Cytoskeleton Organization Research Articles

SLMAP3 is essential for neurulation through mechanisms involving cytoskeletal elements, ABP, and PCP.

SLMAP3 is a tail-anchored membrane protein that targets subcellular organelles and is believed to regulate Hippo signaling. The global loss of SLMAP3 causes late embryonic lethality in mice, with some embryos exhibiting neural tube defects such as craniorachischisis. We show here that SLMAP3 -/- embryos display reduced length and increased width of neural plates, signifying arrested convergent extension. The expression of planar cell polarity (PCP) components Dvl2/3 and the activity of the downstream targets ROCK2, cofilin, and JNK1/2 were dysregulated in SLMAP3 -/- E12.5 brains. Furthermore, the cytoskeletal proteins (γ-tubulin, actin, and nestin) and apical components (PKCζ and ZO-1) were mislocalized in neural tubes of SLMAP3 -/- embryos, with a subsequent decrease in colocalization of PCP proteins (Fzd6 and pDvl2). However, no changes in PCP or cytoskeleton proteins were found in cultured neuroepithelial cells depleted of SLMAP3, suggesting an essential requirement for SLMAP3 for these processes in vivo for neurulation. The loss of SLMAP3 had no impact on Hippo signaling in SLMAP3 -/- embryos, brains, and neural tubes. Proteomic analysis revealed SLMAP3 in an interactome with cytoskeletal components, including nestin, tropomyosin 4, intermediate filaments, plectin, the PCP protein SCRIB, and STRIPAK members in embryonic brains. These results reveal a crucial role of SLMAP3 in neural tube development by regulating the cytoskeleton organization and PCP pathway.

Abstract 4113617: Single-cell Analysis Reveals Endothelial Cell CD45 Deficiency Prevents Endothelial-to-Mesenchymal Transition (EndMT) in Atherosclerosis

Introduction: Protein-tyrosine-phosphatase CD45 is exclusively expressed in all nucleated cells of the hematopoietic system but is rarely expressed in endothelial cells (ECs). However, a recent study indicated that activation of the endogenous CD45 promoter in human endothelial colony forming cells induced expression of EndMT marker genes. Also, the CD45 phosphatase inhibitor blocked EndMT activities in TGFβ1-treated ovine mitral valve endothelial cells. EndMT contributes to atherosclerotic pathobiology and is associated with more complex plaques. We identified CD45-positive ECs undergoing EndMT in atherosclerotic ApoE-deficient (ApoE -/- ) mice. Here, we aim to investigate whether endothelial CD45-specific deletion can prevent EndMT in a mouse model of atherosclerosis. Methods and Results: We generated a tamoxifen-inducible EC-specific CD45-deficient mouse strain (EC-iCD45KO) on an ApoE -/- background (C57BL/6) and fed these mice a Western diet for 16 weeks. We isolated and enriched mouse aortic endothelial cells with CD31 beads to perform single-cell RNA-seq. Cells populated 15 major clusters after integrating the single-cell transcriptomic profiles. In the EC-iCD45KO group, the population of endothelial, resident macrophage, and mesenchymal-endothelial transition cells increased while vascular smooth muscle, EndMT, and fibroblast cells decreased. In EndMT cells, EC markers (Cdh5, Cldn5, Pecam1) increased, but SMC markers (Acta2) and EndMT markers (Col1a2 and Col3a1）decreased. In addition, we characterized EndMT cells into two sub-clusters: 1 and 2. EC-specific CD45 deletions reduced the cell numbers in the EndMT2 sub-cluster in atherosclerotic mice but had less impact on the EndMT1 sub-cluster. FGFR pathways were potentiated while TGFβ1 and Tgfbr2 decreased in EndMT1 sub-cluster， and the genes controlling endothelial cell development were upregulated but the genes controlling actin cytoskeleton organization were downregulated in both EndMT1 and 2 sub-clusters. Conclusion: Our single-cell analysis indicates that the loss of endothelial CD45 protects against EndMT-driven atherosclerosis, inhibiting TGFβ and EndoMT marker expression while stimulating FGFR pathways. Consistently, EC-iCD45KO/ApoE -/- mice showed reduced plaque macrophages, expression of cell adhesion molecules, foam cell formation, and lesion development when compared to ApoE -/- controls. Thus, targeting endothelial CD45 may be a novel therapeutic strategy for EndMT and atherosclerosis.

