Search Results (2,594)

Winter rapeseed (Brassica rapa L.) is an important crop for vegetable oil production in China. However, its productivity is frequently threatened by severe cold waves during winter. To investigate the role of the microtubule cytoskeleton in cold adaptation of winter rapeseed, a microtubule stabilizer paclitaxel (Tax) and a microtubule depolymerizer colchicine (Col) were sprayed on winter rapeseed and transgenic proBrAFP1 Arabidopsis, respectively. The mRNA levels of cold-induced genes, along with cell membrane stability, antioxidant enzyme activities, and hormone levels were assessed under cold stresses of 4 °C and −4 °C. The results showed that low temperature significantly activated the proBrAFP1 promoter activity and increased the mRNA levels of core cold signaling pathway genes, such as C-REPEAT BINDING FACTORS (CBFs), Cyclic Nucleotide-Gated Channel (CNGC), OPEN STOMATA 1 (OST1) and Inducer of CBF EXPRESSION 1 (ICE1). Notably, under low-temperature stress, exogenous application of the microtubule stabilizer Tax markedly suppressed proBrAFP1-driven reporter activity in transgenic Arabidopsis, with consistent inhibition observed across both stem and leaf tissues; meanwhile, the Tax application alleviated reactive oxygen species (ROS) accumulation and mitigated membrane damage. In contrast, under the same low-temperature stress, the Col treatment exacerbated oxidative stress, enhanced lipid peroxidation, and elevated membrane damage. Collectively, these findings establish that microtubule regulators play indispensable roles in the cold stress response of winter rapeseed. It provides new insights into the mechanism by which plant microtubule cytoskeleton regulators mediate the cold response. Full article

Sperm cryopreservation is pivotal for conserving fish germplasm, yet cryodamage-induced quality decline limits its application. This study focused on Sichuan bream (Sinibrama taeniatus), an endemic and economically important fish species in the upper Yangtze River. Based on an established cryopreservation protocol, we evaluated sperm quality using computer-assisted sperm analysis (CASA) and fertility assays, followed by a systematic assessment of structural and functional damage via flow cytometry (membrane integrity, mitochondrial potential, reactive oxygen species, and DNA fragmentation), enzymatic assays (energy metabolism and antioxidant enzymes), Western blotting, and ultrastructural observation. Finally, integrated proteomic and metabolomic analyses were employed to elucidate the underlying physiological mechanisms. The results demonstrated that freeze–thawing significantly impaired sperm motility, fertility, and ultrastructure, concurrently disrupting energy metabolism and the antioxidant system. Crucially, multi-omics revealed that these functional declines were linked to dysregulation in key pathways involving cytoskeleton organization, lipid metabolism, energy homeostasis, and oxidative stress, forming a coherent network from initial molecular perturbation to phenotypic dysfunction. This study provides a comprehensive characterization of sperm cryodamage in Sichuan bream, advancing the understanding of fish sperm cryobiology and informing targeted cryoprotection strategy development. Full article

The aim of this study was to investigate which physicochemical and structural properties of cationic peptides P1–P6 may determine their selective anticancer activity against melanoma cells and their interactions with tumor cell membranes. An integrated approach was applied, including characterization in solution (osmotic pressure, NaCl stability, surface tension); cytotoxicity evaluation against Me45, B16F10, and HaCaT cells; analysis of interactions with phosphatidylglycerol (POPG) model membranes using isothermal titration calorimetry and steady-state fluorescence spectroscopy; membrane permeability assays; and F-actin staining. Anticancer activity depended on positively charged residues, hydrophobic amino acids, and sequence arrangement. Tryptophan-rich peptides P2 and P5 exhibited strong membrane interactions and high efficacy after 72 h. Highly hydrophobic P4, containing long C₁₂ chains with a relatively low net charge, caused nonselective lysis. P3 showed reduced activity due to insufficient amphipathicity, whereas P6, with excessive WWW and KKKK motifs, exhibited weak or nonselective effects. Thermodynamic and fluorescence analyses indicated that P2 and P5 initially bind POPG membranes via entropy-driven electrostatic interactions, followed by hydrophobic insertion of tryptophan residues, evidenced by increased fluorescence intensity and a blue shift of the emission maximum. P2, P4, and P5 induced actin cytoskeleton reorganization and increased membrane permeability, emphasizing the role of balanced amphipathicity and charge–hydrophobicity in designing selective anticancer peptides. Full article

