Search Results (124)

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

In mitotic cell division, cytokinesis is followed by abscission, the final separation of the cytoplasmic canal, to release the two genetically identical daughter cells; however, sometimes chromatin bridges connecting the daughter nuclei appear. Preserving intact chromatin bridges is crucial because their breakage can cause DNA damage, aneuploidy, and cancer predisposition. For this purpose, cells use two main mechanisms: first, they activate the abscission checkpoint, a mechanism that delays the final cut of the cytoplasmic canal to prevent chromatin bridge breakage and secondly, they form accumulations of actin (“actin patches”) at the base of the intercellular canal to stabilize chromatin bridges. Here, we highlight new findings from our laboratory on how human cells “sense” chromatin bridges and remodel the actin cytoskeleton to generate actin patches in cytokinesis. More specifically, we discuss findings showing that the nuclear membrane Sun1/2-Nesprin-2-LINC (linker of nucleoskeleton and cytoskeleton) complex promotes the generation of mechanical tension on daughter nuclei with chromatin bridges. This tension leads to accumulation of Sun1/2 and Nesprin-2, and cytoplasmic accumulation of PDZ RhoGEF (PDZ domain-containing Rho guanine nucleotide exchange factor) at the base of the intercellular canal. In turn, PDZ RhoGEF activates downstream RhoA-ROCK-LIMK-Cofilin and RhoA-mDia1 signaling pathways to promote actin patches and prevent chromatin bridge breakage in cytokinesis. Full article

Cancer metastasis, the spread of tumour cells from the primary site to distant organs, is responsible for over 90% of cancer deaths, yet effective treatments remain elusive due to incomplete understanding of the molecular drivers involved. Podocalyxin (PODXL), a protein overexpressed in many aggressive cancers, links the cell membrane to the internal skeleton through its interaction with Ezrin, an actin cytoskeleton cross-linker. Despite its therapeutic relevance, the PODXL–Ezrin interface remains structurally uncharacterised and pharmacologically intractable. Here, we employed an integrated computational approach combining protein–protein docking, molecular dynamics (MD) simulations, and virtual screening to investigate the structural basis of the PODXL–Ezrin interaction. Using AlphaFold-predicted structures, we modelled PODXL and Ezrin complexes, revealing that PODXL’s cytoplasmic domain stabilises upon Ezrin binding, with Arg495 mediating temporally distinct electrostatic interactions essential for initial complex assembly. Particularly, we characterised the R495W missense mutation in PODXL’s Ezrin-binding domain, demonstrating that substitution of arginine with bulky, hydrophobic tryptophan may allosterically destabilise Ezrin’s dormant conformation. This mutation slightly increases the intramolecular distance between the F3 subdomain and C-terminal domain from 2.59 Å to 3.40 Å, thus leading to potential partial unmasking of the Thr567 phosphorylation site that could plausibly prime Ezrin for activation. Molecular dynamics simulations in the WT state with a total simulation time of 100 ns revealed enhanced structural rigidity and reduced radius of gyration fluctuations in the mutant complex, consistent with a potential “locked,” activation-prone state that amplifies oncogenic signalling. Through virtual screening, we identified NSC305787 as a selective destabiliser of the R495W mutant complex by disrupting key Trp495–pre-C-terminal loop Ezrin interactions and causing steric hindrance to PIP2 recruitment. Our findings identified mutation-dependent changes in drug binding that can guide the development and repurposing of compounds for targeting PODXL-related cancers and improve patient outcomes in PODXL-altered malignancies. Full article

Beyond their canonical role in promoting G1/S progression, the complexes formed by cyclin D and cyclin-dependent kinase (CDK) 4/6 have emerged as contributors to enhanced cell migration. However, a direct link between this complex and cytoskeletal remodeling during cell motility has remained poorly understood. Here, we show that CDK4/6 inhibition in HeLa cells disrupts lamellipodia formation and subsequent focal adhesion assembly, leading to a reduction in cell migration and invasion. Notably, CDK4, but not CDK6, in complex with cyclin D1/D2, localizes to membrane ruffles to facilitate cytoskeletal reorganization. Mechanistically, proteomic and phosphoproteomic analyses revealed that CDK4 inhibition attenuates the transforming growth factor β (TGFβ) pathway via reduced Smad3 phosphorylation at Thr8, downregulating integrin subunits (α5, α6, and β1). Furthermore, CDK4 inhibition significantly decreased focal adhesion kinase (FAK) phosphorylation at Tyr397 and Rac1-GTP levels. Importantly, the resulting migration defect was largely restored by activation of either Rac1 or FAK. Thus, our data support a model in which cyclin D1/D2–CDK4 promotes phosphorylation of Smad3, leading to upregulation of integrin subunits, activation of FAK and Rac1, and consequent lamellipodia formation and cell migration. These findings provide direct evidence that CDK4 regulates actin cytoskeletal reorganization during cell migration and suggest that CDK4/6 inhibitors may dampen cytoskeleton-dependent tumor invasion, in addition to their antiproliferative effects. Full article