ANGI-14. DRUGGABLE GENOME CRISPR SCREEN IN 3D HYDROGEL DEVICES REVEALS TARGETABLE KINASES AND PHOSPHATASES IMPLICATED IN CYTOSKELETAL REORGANIZATION IN INVADING GBM CELLS

Abstract The invasive nature of glioblastoma cells into adjacent brain tissues is a key factor in its unfavorable prognosis. To decipher the mechanisms driving glioblastoma invasion, we developed microdissectable biomimetic 3D hydrogel invasion devices containing hyaluronic acid hydrogels decorated with integrin-binding peptides (RGD) and crosslinked with protease-cleavable crosslinkers. The ability to disassemble these devices and interrogate cells in the core vs. invasive fraction allows for high throughput screens. We thus performed a screen in GBM43 cells expressing CRISPR/Cas9 and a 13,250 gRNA library targeting the 2550 genes in the druggable human genome. Of seven initial hits from this screen, only two could be validated by single gene CRISPR and use of a drug to target the hit - ACP1 (also known as LMW-PTP) and Aurora Kinase B (AURKB). Integration of ChIP-seq and RNA-seq revealed no plausible transcriptional targets of ACP1 and AURKB that could drive invasion. Proximity labeling with the TurboID system identified CTTN (cortactin), an actin binding protein whose phosphorylation is integral to the formation of lamellipodia and invadopodia, as phosphorylated by AURKB and Ephrin A2 (EPHA2), whose dephosphorylation controls cell morphology, adhesion, migration and invasion by modifying the organization of the actin cytoskeleton and influencing the activities of integrins and intercellular adhesion molecules, as dephosphorylated by ACP1. These findings underscore the delicate balance between kinase and phosphatase activities in regulating the actin cytoskeleton broadly and at the cell’s front or “leading edge,” offering new therapeutic targets to disrupt the invasion that is a hallmark of glioblastoma.

Exploring the Role of Rho Kinase Enzyme in the Management of Metabolic Syndrome

Rho kinase [ROCK] enzymes are increasingly recognized for their central role in the pathogenesis of metabolic syndrome [MetS], a cluster of conditions that includes insulin resistance, hypertension, obesity, and dyslipidemia. ROCKs are serine/threonine kinases involved in the regulation of various cellular functions, including smooth muscle contraction, actin cytoskeleton organization, and gene expression. These enzymes are critically implicated in the cardiovascular and metabolic abnormalities that characterize MetS. Elevated ROCK activity has been observed in individuals with MetS, contributing to several pathogenic processes such as endothelial dysfunction, vascular inflammation, oxidative stress, and increased vascular smooth muscle contraction. These mechanisms are key drivers of hypertension and atherosclerosis, which are common complications associated with MetS. Moreover, ROCKs influence adipocyte differentiation and lipid metabolism, linking them directly to obesity and insulin resistance, two core components of the syndrome. The inhibition of ROCKs has emerged as a promising therapeutic strategy for managing MetS. Pharmacological ROCK inhibitors have shown the potential to improve insulin sensitivity, lower blood pressure, and reduce vascular inflammation and remodelling. In addition, by targeting the multiple pathways involved in the development and progression of MetS, ROCK inhibitors offer a comprehensive approach to treatment that addresses the syndrome's multifactorial nature. This therapeutic strategy not only mitigates the metabolic and cardiovascular components of the syndrome but also lowers the risk of associated complications, such as cardiovascular disease and stroke. This review concluded that interrupted Rho kinase activity contributes to the development of MetS in all its manifestations. Overall, these side effects diminish the Rho-kinase method's promise as a novel and significant treatment component.

Purinergic signaling through the P2Y2 receptor regulates osteocytes' mechanosensitivity.

