli showed there were multiple subsets of podocytes, EC and MC, which appear to have different functions and their distributions were changed by diabetes. Understanding the mechanism for the distribution changes of the subsets may provide new insight in the pathogenesis of DN. Disclosure J. Fu: None. Q. Li: None. K. Park: None. Q. Huang: None. I. Wu: None. G.L. King: Research Support; Janssen Research & Development, LLC.

#2519 THE EFFECT OF TRPC5 AND PALLADIN EXPRESSION ON TREATMENT RESPONSE AND RENAL OUTCOME IN FOCAL SEGMENTAL GLOMERULOSCLEROSIS

Abstract Background and Aims Focal segmental glomerulosclerosis (FSGS) is a disease that is among the primary glomerulopathies and often progresses to chronic renal failure. Despite advances in immunosuppressive therapy for the treatment of glomerular diseases, some FSGS patients are resistant to single or combination immunosuppressive regimens containing; glucocorticoids, cyclophosphamide, calcineurin inhibitors, mycophenolate mofetil (MMF), and/or rituximab. FSGS-related nephrotic syndrome, the degree of proteinuria and response to steroid therapy are the most important factors associated with long-term prognosis, independent of histological findings. Failure to respond to immunosuppressive therapy, including corticosteroids, has been evaluated as one of the most important markers of the risk of developing renal failure. Unfortunately, there is no reliable marker that can predict steroid response. One of the earliest manifestations of podocyte damage is cytoskeletal remodeling, known as deletion of foot ridges. It was recently shown that transient receptor potential channel 5 (TRPC5) plays an important role in initiating this process. However, the role of TRPC5 in disease progression is unknown. It is thought that calcium influx caused by sustained TRPC5 activation in the chronic disease setting may lead to calcium toxicity and podocyte death. It is known that the actin regulatory protein palladin, which is highly expressed here, is also highly related to podocyte function and podocytopathies, and plays an important role in the stability of the actin cytoskeleton. The aim of our study was to define the frequency of TRPC5 and palladin renal expressions in podocytes in focal segmental glomerulosclerosis (FSGS) and its relationship with prognosis and treatment response. Method A total of 182 patients were enrolled for baseline clinical and histopathological features and 103 patients with a clinical follow-up for more than 2 years were evaluated for outcomes. Immunohistochemical staining was performed with TRPC5 and palladin antibodies on kidney biopsies and glomerular staining was evaluated. Results Baseline characteristics, and health parameters were similar between TRPC5+/TRPC5− and palladin+/palladin− groups. TRPC5 expression was observed in 69% of the patient biopsies and palladin expression in 73%. We found that TRPC5 and palladin expression was a significant predictor of FSGS severity and poor treatment response (both p<0.05). In addition, TRPC5 expression was significantly associated with the development of end-stage renal disease and ongoing proteinuria. Conclusion The development of proven treatments that delay the progression of FSGS is critical to patient survival. The development of new treatments is possible only by knowing the underlying pathophysiological mechanism of the disease. Podocyte loss is a critical step in the development of irreversible glomerulosclerosis, which causes chronic kidney disease. Therefore, blocking TRPC5 channel and palladin may be an effective treatment strategy, especially in treatment-resistant FSGS patients with positive staining on biopsy. Our study is the first in the literature to show these two antigens in kidney tissue in FSGS and to associate them with disease prognosis. TRPC5 and palladin expression may serve as a biomarker to facilitate diagnosis and choose new treatment regimens of proteinuric kidney diseases in the future.

