European Journal of Cell Biology

Tropomyosins play a central role in the regulation of the actin cytoskeleton by assembling along filaments into rope-like, head-to-tail polymers ("clusters") along filaments that regulate the binding and activity of numerous actin-binding proteins. Importantly, in cells, tropomyosins do not decorate actin filaments uniformly; some filaments are devoid of tropomyosins, whereas others recruit specific isoforms. To explore the diversity of tropomyosin-isoform binding kinetics and identify factors that control their assembly, we designed tropomyosin constructs, bearing a natural N-terminal acetylation and targeted cysteine mutations for fluorescence labelling. We analysed the binding of different tropomyosin isoforms to individual actin filaments reconstituted inside a microfluidic chamber. We show that, despite significant differences in spontaneous nucleation and elongation rates, all tested tropomyosin isoforms can coassemble within the same clusters. In addition, we identified tropomodulin and leiomodin, two pointed-end-binding proteins, to efficiently nucleate the formation of tropomyosin clusters on actin filaments, for all tested tropomyosin isoforms. Notably, while tropomodulin nucleates tropomyosin clusters from the pointed end, leiomodin does so along the entire filament.

The actomyosin cytoskeleton plays important roles in cell-cell adhesion by generating and responding to forces. Actin-binding proteins support actomyosin networks by reinforcing actin filaments, promoting actin remodeling, and transmitting forces to transmembrane adhesion proteins. One family of actin-binding proteins, LIM domain-containing proteins, is recruited to strained actin filaments. Here, we investigate two members of the LIM domain family, Zyxin and Lipoma-Preferred Partner (LPP), using the embryonic epithelium of Xenopus laevis, where actin-associated cell-cell junctions connect cells to promote tissue integrity and barrier function. Specifically, we compare Zyxin and LPP's response to increased tension at tricellular junctions (TCJs), sites of heightened mechanical strain within the tissue. Upon increased tension, Zyxin and LPP mechanoaccumulate to different extents relative to F-actin, suggesting distinct mechanisms. Our results demonstrate that Zyxin's and LPP's LIM domain-containing regions (LCRs) are sufficient for mechanoaccumulation as well as responsible for the difference in mechanoaccumulation, while their N-termini play a regulatory role in their mechanosensitive responses. Docking simulations show that a hydrophobic interaction between the LCR and the "crack site" that forms in F-actin filaments under high force may underlie the LCR's sensing of strained F-actin. Additionally, the docking simulations reveal that previously-identified conserved residues are present at the binding interface. Mutations of conserved residues in Zyxin or LPP reduce the extent of mechanoaccumulation at TCJs, supporting the docking prediction. Together, this study advances our understanding of Zyxin and LPP's sensing of strained actin at TCJs under mechanical challenge.

Skeletal muscle is a dynamic tissue able to regenerate in response to injury. This regeneration relies primarily on the activation of precursor cells, known as muscle stem cells (MuSC). Upon activation, skeletal MuSC re-enter the cell cycle and proliferate as myoblasts, which subsequently either differentiate and fuse to form new fibers or return to quiescence to maintain long-term regenerative capacity. From primary human myoblasts, we generated reserve cells (RCs), quiescent cells similar to MuSC, and investigated their activation after stimulation with a growth medium containing serum. Here, we show that Orai3 calcium channel is primarily expressed in RC, where it promotes RC activation in a calcium-independent manner. We demonstrate that Orai3 does not contribute to store-operated calcium entry (SOCE) or calcium response induced by serum stimulation in RC cells, supporting the hypothesis that Orai3 plays a unique role in RC, distinct from its calcium channel activity. Using a protein proximity assay (BioID), we identified the large scaffold protein AHNAK2 as a potential partner of Orai3. AHNAK2 is expressed at high level in myotubes, and has a similar effect on RC activation than Orai3. Both Orai3 and AHNAK2 are necessary for proper myoblast differentiation and RC activation, while acting through different signaling pathways. Specifically, we show that Orai3 directly influences the RC fate, whereas AHNAK2 facilitates RC activation via signals derived from myotubes. These findings provide new insights into the molecular mechanisms underlying the activation and quiescence of human MuSC.

In our past study on human cilia from primary ciliary dyskinesia (PCD) patients using cryo-electron tomography and mass spectrometry, defects in the DNAH5 gene were proven to cause the loss of the entire outer dynein arm along with other proteins. This phenomenon was not observed in the unicellular green alga Chlamydomonas. In this study, we examined the loss of ciliary components caused by defects in various genes, based on proteomic and structural analyses of motile cilia from multiple PCD patients. The absence of single outer arm dynein genes causes a loss or significant decrease in other inner and outer arm dyneins. Interestingly, some patients with outer dynein defects show decreased intraflagellar transport (IFT) proteins, suggesting an influence of cargo components or assembly on transport. These phenomena were not observed in Chlamydomonas, suggesting a more complex mechanism of ciliogenesis in humans despite the high similarity in the final 3D architecture of the 9+2 axoneme. Defects in certain central pair proteins unexpectedly reduced components on the doublets as well. Our results demonstrate protein-protein interactions during stages of human motile ciliogenesis that are more complex than those in unicellular organisms.