Osteocytes' response to dynamic loading plays a crucial role in regulating the bone mass but quickly becomes saturated such that downstream induction of bone formation plateaus. The underlying mechanisms that downregulate osteocytes' sensitivity and overall response to loading remain unknown. In other cell types, purinergic signaling through the P2Y2 receptor has the potential to downregulate the sensitivity to loading by modifying cell stiffness through actin polymerization and cytoskeleton organization. Herein, we examined the role of P2Y2 activation in regulating osteocytes' mechanotransduction using a P2Y2 knockout cell line alongside conditional knockout mice. Our findings demonstrate that the absence of P2Y2 expression in MLO-Y4 cells prevents actin polymerization while increasing the sensitivity to fluid flow-induced shear stress. Deleting osteocytes' P2Y2 expression in conditional-knockout mice enabled bone formation to increase when increasing the duration of exercise. Overall, P2Y2 activation under loading produces a negative feedback loop, limiting osteocytes' response to continuous loading by shifting the sensitivity to mechanical strain through actin stress fiber formation.

Endomucin regulates the endothelial cytoskeleton independently of VEGF

The endothelial glycocalyx, lining the apical surface of the endothelium, is involved in a host of vascular processes. The glycocalyx is comprised of a network of membrane-bound proteoglycans and glycoproteins along with associated plasma proteins. One such glycoprotein is endomucin (EMCN), which our lab has revealed is a modulator of VEGFR2 function. Intravitreal injection of siEMCN into the eyes of P5 mice impairs vascular development. In vitro silencing of EMCN suppresses VEGF-induced proliferation and migration. Signaling pathways that drive cell migration converge on cytoskeletal remodeling. By coupling co-immunoprecipitation with liquid chromatography/mass spectrometry, we identified interactions between EMCN and proteins associated with actin cytoskeleton organization. The aim of the study was to investigate the influence of EMCN on cytoskeleton dynamics in angiogenesis. EMCN depletion resulted in reduction of F-actin levels, whereas overexpression of EMCN induced increased membrane protrusions in cells that were rich in stress fibers. The reorganization of the actin filaments did not depend on VEGFR2 signaling, suggesting that EMCN connects the cytoskeleton and the glycocalyx.

Proteome of Dried Blood Spots of Cosmonauts during a 6-Month Flight

For successful exploration of near space, construction of planetary bases and exploration of the Solar System planets, it is necessary to fill the gaps in understanding the molecular mechanisms of the human body response to space flight (SF) conditions. Subsequently, this will make it possible to get closer to the discovery of potential molecular targets for protection against adverse processes occurring in the body under the influence of SF conditions. The aim of the work was to assess the effect of a 6-month space flight on the proteome of dried blood spots of cosmonauts. As a result of proteomic analysis, processes with highly and moderately enriched proteins were identified. Among them, processes with reliable dynamics were identified on the 7th day of SF, as well as on the 3rd and 6th months of flight: these are clusters of energy processes, presentation of molecules on the membrane, initiation of immune defense, proteostasis and metabolism. Biological processes were identified in which the representation of proteins decreased most significantly, which was reflected in a weakening of activity in the presentation of molecules on the membrane, the initiation of immune defense, as well as in the mechanisms of proteostasis in the acute period of adaptation to the factors of the initial stage of flight. It has been shown that there is a close interaction with proteins of the cytoskeleton organization that disappear or reappear in the dried blood spots proteome during flight, and that relate to processes whose activity significantly decreased during SF (immune system, proteostasis, metabolism).

Arabidopsis Actin-Binding Protein WLIM2A Links PAMP-Triggered Immunity and Cytoskeleton Organization.

Arabidopsis LIM proteins are named after the initials of three proteins Lin-11, Isl-1, and MEC-3, which belong to a class of transcription factors that play an important role in the developmental regulation of eukaryotes and are also involved in a variety of life processes, including gene transcription, the construction of the cytoskeleton, signal transduction, and metabolic regulation. Plant LIM proteins have been shown to regulate actin bundling in different cells, but their role in immunity remains elusive. Mitogen-activated protein kinases (MAPKs) are a family of conserved serine/threonine protein kinases that link upstream receptors to their downstream targets. Pathogens produce pathogen-associated molecular patterns (PAMPs) that trigger the activation of MAPK cascades in plants. Recently, we conducted a large-scale phosphoproteomic analysis of PAMP-induced Arabidopsis plants to identify putative MAPK targets. One of the identified phospho-proteins was WLIM2A, an Arabidopsis LIM protein. In this study, we investigated the role of WLIM2A in plant immunity. We employed a reverse-genetics approach and generated wlim2a knockout lines using CRISPR-Cas9 technology. We also generated complementation and phosphosite-mutated WLIM2A expression lines in the wlim2a background. The wlim2a lines were compromised in their response to Pseudomonas syringae Pst DC3000 but showed enhanced resistance to the necrotrophic fungus Botrytis cinereae. Transcriptome analyses of wlim2a mutants revealed the deregulation of immune hormone biosynthesis and signaling of salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) pathways. The wlim2a mutants also exhibited altered stomatal phenotypes. Analysis of plants expressing WLIM2A variants of the phospho-dead or phospho-mimicking MAPK phosphorylation site showed opposing stomatal behavior and resistance phenotypes in response to Pst DC3000 infection, proving that phosphorylation of WLIM2A plays a crucial role in plant immunity. Overall, these data demonstrate that phosphorylation of WLIM2A by MAPKs regulates Arabidopsis responses to plant pathogens.