Mechanical and contractile forces in the vascular wall regulate smooth muscle cell migration. We previously demonstrated the presence of C3 complement products in atherosclerotic lesions of human aortas and showed that that C3-derived fragments promote key cellular processes, such as actin cytoskeleton organization and cell migration, in lipid-loaded human vascular smooth muscle cells (hVSMCs). In the present study, we aimed to investigate gene expression profiles related to cytoskeletal remodeling and cell adhesion in migrating hVSMCs with a particular focus on modulatory effect of the C3 complement pathway on these processes. We analyzed gene expression in migrating and non-migrating hVSMCs using real-time PCR and in silico network analysis. Additionally, we investigated cytoskeletal remodeling through Western blotting and confocal microscopy. PCR profiling revealed 30 genes with significantly altered expression in migrating hVSMCs compared to non-migrating control cells. In silico analysis identified six of these genes—PXN, AKT1, RHOA, VCL, CTNNB1, and FN1—as being associated with cytoskeletal remodeling and focal adhesion, with PXN occupying a central position in the interaction network. PXN expression was reduced at both the transcript and protein levels and showed altered subcellular localization in migrating lipid-loaded hVSMCs. Protein–protein interaction analysis using STRING predicted an association between PXN and the integrin complex αMβ2 (comprising ITGAM (CD11b) and ITGB2 (CD18)), which functions as receptors for the iC3b complement fragment. Confocal imaging of cell adhesion structures revealed that lipid-loaded hVSMCs stimulated with iC3b displayed a more diffuse PXN distribution and significantly increased PXN–F-actin colocalization in active cytoplasmic regions compared to lipid-loaded control cells. PXN–F-actin colocalization increased from 1.26% to 19.68%. Subcellular fractionation further confirmed enhanced PXN enrichment in the membrane fraction, with no significant changes observed in the cytosolic or cytoskeletal compartments. In conclusion, iC3b-mediated molecular signaling in lipid-loaded hVSMCs alters PXN distribution and enhances cytoskeletal remodeling, revealing novel molecular interactions in vascular remodeling and the progression of atherosclerotic lesions. Full article

Transmembrane protein 43 (TMEM43 or LUMA) encodes a highly conserved protein found in the nuclear and endoplasmic reticulum membranes of many cell types and the intercalated discs and adherens junctions of cardiac myocytes. TMEM43 is involved in facilitating intra/extracellular signal transduction to the nucleus via the linker of the nucleoskeleton and cytoskeleton complex. Genetic mutations may result in reduced TMEM43 expression and altered TMEM43 protein cellular localization, resulting in impaired cell polarization, intracellular force transmission, and cell–cell connections. The p.S358L mutation causes arrhythmogenic right ventricular cardiomyopathy type-5 and is associated with increased absorption of lipids, fatty acids, and cholesterol in the mouse small intestine, which may promote fibro-fatty replacement of cardiac myocytes. Mutations (p.E85K and p.I91V) have been identified in patients with Emery–Dreifuss Muscular Dystrophy-related myopathies. Other mutations also lead to auditory neuropathy spectrum disorder-associated hearing loss and have a negative association with cancer progression and tumor cell survival. This review explores the pathogenesis of TMEM43 mutation-associated diseases in humans, highlighting animal and in vitro studies that describe the molecular details of disease processes and clinical, histologic, and molecular manifestations. Additionally, we discuss TMEM43 expression-related conditions and how each disease may progress to severe and life-threatening states. Full article