Septins are cytoskeletal GTPases that assemble into diverse higher-order structures with context-dependent mechanical and signaling functions. Recent work identified a septin-dependent pro-survival pathway in metastatic cancer cells, termed bleb signaling, in which plasma membrane blebbing promotes the formation of septin signaling hubs that support survival during cellular detachment and dissemination. Disrupting these hubs may offer a novel therapeutic strategy targeting disseminated cancer cells. Here, we compare the effects of three septin-targeting compounds, forchlorfenuron (FCF), UR214-9, and REM127, on septin organization and viability in melanoma cells using confocal microscopy and quantitative computer vision analysis. FCF and UR214-9 both effectively destabilized septin hubs, while REM127 had no appreciable effect. FCF also reproduced the context-specific viability reduction previously seen in detached melanoma cells, while UR214-9 caused broad off-target toxicity and cell death in all growth conditions. As with hub disruption, REM127 produced no effect on viability. Surprisingly, though none of the compounds caused an increase in higher-order septin structures during detachment, all three increased end-on-end polymerization of filamentous septins at stress fibers when grown adhered to substrate. These findings demonstrate that septin-targeting compounds exert divergent effects on different classes of septin higher-order structures within the same cell type, promoting filamentous assembly while disrupting or failing to promote membrane-associated signaling hubs. This highlights not only the structural distinctiveness of such classes, but that pharmacological targeting can produce opposite effects on them. Moreover, this study emphasizes the need for next-generation septin inhibitors capable of selectively targeting disease-relevant septin assemblies.

RASAL3 acts as a negative regulator of small cellular GTPases in hematopoietic cells. In immune cells, it primarily modulates the RAS/MAPK signaling pathway and affects cellular events including proliferation, differentiation, survival, and migration. Due to its inhibitory role in T cells, RASAL3 may represent a potential modulatory target for improving therapeutic strategies such as cell-based immunotherapy. However, most existing knowledge about RASAL3 function is derived from murine models, and its role in human T-cell signaling remains insufficiently characterized. To address this gap, we systematically investigated the function of RASAL3 in human primary T cells and T-cell line. For this purpose, we employed RASAL3 overexpression, CRISPR/Cas9-mediated deletion, and siRNA-mediated knockdown to thoroughly analyze the effects of RASAL3 on T-cell signaling, proliferation, and migration. Our data demonstrate that RASAL3 modulates primarily CDC42 and RAC1/RAC2 GTPases activity, SAPK/JNK phosphorylation, c-Fos and c-Jun expression, and IL-2 gene promoter activation. In addition, RASAL3 regulates actin polymerization and T-cell migration. Notably, loss of RASAL3 increases Jurkat T cells motility in vivo and potentiates their homing to the spleen. Collectively, these findings identify RASAL3 as an important regulator of human T-cell activation and motility and highlight its application potential for improving CAR-T cell therapy.

Long noncoding RNAs encode for microproteins that regulate cellular functions. Small regulatory peptide of amino acid response (SPAR) is a microprotein in the lysosome that responds to amino acid availability of the cell. In this study, we investigated the interactions between SPAR and SLC38A9, a lysosomal amino acid transporter and receptor involved in the mechanistic target of rapamycin 1 (mTORC1) pathway. We found that SPAR binds SLC38A9 and inhibits arginine transport in SLC38A9. Moreover, the downstream recruitment of Rag GTPases is also inhibited when SPAR is present in SLC38A9 liposomes. Docking model shows potential interactions between SPAR and SLC38A9. Together, these findings reveal the mechanism of mTORC1 inhibition through microprotein SPAR and illustrates the power of non long coding RNAs in altering cellular functions. Statement of SignificanceMicroproteins encoded from long noncoding RNAs are emerging as critical regulators of many pathways. This study investigates a novel mechanism of SPAR microprotein that directly regulates the mechanistic target of rapamycin complex1 (mTORC1) signaling pathway through the lysosomal amino acid transporter SLC38A9. SPAR blocks both arginine transport and the downstream recruitment of Rag GTPases. These findings provide critical results in how SPAR controls cellular amino acid availability, while broadly highlighting the powerful regulatory mechanism of microproteins in cellular processes.