ARP2/3 complex affects myofibroblast differentiation and migration in pancreatic ductal adenocarcinoma.

The ARP2/3 complex, which orchestrates actin cytoskeleton organization and lamellipodia formation, has been implicated in the initiation of pancreatic ductal adenocarcinoma (PDAC). This study aims to clarify its impact on the activity of cancer-associated fibroblasts (CAFs), key players in PDAC progression, and patient outcomes. Early pancreatic carcinogenesis was modeled in p48Cre; LSL-KrasG12D mice with caerulein-induced pancreatitis, complemented by in vitro studies on human immortalized pancreatic stellate cells (PSCs) and primary PDAC-derived CAFs. Data were gained from microarray analysis, RNA sequencing (RNA-seq), and single-cell RNA sequencing (sc-RNA-seq), with subsequent bioinformatics analysis. We uncovered a specific transcriptional signature associated with fibroblast migration in early pancreatic carcinogenesis and linked it to poor survival in patients with PDAC. A pivotal role of the ARP2/3 complex in CAF migration was identified. Inhibition of the ARP2/3 complex markedly decreased CAF motility and induced significant morphological changes in vitro. Furthermore, its inhibition also hindered TGFβ1-mediated myofibroblastic CAF differentiation but had no effect on IL-1-mediated inflammatory CAF differentiation. Our findings position the ARP2/3 complex as central to the migration and differentiation of myofibroblastic CAF. Targeting this complex presents a promising new therapeutic avenue for PDAC treatment.

AAV-based TCAP delivery rescues mitochondria dislocation in limb-girdle muscular dystrophy R7.

Limb-girdle muscular dystrophy R7 is a rare genetic disease caused by homozygous or compound heterozygous variants in the titin-cap (TCAP) gene that results in the absence of the protein telethonin. The primary pathological features of limb-girdle muscular dystrophy R7 are fiber size variation, nuclear centralization, and abnormal mitochondrial distribution. The mechanisms underlying this disease are unclear, and there is currently no specific treatment for limb-girdle muscular dystrophy R7. This study established a Tcap-deficient mouse model to explore the disease mechanism of mitochondria dislocation and potential therapeutic strategies. We use methods such as proteomics, immunofluorescence, histopathological staining, and western blotting to explore the mechanism of mitochondrial dislocation. Moreover, in the quest for a prospective therapeutic intervention for this disorder, the adeno-associated virus serotype 2/9 was employed to deliver the Tcap gene into the muscles of these mice, facilitating preclinical experimentation. After 2 months and 7 months, the muscular phenotype was evaluated and selected mice were humanely euthanized for subsequent molecular and histological analysis. The phenotype of Tcap-/- mice mimicked that observed in individuals diagnosed with limb-girdle muscular dystrophy R7. This study elucidated the mechanism of mitochondrial dislocation in limb-girdle muscular dystrophy R7. Through our in vitro experiments, we discovered that telethonin aids in preserving the integrity of desmin by preventing truncation at the N-terminus. Additionally, telethonin combined with desmin and colocalized at the Z-disc. Research has shown that the Tcap gene plays a crucial role in controlling the desmin cytoskeleton organization. The absence of telethonin leads to a collapsed desmin cytoskeleton. This causes disorganization of the mitochondrial network, leading to mitochondrial dysfunction. In addition, the study investigated the efficacy of adeno-associated virus (AAV)-mediated Tcap replacement in Tcap-/- mice. By intramuscular delivery of AAV, we observed dramatic improvements in muscle phenotype, muscle pathology, CK levels, muscle magnetic resonance imaging, mitochondrial network organization, and mitochondrial function. The results of this study demonstrated that telethonin deficiency led to desmin cytoskeleton collapse that caused mitochondrial dislocation. AAV-mediated replacement therapy could be a promising safe and efficient treatment option for limb-girdle muscular dystrophy R7. The study highlights the potential of AAV-mediated replacement therapy for specific types of limb-girdle muscular dystrophy.