The study of neuronal electrical activity and spatial organization is essential for uncovering the mechanisms that regulate neuronal electrophysiology and function. Mathematical models have been utilized to analyze the structural properties of neuronal networks, predict connectivity patterns, and examine how morphological changes impact neural network function. In this study, we aimed to explore the role of the actin cytoskeleton in neuronal signaling via primary cilia and to elucidate the role of the actin network in conjunction with neuronal electrical activity in shaping spatial neuronal formation and organization, as demonstrated by relevant mathematical models. Our proposed model is based on the polygamma function, a mathematical application of ramification, and a geometrical definition of the actin cytoskeleton via complex numbers, ring polynomials, homogeneous polynomials, characteristic polynomials, gradients, the Dirac delta function, the vector Laplacian, the Goldman equation, and the Lie bracket of vector fields. We were able to reflect the effects of neuronal electrical activity, as modeled by the Van der Pol equation in combination with the actin cytoskeleton, on neuronal morphology in a 2D model. In the next step, we converted the 2D model into a 3D model of neuronal electrical activity, known as a core-shell model, in which our generated membrane potential is compatible with the neuronal membrane potential (in millivolts, mV). The generated neurons can grow and develop like an organoid brain based on the developed mathematical equations. Furthermore, we mathematically introduced the signal transduction of primary cilia in neurons. Additionally, we proposed a geometrical model of the neuronal branching pattern, which we described as ramification, that could serve as an alternative mathematical explanation for the branching pattern emanating from the neuronal soma. In conclusion, we highlighted the relationship between the actin cytoskeleton and the signaling processes of primary cilia. We also developed a 3D model that integrates the geometric organization unique to neurons, which contains soma and branches, such that the mathematical model represents the interaction between the actin cytoskeleton and neuronal electrical activity in generating action potentials. Next, we could generalize the model into a cluster of neurons, similar to an organoid brain model. This mathematical framework offers promising applications in artificial intelligence and advancements in neural networks. Full article

During radiotherapy, X-rays can deliver significant doses of ionising radiation to both cancerous and healthy tissue, often leading to undesirable side effects that compromise patient outcomes. While the cellular effects of such therapeutic X-ray exposures are well studied, the impact on extracellular matrix (ECM) proteins remains poorly understood. This study characterises the response of ECM proteins, including the major tissue components collagen I and fibronectin (FN), to X-ray doses similar to those used in clinical practice (50 Gy, as employed in breast radiotherapy, and 100 Gy), using a combination of gel electrophoresis, biochemical assays, and mass spectrometry-based peptide location fingerprinting (PLF) analysis. In purified protein solutions, 50 Gy X-ray exposure led to the fragmentation of constituent collagen I α chains. Irradiation of purified plasma FN (pFN) induced localised changes in peptide yields (detected by liquid chromatography and tandem mass spectrometry (LC-MS/MS) and PLF) and enhanced its binding to collagen I. In complex environments, such as newly synthesised fibroblast-derived ECM and mature ex vivo breast tissue, X-ray exposure induced peptide yield changes in not only collagen I and FN but also key basement membrane proteins, including collagen IV, laminin, and perlecan. Intracellular proteins associated with gene expression (RPS3, MeCP2), the cytoskeleton (moesin, plectin), and the endoplasmic reticulum (calnexin) were also found to be impacted. These X-ray-induced structural changes may impair the ECM integrity and alter cell–ECM interactions, with potential implications for tissue stiffening, fibrosis, and impaired wound healing in irradiated tissues. Full article

The Spodoptera litura, a Lepidopteran pest known for its high fecundity, undergoes a complete metamorphosis, including a distinctive process during which the male testes fuse from two separate organs into a single entity, significantly enhancing its fertility. To elucidate the molecular mechanisms underlying this testicular fusion, this study employed an integrated multi-omics approach to investigate concurrent changes at the transcriptomic and proteomic levels. We identified a series of synchronized alterations on the peritestic larval membrane, including heme binding, peptidase activity, hydrolase activity, metal ion transport, redox reactions, and chitin metabolism, all of which are substantially enriched at specific temporal points during testicular fusion. Nine genes/proteins co-expressed at the mRNA and protein levels were selected for targeted quantitative proteomics (PRM) and quantitative PCR (qPCR) validation, leading to the identification of five genes potentially involved in the testicular fusion process: Sl3030, ARCP, PSLRE, Obstructor-E, and Osris9B. Notably, the gene Sl3030, once knocked out, not only disrupted the normal fusion process but also resulted in reduced testis size, thickened peritestic membranes, and abnormal sperm development. Transcriptomic sequencing of the Sl3030 knockout mutant revealed its primary influence on the fusion process by affecting the assembly of the microtubule system and cytoskeleton. This research, for the first time, provides a multi-omics perspective on the response of key signaling pathways and molecular changes during the testicular fusion of S. litura and validates the role of the previously uncharacterized gene Sl3030 in this process, offering valuable insights into the complex mechanisms of testicular fusion in this species. Full article