The cuticular plate, a dense F-actin meshwork anchoring stereocilia in auditory hair cells (HCs), undergoes dynamic remodeling during development, but its structural transitions remain poorly understood. Here, we identified two distinct structural domains associated with this maturation. First, a transient F-actin-free area emerges within the lateral periphery of the developing cuticular plate, presenting as a crescent-shaped region that disappears upon HC maturation. Second, the lateral margin of the mature cuticular plate itself remodels into a persistent step-like structure, exhibiting cell-type-specific geometries in inner versus outer HCs. The consistent coincidence between Gαi-GPSM2 complex disruption and aberrant development of both structures in mutant mice implies a role for this complex in their formation. Additionally, microtubules spatially complemented F-actin distribution, suggesting coordinated cytoskeletal regulation. These findings revealed a sophisticated developmental program for cuticular plate maturation. Full article

Steroid-resistant nephrotic syndrome (SRNS) in childhood frequently reflects monogenic podocytopathies in which immunosuppression is ineffective. Biallelic variants in MYO1E, encoding the class I myosin Myo1E, cause a distinctive form of focal segmental glomerulosclerosis (FSGS) often accompanied by “Alport-like” multilamination of the glomerular basement membrane (GBM). Early recognition has therapeutic and prognostic implications. A previously healthy 4-year-old boy presented with generalized edema and nephrotic-range proteinuria. Glucocorticoids induced no remission; sequential calcineurin inhibition (cyclosporine, then tacrolimus) and a single dose of ofatumumab yielded only transient, partial reductions in proteinuria. A first biopsy elsewhere showed FSGS with nonspecific IgM/C3 trapping; electron microscopy (EM) was not performed. At age 10, repeat biopsy with EM revealed ~30% segmental foot-process effacement, focal GBM thickening (to 1740 nm), irregular lamina densa multilamination, and lamellar duplications without immune-complex deposits—features highly suggestive of hereditary GBM disease. Targeted sequencing identified compound-heterozygous MYO1E variants segregating in trans: a canonical splice-donor change (c.2785+1G>A) and a frameshift (c.3094_3097del; p.Thr1032Profs*73). Each parent was an unaffected heterozygous carrier; the sibling was negative. Supportive therapy with ramipril was continued. At last follow-up (January 2025), renal function was normal (serum creatinine 0.5 mg/dL; creatinine clearance 122 mL/min) with stable sub-nephrotic proteinuria (0.52 g/day; 16 mg/m² per hour) and normotension. This case broadens clinicopathologic recognition of MYO1E-associated nephropathy and highlights the teaching point that Alport-like GBM changes are not pathognomonic for type IV collagen disorders but may signal defects in podocyte cytoskeletal anchoring. Full article

The goal of this review is to bring to the attention of the scientific community the opportunities of transmission electron microscopy for analyses of biological subjects and resolving complicated cases of data interpretation. Although procedures for electron microscopy are in general more elaborate (particularly for simultaneous immunolabeling of multiple antigens) compared to fluorescent microscopy, they can help view cellular morpho-functional features undetectable using other methods. In this review, we consider several unexpected and serendipitous discoveries made in our laboratory and fulfilled using unique opportunities provided by electron microscopy of ultrathin sections. We are deliberating the following topics: interpretation of unusual results of immunolabeling; a novel method for in situ identification of cells undergoing mitochondrial disorder and necrosis-like death; the sequence of organelles’ reorganization in dying cells; simultaneous rupture of nuclear and plasma membranes in migrating neurons; and the role of cytoskeleton in lateral expansion of the cerebral cortex. Full article

Platelets are small circulating blood cells that mediate haemostasis and thrombosis. Platelets respond to vascular damage by adhesion, granule release, and aggregation. Healthy endothelial cells inhibit platelets through prostacyclin-induced cAMP signalling. Intracellular cAMP activates protein kinase A (PKA), a tetrameric kinase composed of two regulatory (R) and two catalytic (C) subunits. cAMP-binding triggers dissociation of C subunits from the PKA complex and phosphorylation of substrate proteins, which mediate platelet inhibition. The R subunits of PKA are known to be attached to A-kinase anchoring proteins (AKAPs), which enable subcellular compartmentalisation of cAMP signalling. Proteomics have identified 22 AKAPs in platelets, but only a few of these have been studied in detail. This review summarises current knowledge about platelet AKAPs, including studies done regarding other cells. Possible integration of AKAPs into platelet signalling is explored with a focus on subcellular localisation, interaction partners, and PKA-mediated substrate phosphorylation. As main platelet compartments, the plasma membrane, endosomes, mitochondria, the Golgi, the dense tubular system, and the cytoskeleton are considered. Potential roles of individual AKAPs in platelet inhibition are discussed, and open questions in the field are defined. Full article