Comparative proteomic analysis of male and female androgenetic alopecia: elucidating gender-specific molecular patterns.

This study presents a comprehensive comparative proteomic analysis aimed at elucidating the molecular mechanisms underlying male androgenetic alopecia (AGA) and female AGA. Scalp samples from both male AGA and female AGA patients, along with their respective normal controls, were subjected to proteomic analysis, followed by bioinformatics investigations. Our findings revealed distinct proteomic profiles between male AGA and female AGA, with a total of 68 differentially expressed proteins identified in male AGA and 84 in female AGA. Among these, specific proteins were altered in male AGA and female AGA, highlighting the sex-specific molecular pathways involved in the pathogenesis of pattern hair loss. Protein-protein interaction network analyses further delineated the most impacted biological processes, including cytoskeleton organization, stress response, and metabolic pathways, with particular emphasis on the differing altered stress responses and metabolic states associated with hair loss between sexes. Our study not only uncovered the complex molecular landscape of male AGA and female AGA but also identified potential biomarkers and therapeutic targets, offering new insights into the sex-specific pathogenesis of pattern hair loss.

Gene co-expression analysis reveals conserved and distinct gene networks between resistant and susceptible Lens ervoides challenged by hemibiotrophic and necrotrophic pathogens

As field crops are likely to be challenged by multiple pathogens during their development, the investigation of broad-spectrum resistance in the host is of great interest for crop genetic enhancement. In this study, we attempted to address this question by adopting a weighed gene co-expression approach to study the temporal transcriptome dynamics of resistant and susceptible recombinant inbred lines (RILs) derived from an intraspecific Len ervoides cross during the infection process with either the necrotrophic pathogens Ascochyta lentis or Stemphylium botryosum, or the hemibiotrophic pathogen Colletotrichum lentis. By comparing networks of resistant and susceptible RILs, seven network module pairs were found to possess high correlation coefficients (R > 0.70) and large number of overlapping genes (n > 100). The conserved co-regulation of genes in these network module pairs were involved in plant cell wall synthesis, cell division, cytoskeleton organization, and protein ubiquitin related processes and appeared to be common disease responses against these pathogens. On the other hand, we also identified eight modules with low correlation between resistance and susceptibility networks. Among those, a stronger gene co-expression in R-genes and small RNA processes in the resistant hosts may be enhancing L. ervoides resistance against A. lentis, C. lentis, and S. botryosum, whereas the higher level of synergistic regulation in the synthesis of arginine and glutamine and phospholipid and glycerophospholipids in the susceptible hosts may contribute to increased susceptibility in L. ervoides.

Single-cell transcriptome sequencing reveals that Wolbachia induces gene expression changes in Drosophila ovary cells to favor its own maternal transmission.

Wolbachia is an obligate endosymbiont that is maternally inherited and widely distributed in arthropods and nematodes. It remains in the mature eggs of female hosts over generations through multiple strategies and manipulates the reproduction system of the host to enhance its spreading efficiency. However, the transmission of Wolbachia within the host's ovaries and its effects on ovarian cells during oogenesis, have not been extensively studied. We used single-cell RNA sequencing to comparatively analyze cell-typing and gene expression in Drosophila ovaries infected and uninfected with Wolbachia. Our findings indicate that Wolbachia significantly affects the transcription of host genes involved in the extracellular matrix, cytoskeleton organization, and cytomembrane mobility in multiple cell types, which may make host ovarian cells more conducive for the transmission of Wolbachia from extracellular to intracellular. Moreover, the genes nos and orb, which are related to the synthesis of ribonucleoprotein complexes, are specifically upregulated in early germline cells of ovaries infected with Wolbachia, revealing that Wolbachia can increase the possibility of its localization to the host oocytes by enhancing the binding with host ribonucleoprotein-complex processing bodies (P-bodies). All these findings provide novel insights into the maternal transmission of Wolbachia between host ovarian cells.IMPORTANCEWolbachia, an obligate endosymbiont in arthropods, can manipulate the reproduction system of the host to enhance its maternal transmission and reside in the host's eggs for generations. Herein, we performed single-cell RNA sequencing of ovaries from Drosophila melanogaster and observed the effects of Wolbachia (strain wMel) infection on different cell types to discuss the potential mechanism associated with the transmission and retention of Wolbachia within the ovaries of female hosts. It was found that the transcriptions of multiple genes in the ovary samples infected with Wolbachia are significantly altered, which possibly favors the maternal transmission of Wolbachia. Meanwhile, we also discovered that Wolbachia may flexibly regulate the expression level of specific host genes according to their needs rather than rigidly changing the expression level in one direction to achieve a more suitable living environment in the host's ovarian cells. Our findings contribute to a further understanding of the maternal transmission and possible universal effects of Wolbachia within the host.