Defining TLR4-independent molecular pathways activated during endotoxemia

Abstract Sepsis is the leading cause of nosocomial mortality, accounting for almost 20% of annual global deaths. Patients who recover from septic shock maintain a significantly increased risk for mortality due to secondary infection. Toll-like receptor 4 (TLR4) recognizes Gram-negative bacterial lipopolysaccharides (LPS) and initiates robust immune activation that can be detrimental to the host. Towards the end of septicemia, cells self-regulate and remove surface TLR4 which contributes to immune ataxia. TLR4 deficient (TLR4 −/−) C57BL6 mice are used as a model for the immune refractory period following septic shock and compared to wildtype (WT) mice. In the serum of both WT and TLR4 −/−mice, 51 proteins were previously found to be dysregulated over the course of endotoxemia. We hypothesize that these 51 proteins represent TLR4 independent activation mechanisms. This study further investigates protein and mRNA distribution during endotoxemia. Liver, spleen, kidney, heart, and brain were collected at 0, 6, 12, and 18 hours post-intraperitoneal injection of 30 mg/kg E. coli LPS. Using qPCR and Western Blot to quantify gene and protein expression, we observed down regulation of genes involved with metabolism (Adsl, Mdh1) and erythrocyte cytoskeleton stabilization (Dmtn). In the same samples we observe activation of genes and proteins involved in T-cell function (Coro1a), and the complement cascades (Masp1, C1qb, Cfd, F5). These results further define TLR4 independent activation during endotoxemia as a model for immune ataxia that occurs after primary septic shock. This project could provide proof of principle to support the development of more targeted treatments to address immune activation during secondary infection after septic shock. This project was supported by a Towson University FDRC grant to EMH and a Fisher College of Science and Mathematics undergraduate research award to JCH, HA, PC, and AA.

Multi-omics Investigation of Freeze Tolerance in the Amur Sleeper, an Aquatic Ectothermic Vertebrate.

Freeze tolerance, the ability of an organism to survive internal ice formation, is a striking survival strategy employed by some ectotherms living in cold environments. However, the genetic bases of this remarkable adaptation are largely unknown. The Amur sleeper (Perccottus glenii), the only known freeze-tolerant fish species, can overwinter with its entire body frozen in ice. Here, we sequenced the chromosome-level genome of the Amur sleeper and performed comparative genomic, transcriptomic, and metabolomic analyses to investigate its strategies for surviving freezing. Evolutionary analysis suggested that the Amur sleeper diverged from its closest non-cold-hardy relative about 15.07 million years ago and has experienced a high rate of protein evolution. Transcriptomic and metabolomic data identified a coordinated and tissue-specific regulation of genes and metabolites involved in hypometabolism, cellular stress response, and cryoprotectant accumulation involved in freezing and thawing. Several genes show evidence of accelerated protein sequence evolution or family size expansion were found as adaptive responses to freezing-induced stresses. Specifically, genetic changes associated with cytoskeleton stability, cryoprotectant synthesis, transmembrane transport, and neuroprotective adaptations were identified as potentially key innovations that aid in freezing survival. Our work provides valuable resources and opportunities to unveil the molecular adaptations supporting freeze tolerance in ectothermic vertebrates.

Tubulin-mediated anatomical and functional changes caused by Ca2+ in human erythrocytes.

In previous research, we observed that tubulin can be found in three fractions within erythrocytes, i.e., attached to the membrane, as a soluble fraction, or as part of a structure that can be sedimented by centrifugation. Given that its differential distribution within these fractions may alter several hemorheological properties, such as erythrocyte deformability, the present work studied how this distribution is in turn affected by Ca2+, another key player in the regulation of erythrocyte cytoskeleton stability. The effect of Ca2+ on some hemorheological parameters was also assessed. The results showed that when Ca2+ concentrations increased in the cell, whether by the addition of ionophore A23187, by specific plasma membrane Ca2+_ATPase (PMCA) inhibition, or due to arterial hypertension, tubulin translocate to the membrane, erythrocyte deformability decreased, and phosphatidylserine exposure increased. Moreover, increased Ca2+ was associated with an inverse correlation in the distribution of tubulin and spectrin, another important cytoskeleton protein. Based on these findings, we propose the existence of a mechanism of action through which higher Ca2+ concentrations in erythrocytes trigger the migration of tubulin to the membrane, a phenomenon that results in alterations of rheological and molecular aspects of the membrane itself, as well as of the integrity of the cytoskeleton.

Comparison of Rheological Properties of Healthy versus Dupuytren Fibroblasts When Treated with a Cell Contraction Inhibitor by Atomic Force Microscope.