Cervical Intervertebral Disc Degeneration (CIVDD) involves significant microenvironmental physical stiffening, forcing nucleus pulposus cells (NPCs) into a rigid phenotype via F-actin over-assembly. It remains unclear if cyclic tensile strain (CTS) can reverse this physical stiffening, particularly in severe degeneration. This study stratified 18 patients into Mild, Moderate, and Severe cohorts based on MRI. Primary NPCs were subjected to physiological 5% CTS (1 Hz, 24 h). Atomic Force Microscopy (AFM) and immunofluorescence were utilized to evaluate Young’s modulus and cytoskeletal remodeling. Results demonstrated that baseline cellular stiffness increased significantly with degeneration severity. Following CTS, all groups exhibited universal de-stiffening and F-actin depolymerization. Crucially, a “Degeneration Paradox” emerged: the Severe group displayed the highest relative elastic modulus recovery rate, significantly surpassing the Mild group. This microscopic recovery correlated inversely with preoperative disc height loss and range of motion. We conclude that severely degenerated cells are not metabolically quiescent but “physically locked” by a rigid cytoskeleton. Physiological CTS restores compliance via mechanical unloading, confirming that severe cells retain superior relative mechanoplasticity and may benefit from mechanotherapy-based “unlocking” strategies. Full article

Nuclear mechanics and mechanotransduction are involved in the migration and invasion process, such as those in which the cells need to deform themselves to pass through constrictions. Specifically, properties like nuclear softness, viscoelasticity, plasticity (like nuclear pore complexes) and deformability are critical in cancer and its malignant progression. The nucleus represents a physical barrier for the migration and invasion in dense 3D extracellular matrix (ECM) scaffolds. Therefore, the deformability of the nucleus seems to determine the migration limit in circumstances where the enzymatic remodeling of the surroundings is impaired. There are still significant knowledge gaps regarding effects of nuclear deformation during cancer dissemination. It seems that nuclear deformation can alter gene transcription, induce alternative splicing processes, impact nuclear envelope rupture, nuclear pore complex dilatation, damage the DNA, and increase the genomic instability. These mechanically induced alterations can in turn impact the migratory behavior of the cancer cells. The stiffness of the nucleus relies on the condensation of chromatin, and the nuclear lamina, which consists of a network of intermediate filaments underneath the nuclear envelope. All of this is discussed in the review and it is argued that nuclear deformability is universally found in various cancer types. Another focus is placed on the nuclear envelope proteins like emerin, and the SUN-KASH complex and how they contribute to the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, which consequently couples the nucleus and the cytoskeleton. It is argued that this connection is crucial for force transmission, which governs nuclear stiffness dynamically, depending on the force applied. In this review, recent findings are described that couple ECM-induced nuclear mechanosensing and mechanotransduction with the migration and invasion of cancer cells. Moreover, it is suspected that changes in the mechanosensory characteristics of the cell nucleus could play a pivotal part in the malignancy of cancer cells and the heterogeneity of tumors. Finally, it is discussed what impact the individual elements of the nucleus offer to mechanically alter cellular migration and invasion in cancer and its malignant progression. Full article

Different components of the cytoskeleton are very important determinants of brain development. They orchestrate multiple cellular processes involved in all phases of cerebral cortex development. In this review, we summarize current knowledge on the components of the cytoskeleton—microtubules, actin filaments, and intermediate filaments—and their roles in cortical development. We provide a detailed analysis of how cytoskeleton molecules control neuronal progenitor proliferation, neuronal migration, polarization, axon and dendrite specification and outgrowth, and synaptogenesis. We further examine how pathogenic variants in genes encoding cytoskeletal proteins or their regulators disrupt particular steps of neurogenesis and contribute to major neurodevelopmental disorders (NDDs). Focusing on NDDs such as microcephaly, lissencephaly, corpus callosum agenesis, and synaptopathies, we discuss consequences of cytoskeletal dysfunctions causing altered cellular behavior and clinical phenotypes. By linking molecular defects to developmental and phenotypic consequences, this review highlights the cytoskeleton as a central element in neurodevelopmental pathologies and underscores its potential as a target for future therapeutic strategies. Full article