Dysregulation of protein SUMOylation networks in Huntington's disease R6/2 mouse striatum.

Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded CAG repeat mutation in the Huntingtin (HTT) gene. The mutation impacts neuronal protein homeostasis and cortical/striatal circuitry. SUMOylation is a post-translational modification with broad cellular effects including via modification of synaptic proteins. Here, we used an optimised SUMO protein-enrichment and mass spectrometry method to identify the protein SUMOylation/SUMO interaction proteome in the context of HD using R6/2 transgenic and non-transgenic (NT) mice. Significant changes in enrichment of SUMOylated and SUMO-interacting proteins were observed, including those involved in presynaptic function, cytomatrix at the active zone scaffolding, cytoskeleton organization, and glutamatergic signaling. Mitochondrial and RNA-binding proteins also showed altered enrichment. Modified SUMO-associated pathways in HD tissue include clathrin-mediated endocytosis signaling, synaptogenesis signaling, synaptic long-term potentiation, and SNARE signaling. To evaluate how modulation of SUMOylation might influence functional measures of neuronal activity in HD cells in vitro, we utilised primary neuronal cultures from R6/2 and NT mice. A receptor internalization assay for the metabotropic glutamate receptor 7 (mGLUR7), a SUMO enriched protein in the mass spec, showed decreased internalization in R6/2 neurons compared to NT. siRNA-mediated knockdown of the E3 SUMO ligase Protein Inhibitor of Activated STAT1 (Pias1), which can SUMO modify mGLUR7, prevented this HD phenotype. In addition, microelectrode array analysis of primary neuronal cultures indicated early hyperactivity in HD cells, while later timepoints demonstrated deficits in several measurements of neuronal activity within cortical neurons. HD phenotypes were rescued at selected timepoints following knockdown of Pias1. Collectively, our results provide a mouse brain SUMOome resource and show that significant alterations occur within the post-translational landscape of SUMO-protein interactions of synaptic proteins in HD mice, suggesting that targeting of synaptic SUMO networks may provide a proteostatic systems-based therapeutic approach for HD and other neurological. Disorders.

Pathogenic Huntingtin aggregates alter actin organization and cellular stiffness resulting in stalled clathrin mediated endocytosis.

Aggregation of mutant forms of Huntingtin is the underlying feature of neurodegeneration observed in Huntington's disorder. In addition to neurons, cellular processes in non-neuronal cell types are also shown to be affected. Cells expressing neurodegeneration-associated mutant proteins show altered uptake of ligands, suggestive of impaired endocytosis, in a manner as yet unknown. Using live cell imaging, we show that clathrin-mediated endocytosis (CME) is affected in Drosophila hemocytes and mammalian cells containing Huntingtin aggregates. This is also accompanied by alterations in the organization of the actin cytoskeleton resulting in increased cellular stiffness. Further, we find that Huntingtin aggregates sequester actin and actin-modifying proteins. Overexpression of Hip1 or Arp3 (actin-interacting proteins) could restore CME and cellular stiffness in cells containing Huntingtin aggregates. Neurodegeneration driven by pathogenic Huntingtin was also rescued upon overexpression of either Hip1 or Arp3 in Drosophila. Examination of other pathogenic aggregates revealed that TDP-43 also displayed defective CME, altered actin organization and increased stiffness, similar to pathogenic Huntingtin. Together, our results point to an intimate connection between dysfunctional CME, actin misorganization and increased cellular stiffness caused by alteration in the local intracellular environment by pathogenic aggregates.