Mechanical properties of healthy and Dupuytren fibroblasts were investigated by atomic force microscopy (AFM). In addition to standard force curves, rheological properties were assessed using an oscillatory testing methodology, in which the frequency was swept from 1 Hz to 1 kHz, and data were analyzed using the structural damping model. Dupuytren fibroblasts showed larger apparent Young's modulus values than healthy ones, which is in agreement with previous results. Moreover, cell mechanics were compared before and after ML-7 treatment, which is a myosin light chain kinase inhibitor (MLCK) that reduces myosin activity and hence cell contraction. We employed two different concentrations of ML-7 inhibitor and could observe distinct cell reactions. At 1 µM, healthy and scar fibroblasts did not show measurable changes in stiffness, but Dupuytren fibroblasts displayed a softening and recovery after some time. When increasing ML-7 concentration (3 µM), the majority of cells reacted, Dupuytren fibroblasts were the most susceptible, not being able to recover from the drug and dying. These results suggested that ML-7 is a potent inhibitor for MLCK and that myosin II is essential for cytoskeleton stabilization and cell survival.

Pseudo-chromosome-length genome assembly for a deep-sea eel Ilyophis brunneus sheds light on the deep-sea adaptation.

High hydrostatic pressure, low temperature, and scarce food supply are the major factors that limit the survival of vertebrates in extreme deep-sea environments. Here, we constructed a high-quality genome of the deep-sea Muddy arrowtooth eel (MAE, Ilyophis brunneus, captured below a depth of 3,500 m) by using Illumina, PacBio, and Hi-C sequencing. We compare it against those of shallow-water eel and other outgroups to explore the genetic basis that underlies the adaptive evolution to deep-sea biomes. The MAE genome was estimated to be 1.47 Gb and assembled into 14 pseudo-chromosomes. Phylogenetic analyses indicated that MAE diverged from its closely related shallow-sea species, European eel, ∼111.9 Mya and experienced a rapid evolution. The genome evolutionary analyses primarily revealed the following: (i) under high hydrostatic pressure, the positively selected gene TUBGCP3 and the expanded family MLC1 may improve the cytoskeleton stability; ACOX1 may enhance the fluidity of cell membrane and maintain transport activity; the expansion of ABCC12 gene family may enhance the integrity of DNA; (ii) positively selected HARS likely maintain the transcription ability at low temperatures; and (iii) energy metabolism under a food-limited environment may be increased by expanded and positively selected genes in AMPK and mTOR signaling pathways.

Environmental microplastic accumulation exacerbates liver ischemia-reperfusion injury in rat: Protective effects of melatonin

Ischemia-reperfusion (IR) injury is an inevitable complication of liver transplantation and partial hepatectomy. Although the hazards of environmental microplastics (EMPs) have been well explored, data underlying their impact on IR-induced hepatotoxicity and how to alleviate these damages remain largely undefined. In this study, the involvement of melatonin (MT) in modulating EMPs toxicity in the liver undergoing ischemia-reperfusion injury was investigated. Male Wistar rats were exposed to MPs for 7 days and then subjected to 1 h of partial warm ischemia (70 %) followed by 24 h of reperfusion. We analyzed some parameters as the oxidative stress, the stability of cytoskeleton as well as inflammation, and autophagy. Our data suggested that EMPs elicited liver injury in ischemic animals. Data revealed several histological alterations caused by EMP and IRI, including cellular disorientation, cell necrosis, and microvacuolar steatosis, as well as inflammatory cell infiltration. EMPs increased blood transaminase (AST and ALT) and oxidative stress levels in the ischemic liver. In addition, RT-qPCR, immunofluorescence, and western blot analyses highlighted an increased expression of α-tubulin, IL-18, NFkB, and LC3. However, the ability of MT to reduce MPs and IRI toxicity was consistent with a significant decrease in the evaluated markers. The combined data not only document that melatonin is an effective agent to protect against hepatic IRI but also reduces cellular dysfunction caused by EMPs.