The type II cadherin Cadherin-19 (CDH19) plays a crucial role in neural crest development. CDH19 regulates cell–cell junctions and migration by forming catenin–cytoskeleton complexes. Although anti-CDH19 monoclonal antibodies (mAbs) are used for specific applications such as Western blotting and immunohistochemistry (IHC), suitable anti-CDH19 mAbs for flow cytometry are limited. Therefore, developing mAbs that specifically recognize cell-surface-expressed CDH19 is essential for advancing both basic research and therapeutic strategies. Here, novel anti-human CDH19 mAbs (Ca₁₉Mabs) were created using flow cytometry-based high-throughput screening. One clone, Ca₁₉Mab-8 (IgG₁, κ), specifically recognized CDH19-overexpressed Chinese hamster ovary-K1 cells but did not bind to other 21 CDHs (including both type I and type II CDHs) in flow cytometry. Additionally, Ca₁₉Mab-8 recognized endogenous CDH19 in the human glioblastoma cell line LN229. The dissociation constant (K_D) of Ca₁₉Mab-8 for LN229/CDH19 was 9.0 × 10⁻⁹ M. Ca₁₉Mab-8 also detected endogenous CDH19 in Western blotting. Furthermore, Ca₁₉Mab-8 can detect CDH19 in IHC using human melanoma tissue. These findings suggest that Ca₁₉Mab-8 is a novel mAb that detects cell-surface-expressed CDH19 with high specificity and is suitable for various applications in basic research. Therefore, Ca₁₉Mab-8 has potential for clinical diagnosis and tumor therapy. Full article

Fat deposition plays a crucial role in regulating the production performance and meat quality of broilers. Although the heterogeneity of mammalian adipocytes has been extensively studied, research on the molecular mechanisms underlying differences in lipid droplet accumulation in avian adipocytes remains limited. This study confirmed a significant positive correlation (R² > 0.81, p < 0.001) between the SSC signal and lipid droplet content via fluorescence staining of lipid droplets, Oil Red O staining, and triglyceride (TG) quantification. Based on this, a label-free sorting strategy using SSC signals was established to sort differentiated chicken preadipocytes, obtaining high lipid droplet (H) and low lipid droplet (L) subpopulations, which were subsequently subjected to transcriptome sequencing and differential gene expression (DEG) analysis, followed by GO and KEGG enrichment analysis. The results indicated no significant differences in the expression of adipogenesis marker genes (PPARG, LPL, CD36, PLIN1, PLIN2) between the high lipid droplet (H) and low lipid droplet (L) groups, suggesting that both groups are at similar stages of differentiation. KEGG analysis revealed that both the H vs. NC and L vs. NC comparisons were enriched in common pathways, including the PPAR signaling pathway, ECM–receptor interaction, focal adhesion, cytokine–receptor interaction, and calcium–Apelin signaling pathway, suggesting that both groups of cells had activated the adipogenesis program. GO analysis showed that, in both H vs. NC and L vs. NC comparisons, differentially expressed genes (DEGs) were enriched in biological processes (BPs) related to cell adhesion, nucleosome assembly, chromatin remodeling, and receptor activity, as well as cellular components (CCs) such as the extracellular matrix, cytoskeleton, and nucleosome organization, indicating extensive gene reprogramming and activation of signaling transduction during differentiation. In the H vs. L comparison, enriched pathways included ABC transporters, ECM–receptor interaction, focal adhesion, gap junctions, microtubule-related processes, and neuroactive ligand–receptor interactions, involving lipid transmembrane transport, cytoskeleton stabilization, and signal transduction regulation, suggesting that high lipid droplet cells are more mature in lipid droplet transport, storage, and homeostasis maintenance. GO enrichment results further supported this conclusion, as H vs. L specifically enriched processes related to microtubule-related processes, cell cycle, and redox reactions (BPs), as well as chromosome organization, cytoskeleton, and motor activity (CC/MF), indicating that high lipid droplet cells maintain lipid droplet fusion and metabolic homeostasis via enhanced microtubule transport and antioxidant regulation. Differential gene analysis revealed that the L group upregulated genes associated with fatty acid synthesis and elongation (ACACA, FASN, SCD, FADS2, ELOVL1), cholesterol and isoprenoid biosynthesis (HMGCR, SQLE, MSMO1, DHCR7, DHCR24, FDPS, LSS), and fatty acid oxidation (PPARA, PPARD, ACAD11, SIRT5), reflecting a metabolic characteristic of concurrent lipid synthesis and mobilization; the H group, conversely, upregulated genes associated with lipid droplet formation and storage (G0S2, MOGAT1, GPAT4, PLIN4, AUP1), lipid transport (ABCA1, ABCA2, ABCG1, OSBPL3, VLDLR), and antioxidant defense (GPX3, GPX4, HMOX1), exhibiting a storage and homeostasis-oriented metabolic state. In the NC, L, and H groups, the expression of five genes—GEM, SPP1, ABCA1, PDLIM3, and ITGA8—showed a gradual increase, suggesting that these genes were associated with preadipocyte differentiation and lipid droplet deposition. In summary, although the high and low lipid droplet subpopulations of chicken preadipocytes exhibit similar differentiation states, they form distinct metabolic orientations. The L group is characterized by active lipid synthesis, fatty acid oxidation, and membrane lipid remodeling, while the H group predominantly features lipid droplet storage, lipid transport, and antioxidant homeostasis. This study highlights the molecular mechanisms underlying the metabolic heterogeneity of avian adipocytes and provides a theoretical basis for poultry fat deposition regulation and genetic improvement. Full article