So far, synthetic biology approaches for the construction of artificial microorganisms have fostered the transformation of acceptor cells with genomes from donor cells. However, this strategy seems to be limited to closely related bacterial species only, due to the need for a “fit” between donor and acceptor proteomes and structures. “Fitting” of cellular regulation of metabolite fluxes and turnover between donor and acceptor cells, i.e. cybernetic heredity, may be even more difficult to achieve. The bacterial transformation experiment design 1.0, as introduced by Frederick Griffith almost one century ago, may support integration of DNA, macromolecular, topological, cybernetic and cellular heredity: (i) attenuation of donor Pneumococci of (S) serotype fosters release of DNA, and hypothetically of non-DNA structures compatible with subsequent transfer to and transformation of acceptor Pneumococci from (R) to (S) serotype; (ii) use of intact donor cells rather than of subcellular or purified fractions may guarantee maximal diversity of the structural and cybernetic matter and information transferred; (iii) “Blending” or mixing and fusion of donor and acceptor Pneumococci may occur under accompanying transfer of metabolites and regulatory circuits. A Griffith transformation experiment design 2.0 is suggested, which may enable efficient exchange of DNA as well as non-DNA structural and cybernetic matter and information, leading to unicellular hybrid microorganisms with large morphological/metabolic phenotypic differences and major features compared to predeceding cells. The prerequisites of horizontal gene and somatic cell nuclear transfer, the molecular mechanism of transformation, the machineries for the biogenesis of bacterial cytoskeleton, micelle-like complexes and membrane landscapes are briefly reviewed on the basis of underlying conceptions, ranging from Darwin’s “gemmules” to “stirps”, cytoplasmic and “plasmon” inheritance, “rhizene agency”, “communicology”, “transdisciplinary membranology” to up to Kirschner’s “facilitated variation”. Full article

Organisms have evolved various protective mechanisms to survive in complex and dynamic environments. Phase separation is a ubiquitous mechanism in plants, animals, and microorganisms. It facilitates the aggregation of biomolecules through weak interactions, forming membrane-less organelles that help organisms respond effectively to stress signals. These biomolecular condensates include DNA, RNA, and proteins. Proteins are categorized into scaffold and client proteins, whose coordinated actions ensure the compartmentalization of cellular signals, thereby regulating various biological processes. Studies indicate that, in response to stress, phase separation modulates gene expression, signal transduction, cytoskeleton dynamics, and protein homeostasis, ensuring the precise spatiotemporal control of cellular functions. These insights underscore the crucial role of phase separation in maintaining cellular integrity and adaptability. Full article

The cell wall integrity (CWI) pathway is responsible for transcriptional regulation of cell wall remodeling in response to cell wall stress. How cell wall remodeling mediated by the CWI pathway is effected by inputs from other signaling pathways is not well understood. Here, we demonstrate that the Mck1 kinase cooperates with Slt2, the MAP kinase of the CWI pathway, to promote cell wall thickening in glucose-starved cells. Integrative analyses of the transcriptome, proteome and metabolic profiling indicate that Mck1 is required for the accumulation of UDP-glucose (UDPG), the substrate for β-glucan synthesis, through the activation of two regulons: the Msn2/4-dependent stress response and the Cat8-/Adr1-mediated metabolic reprogram dependent on the SNF1 complex. Analysis of the phosphoproteome suggests that similar to mammalian Gsk-3 kinases, Mck1 is involved in the regulation of cytoskeleton-dependent cellular processes, metabolism, signaling and transcription. Specifically, Mck1 may be implicated in the Snf1-dependent metabolic reprogram through PKA inhibition and SAGA (Spt-Ada-Gcn5 acetyltransferase)-mediated transcription activation, a hypothesis further underscored by the significant overlap between the Mck1- and Gcn5-activated transcriptomes. Phenotypic analysis also supports the roles of Mck1 in actin cytoskeleton-mediated exocytosis to ensure plasma membrane homeostasis and cell wall remodeling in cell wall-stressed cells. Together, these findings not only reveal the novel functions of Mck1 in metabolic reprogramming and polarized growth but also provide valuable omics resources for future studies to uncover the underlying mechanisms of Mck1 and other Gsk-3 kinases in cell growth and stress response. Full article