PTBP1 mediates Sertoli cell actin cytoskeleton organization by regulating alternative splicing of actin regulators.

Spermatogenesis is a biological process within the testis that produces haploid spermatozoa for the continuity of species. Sertoli cells are somatic cells in the seminiferous epithelium that orchestrate spermatogenesis. Cyclic reorganization of the Sertoli cell actin cytoskeleton is vital for spermatogenesis, but the underlying mechanism remains largely unclear. Here, we report that the RNA-binding protein PTBP1 controls Sertoli cell actin cytoskeleton reorganization by programming alternative splicing of actin cytoskeleton regulators. This splicing control enables ectoplasmic specializations, the actin-based adhesion junctions, to maintain the blood-testis barrier and support spermatid transport and transformation. Particularly, we show that PTBP1 promotes actin bundle formation by repressing the inclusion of exon 14 of Tnik, a kinase present at the ectoplasmic specialization. Our results thus reveal a novel mechanism wherein Sertoli cell actin cytoskeleton dynamics are controlled post-transcriptionally by utilizing functionally distinct isoforms of actin regulatory proteins, and PTBP1 is a critical regulatory factor in generating such isoforms.

Local Shear Stress and Dyslipidemia Interfere with Actin Cyto-Skeleton and Lysosomal Organization Contributing to Vascular Fragility

Shear stress is one of the major hemodynamic forces acting on the endothelium. However, it is not well known how endothelial cells (EC) respond mechanically to these stimuli in vivo. Here we investigated whether changes in biomechanics properties and shear stress could increase cell susceptibility to injury, contributing to vascular fragility. We surgically implanted a shear stress modifier device on the carotid artery of ApoE-knockout mice (ApoE−/−), which, due to its shape, causes a gradual stenosis in the vessel, resulting in distinct shear stress patterns. Our data show actin fibers accumulation in areas with higher lipid deposition in ApoE−/−, indicating that dyslipidemia might interfere with EC actin cytoskeleton organization. We also showed that both shear stress and dyslipidemia were important for EC susceptibility to injury. Furthermore, lysosomal distribution, an important organelle for plasma membrane repair, was altered in ApoE−/−, which could compromise EC’s ability to repair from damage. Therefore, dyslipidemia and variations in shear stress patterns not only affect cellular mechanics by compromising the actin cytoskeleton organization, but also enhance cell susceptibility to injury and alter vesicle trafficking in vascular cells. This may likely contribute to vascular fragility and thus to the initial steps of atherosclerosis development.

Quantitative proteomics and phosphoproteomics profiling of meiotic divisions in the fission yeast Schizosaccharomyces pombe

In eukaryotes, chromosomal DNA is equally distributed to daughter cells during mitosis, whereas the number of chromosomes is halved during meiosis. Despite considerable progress in understanding the molecular mechanisms that regulate mitosis, there is currently a lack of complete understanding of the molecular mechanisms regulating meiosis. Here, we took advantage of the fission yeast Schizosaccharomyces pombe, for which highly synchronous meiosis can be induced, and performed quantitative proteomics and phosphoproteomics analyses to track changes in protein expression and phosphorylation during meiotic divisions. We compared the proteomes and phosphoproteomes of exponentially growing mitotic cells with cells harvested around meiosis I, or meiosis II in strains bearing either the temperature-sensitive pat1-114 allele or conditional ATP analog-sensitive pat1-as2 allele of the Pat1 kinase. Comparing pat1-114 with pat1-as2 also allowed us to investigate the impact of elevated temperature (25 °C versus 34 °C) on meiosis, an issue that sexually reproducing organisms face due to climate change. Using TMTpro 18plex labeling and phosphopeptide enrichment strategies, we performed quantification of a total of 4673 proteins and 7172 phosphosites in S. pombe. We found that the protein level of 2680 proteins and the rate of phosphorylation of 4005 phosphosites significantly changed during progression of S. pombe cells through meiosis. The proteins exhibiting changes in expression and phosphorylation during meiotic divisions were represented mainly by those involved in the meiotic cell cycle, meiotic recombination, meiotic nuclear division, meiosis I, centromere clustering, microtubule cytoskeleton organization, ascospore formation, organonitrogen compound biosynthetic process, carboxylic acid metabolic process, gene expression, and ncRNA processing, among others. In summary, our findings provide global overview of changes in the levels and phosphorylation of proteins during progression of S. pombe cells through meiosis at normal and elevated temperatures, laying the groundwork for further elucidation of the functions and importance of specific proteins and their phosphorylation in regulating meiotic divisions in this yeast.