Neuronal function relies on the precise coordination between intracellular calcium (Ca²⁺) signaling and the cytoskeletal architecture that underpins synaptic transmission, plasticity, and structural stability. Disruption of this calcium–cytoskeleton interplay has been noted in numerous neurodegenerative diseases. We discuss how Ca²⁺-dependent cytoskeletal remodeling governs long-term potentiation and depression, dendritic spine morphology, and presynaptic function, highlighting the functions of end-binding proteins, STIM (Stromal Interaction Molecule)/Orai-mediated store-operated calcium entry, and the spine apparatus. Disease-specific manifestations of cytoskeletal–calcium dysregulation are reviewed across Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, tauopathies, and prion disorders. Finally, we evaluate emerging therapeutic strategies targeting calcium homeostasis, cytoskeletal dynamics, and their downstream effectors, including multi-target approaches. Full article

Rho GTPases are a group of guanosine triphosphate (GTP)-binding proteins with a relative molecular weight of about 20–30 kD, and 22 different Rho GTPases have been identified in mammalian cells, among which RhoA, Rac1 and Cdc42 are the most well-studied. Rho-associated coiled coil forming protein kinase (ROCK) is the most well-researched downstream effector of Rho GTPases. The Rho/ROCK signaling pathway widely participates in the reorganization of the cytoskeleton through cascade phosphorylation/dephosphorylation reactions and modulates cellular biological behaviors including cell adhesion, migration and phenotypic transformation. Abnormal activation of the Rho/ROCK signaling pathway is closely associated with the occurrence and progression of acute kidney injury, diabetic nephropathy, hypertension-related nephropathy and chronic allograft nephropathy, which contributes to podocyte injury, renal tubular epithelial-to-mesenchymal transition (EMT), mesangial cell proliferation and inflammatory infiltration in the kidney. This review focuses on the research progress and regulatory mechanisms of the Rho/ROCK signaling pathway in the above four major kidney diseases and discusses the therapeutic potential of targeting this pathway for kidney disease treatment, aiming to provide new insights for elucidating the pathogenesis of kidney diseases and developing novel therapeutic strategies. Full article

Background/Objectives: Methadone maintenance treatment (MMT) is one of the major pharmacotherapies for opioid use disorder. The underlying mechanisms of addiction and the treatment response are only partially understood. The study’s main goal was to identify differential DNA CpG methylation that occurred in response to MMT. Methods: Toward this goal, we have conducted a longitudinal epigenome-wide study of blood samples from 64 patients at the beginning and after 1–3 years of MMT, using a linear mixed model. Results: A total of 1881 differentially methylated probes (DMPs) were identified (FDR < 0.05), controlling for sex, age, estimates of blood cell proportions, and the first two principal components based on genome-wide SNP genotypes. Among the genes annotated to the top DMPs are DGLUCY, NXNL2, SOX10, and NPAS3. Several genes associated with substance use disorder were annotated by the identified DMPs, including ADORA2A, BDNF, CACNA1D, CREB1, CRHR1, CRY1, DNMT3B, GABRD, GNAS, GRIP1, OXR1, PRKACB, SCN2A, and SCN3A. The most overrepresented pathway is the small GTPase-mediated signal transduction pathway, and the most overrepresented process is the actin cytoskeleton organization. Conclusions: The study provides preliminary insight into the epigenetic effect of MMT. Future studies will have to confirm the DMPs, assess their impact on gene expression, and determine their clinical relevance. Full article