Recent discoveries revealed mechanistic insights into the control of adipogenesis by the Constitutive Photomorphogenesis 9 Signalosome (CSN) and its variants, CSN^CSN7A and CSN^CSN7B, which differ in the paralog subunits, CSN7A and CSN7B. CSN^CSN7A and CSN^CSN7B variants form permanent complexes with cullin-RING-ubiquitin ligases 3 and 4A (CRL3 and CRL4A), respectively. These complexes can be found in most eukaryotic cells and represent a critical reservoir for cellular functions. In an early stage of adipogenesis, mitotic clonal expansion (MCE), CSN-CRL1, and CSN^CSN7B-CRL4A are blocked to ubiquitinate the cell cycle inhibitor p27^KIP, leading to cell cycle arrest. In addition, in MCE CSN-CRL complexes rearrange the cytoskeleton for adipogenic differentiation and CRL3^KEAP1 ubiquitylates the inhibitor of adipogenesis C/EBP homologous protein (CHOP) for degradation by the 26S proteasome, an adipogenesis-specific proteolysis. During terminal adipocyte differentiation, the CSN^CSN7A-CRL3 complex is recruited to a lipid droplet (LD) membrane by RAB18. Currently, the configuration of the substrate receptors of CSN^CSN7A-CRL3 on LDs is unclear. CSN^CSN7A-CRL3 is activated by neddylation on the LD membrane, an essential adipogenic step. Damage to CSN/CUL3/CUL4A genes is associated with diverse diseases, including obesity. Due to the tremendous impact of CSN-CRLs on adipogenesis, we need strategies for adequate treatment in the event of malfunctions. Full article

The actin cytoskeleton plays an important role in morphological changes of ameloblasts during the formation of enamel, which is indispensable for teeth to withstand wear, fracture and caries progression. This study reveals that the actin nucleator Cobl is expressed in ameloblasts of mandibular molars during amelogenesis. Cobl expression was particularly pronounced during the secretory phase of the enamel-forming cells. Cobl colocalized with actin filaments at the cell cortex. Importantly, our analyses show an influence of Cobl on both ameloblast morphology and cytoskeletal organization as well as on enamel composition. At P0, Cobl knock-out causes an increased height of ameloblasts and an increased F-actin content at the apical membrane. During the maturation phase, the F-actin density at the apical membrane was instead significantly reduced when compared to WT mice. At the same time, Cobl-deficient mice showed an increased carbon content of the enamel and an increased enamel surface of mandibular molars. These findings demonstrate a decisive influence of the actin nucleator Cobl on the actin cytoskeleton and the morphology of ameloblasts during amelogenesis. Our work thus expands the understanding of the regulation of the actin cytoskeleton during amelogenesis and helps to further elucidate the complex processes of enamel formation during tooth development. Full article

The nuclear envelope (NE), a protective membrane bordering the nucleus, is composed of highly specialized proteins that are indispensable for normal cellular activity. Lamina-associated polypeptide 1 (LAP1) is a NE protein whose functions are just beginning to be unveiled. The fact that mutations causing LAP1 deficiency are extremely rare and pathogenic is indicative of its paramount importance to preserving human health, anticipating that LAP1 might have a multifaceted role in the cell. Mapping the LAP1 protein interactome is, thus, imperative to achieve an integrated view of its potential biological properties. To this end, we employed in silico- and mass spectrometry-based approaches to identify candidate LAP1-interacting proteins, whose functional attributes were subsequently characterized using bioinformatics tools. Our results reveal the complex and multifunctional network of protein–protein interactions associated to LAP1, evidencing a strong interconnection between LAP1 and cellular processes as diverse as chromatin and cytoskeleton organization, DNA repair, RNA processing and translation, as well as protein biogenesis and turnover, among others. Novel interactions between LAP1 and DNA repair proteins were additionally validated, strengthening the previously proposed involvement of LAP1 in the maintenance of genomic stability. Overall, this study reaffirms the biological relevance of LAP1 and the need to deepen our knowledge about this NE protein, providing new insights about its potential functional partners that will help guiding future research towards a mechanistic understanding of LAP1’s functioning. Full article