Intestinal Apc-inactivation induces HSP25 dependency.

The majority of colorectal cancers (CRCs) present with early mutations in tumor suppressor gene APC. APC mutations result in oncogenic activation of the Wnt pathway, which is associated with hyperproliferation, cytoskeletal remodeling, and a global increase in mRNA translation. To compensate for the increased biosynthetic demand, cancer cells critically depend on protein chaperones to maintain proteostasis, although their function in CRC remains largely unexplored. In order to investigate the role of molecular chaperones in driving CRC initiation, we captured the transcriptomic profiles of murine wild type and Apc-mutant organoids during active transformation. We discovered a strong transcriptional upregulation of Hspb1, which encodes small heat shock protein 25 (HSP25). We reveal an indispensable role for HSP25 in facilitating Apc-driven transformation, using both invitro organoid cultures and mouse models, and demonstrate that chemical inhibition of HSP25 using brivudine reduces the development of premalignant adenomas. These findings uncover a hitherto unknown vulnerability in intestinal transformation that could be exploited for the development of chemopreventive strategies in high-risk individuals.

Age-dependent increase of cytoskeletal components in sensory axons in human skin.

Aging is a complex process characterized by several molecular and cellular imbalances. The composition and stability of the neuronal cytoskeleton is essential for the maintenance of homeostasis, especially in long neurites. Using human skin biopsies containing sensory axons from a cohort of healthy individuals, we investigate alterations in cytoskeletal content and sensory axon caliber during aging via quantitative immunostainings. Cytoskeletal components show an increase with aging in both sexes, while elevation in axon diameter is only evident in males. Transcriptomic data from aging males illustrate various patterns in gene expression during aging. Together, the data suggest gender-specific changes during aging in peripheral sensory axons, possibly influencing cytoskeletal functionality and axonal caliber. These changes may cumulatively increase susceptibility of aged individuals to neurodegenerative diseases.

TRIOBP-1 Protein Aggregation Exists in Both Major Depressive Disorder and Schizophrenia, and Can Occur through Two Distinct Regions of the Protein.

The presence of proteinopathy, the accumulation of specific proteins as aggregates in neurons, is an emerging aspect of the pathology of schizophrenia and other major mental illnesses. Among the initial proteins implicated in forming such aggregates in these conditions is Trio and F-actin Binding Protein isoform 1 (TRIOBP-1), a ubiquitously expressed protein involved in the stabilization of the actin cytoskeleton. Here we investigate the insolubility of TRIOBP-1, as an indicator of aggregation, in brain samples from 25 schizophrenia patients, 25 major depressive disorder patients and 50 control individuals (anterior cingulate cortex, BA23). Strikingly, insoluble TRIOBP-1 is considerably more prevalent in both of these conditions than in controls, further implicating TRIOBP-1 aggregation in schizophrenia and indicating a role in major depressive disorder. These results were only seen using a high stringency insolubility assay (previously used to study DISC1 and other proteins), but not a lower stringency assay that would be expected to also detect functional, actin-bound TRIOBP-1. Previously, we have also determined that a region of 25 amino acids in the center of this protein is critical for its ability to form aggregates. Here we attempt to refine this further, through the expression of various truncated mutant TRIOBP-1 vectors in neuroblastoma cells and examining their aggregation. In this way, it was possible to narrow down the aggregation-critical region of TRIOBP-1 to just 8 amino acids (333–340 of the 652 amino acid-long TRIOBP-1). Surprisingly our results suggested that a second section of TRIOBP-1 is also capable of independently inducing aggregation: the optionally expressed 59 amino acids at the extreme N-terminus of the protein. As a result, the 597 amino acid long version of TRIOBP-1 (also referred to as “Tara” or “TAP68”) has reduced potential to form aggregates. The presence of insoluble TRIOBP-1 in brain samples from patients, combined with insight into the mechanism of aggregation of TRIOBP-1 and generation of an aggregation-resistant mutant TRIOBP-1 that lacks both these regions, will be of significant use in further investigating the mechanism and consequences of TRIOBP-1 aggregation in major mental illness.