Characterization of Ksg1 protein kinase-dependent phosphoproteome in the fission yeast S. pombe

Ksg1 is an essential protein kinase of the fission yeast S. pombe that belongs to the AGC kinase family and is homologous to the mammalian PDPK1 kinase. Previous studies have shown that Ksg1 functions in the nutrient-sensing TOR signaling pathway and is involved in the phosphorylation and activation of other AGC kinases, thereby affecting various downstream targets related to metabolism, cell division, stress response, and gene expression. To date, the molecular function of Ksg1 has been analyzed using its temperature sensitive mutants or mutants expressing its truncated isoforms, which are not always suitable for functional studies of Ksg1 and the identification of its targets. To overcome these limitations, we employed a chemical genetic strategy and used a conditional ksg1as mutant sensitive to an ATP analog. Combining this mutant with quantitative phosphoproteomics analysis, we identified 1986 phosphosites that were differentially phosphorylated when Ksg1as kinase was inhibited by an ATP analog. We found that proteins whose phosphorylation was dysregulated after inhibition of Ksg1as kinase were mainly represented by those involved in the regulation of cytokinesis, contractile ring contraction, cell division, septation initiation signaling cascade, intracellular protein kinase cascade, barrier septum formation, protein phosphorylation, intracellular signal transduction, cytoskeleton organization, cellular response to stimulus, or in RNA, ncRNA and rRNA processing. Importantly, proteins with significantly down-regulated phosphorylation were specifically enriched for R-X-X-S and R-X-R-X-X-S motifs, which are typical consensus substrate sequences for phosphorylation by the AGC family of kinases. The results of this study provide a basis for further analysis of the role of the Ksg1 kinase and its targets in S. pombe and may also be useful for studying Ksg1 orthologs in other organisms.

Unveiling mitochondria as central components driving cognitive decline in alzheimer's disease through cross-transcriptomic analysis of hippocampus and entorhinal cortex microarray datasets

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder characterized by symptoms such as memory loss and impaired learning. This study conducted a cross-transcriptomic analysis of AD using existing microarray datasets from the hippocampus (HC) and entorhinal cortex (EC), comparing them with age-matched non-AD controls. Both of these brain regions are critical for learning and memory processing and are vulnerable areas that exhibit abnormalities in early AD. The cross-transcriptomic analysis identified 564 significantly differentially expressed genes in HC and 479 in EC. Among these, 151 genes were significantly differentially expressed in both tissues, with functions related to synaptic vesicle clustering, synaptic vesicle exocytosis/endocytosis, mitochondrial ATP synthesis, hydrogen ion transmembrane transport, and structural constituent of cytoskeleton, suggesting a potential association between cognitive decline in AD, synaptic vesicle dynamics, dysregulation of cytoskeleton organization, and mitochondrial dysfunction. Further gene ontology analysis specific to the HC revealed the gene ontology enrichment in aerobic respiration, synaptic vesicle cycle, and oxidative phosphorylation. The enrichment analysis in CA1 of HC revealed differentiation in gene expression related to mitochondrial membrane functions involved in bioenergetics, mitochondrial electron transport, and biological processes associated with microtubule-based process, while analysis in the EC region showed enrichment in synaptic vesicle dynamics which is associated with neurotransmitter release and the regulation of postsynaptic membrane potential and synaptic transmission of GABAergic and glutamatergic synapse. Protein-protein interaction analysis highlighted central hub proteins predominantly expressed in mitochondria, involved in regulation of oxidative stress and ATP synthesis. These hub proteins interact not only within the mitochondria but also with proteins in the vesicular membrane and neuronal cytoskeleton, indicating a central role of mitochondria. This finding underscores the association between clinical symptoms and mitochondrial dysregulation of synaptic vesicle dynamics, cytoskeleton organization, and mitochondrial processes in both the HC and EC of AD. Therefore, targeting these dysregulated pathways could provide promising therapeutic targets aimed at cognitive decline and memory impairment in early AD stages.