One of the first genes identified to regulate the spacing of nuclei in the multinucleated myofiber was Kinesin-1. However, the mechanism by which Kinesin-1 or other kinesins regulate myonuclear spacing is not known. Critically, the myofiber lacks centrosomes, and the many myonuclei act as the primary microtubule organizing centers of the cell. Because of this unique re-structuring, we hypothesized that the kinesins that drive centrosomes apart during mitotic spindle elongation may play a similar role in spacing myonuclei. We found that the bipolar Kinesin-5 (Klp61f) and the (-)-end directed Kinesin-14 (ncd) were both necessary for myonuclear spacing at different times, with both being necessary during embryogenesis, but only ncd being necessary in the fully differentiated myofiber. To investigate the shared mechanisms during embryogenesis, we used live-imaging and found that, similar to the mitotic spindle, Klp61f acts as an accelerator for myonuclear movement, whereas ncd acts as a brake contrary to this movement. To investigate these mechanisms and test the hypothesis that this is dependent on microtubule-sliding, we used super-resolution microscopy to visualize and quantify the microtubule network in animals with disrupted Klp61f or ncd. We found that in both cases, there was a decrease in the amount of microtubule overlap between neighboring myonuclei. Furthermore, we found that disrupting ncd led to severe changes in microtubule network organization, supporting our hypotheses that microtubule-sliding is necessary to space myonuclei, and that ncd likely functions through a unique mechanism in the differentiated myofiber to maintain myonuclear spacing. Together, our data supports a model where myonuclear spacing is regulated by a counteracting force generated by different kinesins during embryonic development. Furthermore, one kinesin, ncd, is repurposed in the differentiated myofiber to dynamically crosslink microtubules, a function necessary to anchor nuclei in place. Thus, kinesin motors regulate myonuclear spacing across developmental time by leveraging opposing forces through diverse mechanisms.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent inherited muscular dystrophies, for which there are no disease-modifying therapies. Metabolic perturbation, mitochondrial dysfunction, and oxidative stress are key contributors to FSHD pathology. Here, the effects of the metabolic regulator and anti-diabetic drug Metformin on myogenesis and muscle function in human and murine models of FSHD were investigated. Metformin did not affect the proliferation rate of human control or patient-derived FSHD myoblasts but promoted their myogenic differentiation, increasing myotube formation and maturation. Metformin also enhanced the metabolic health and viability of myotubes. Mechanistic interrogation revealed reduced levels of mitochondrial reactive oxygen species and modified mitochondrial turnover. These cellular investigations were complemented with in vivo functional assessment in a murine model of FSHD, in which Metformin treated mice exhibited significantly improved muscle strength. Collectively, these findings identify metabolic regulation as a therapeutically tractable feature of FSHD and demonstrate that Metformin improves muscle function in multiple models of FSHD via reduction of oxidative stress and augmentation of cellular metabolic fitness. These results provide insight into the therapeutic actions of Metformin and pre-clinical data to support its testing for repurposing in FSHD.

The methyltransferase EZH2 (Enhancer of Zest Homolog 2) and the demethylase KDM6B (Lysine Demethylase 6B) have been associated with epithelial to mesenchymal transition (EMT) and poor prognosis in various cancers. These enzymes methylate and demethylate H3K27me3 and regulate distinct sets of genes controlling EMT induction, despite having opposite catalytic activities. This could be due to their recruitment or the modulation of their activity by partner proteins on specific loci. This work sought to identify proteins associated with chromatin and interacting with EZH2 or with KDM6B during EMT. To do so, co-immunoprecipitation and mass spectroscopy was used under TGF{beta} (Tumor growth factor {beta}) and TNF (Tumor necrosis factor ) treatment to induce EMT in A549 lung cancer cells. Surprisingly, numerous proteins related to focal adhesions were identified to interact with EZH2 or KDM6B. These proteins are part of a nuclear protein interaction network previously described as nucleo-adhesome. Among these proteins, TGFB1I1 (transforming growth factor induced peptide 1) and CSRP2 (cysteine and glycine rich protein 2) were further confirmed to interact with KDM6B in the nucleus and even more so during EMT. The target genes of these complexes were then sought by knocking down KDM6B, TGFB1I1 or CSRP2. Three genes (coding Integrin alpha 5, Laminin y2 and Matrix Metalloproteinase 9) were confirmed to be regulated by KDM6B, TGFB1I1 and CSRP2. These findings may have clinical relevance, as immunohistochemistry analyses performed on a cohort of lung cancer patients revealed increased nuclear localization of TGFB1I1 and CSRP2 in cells undergoing EMT.

Background: Glucocorticoids cause skeletal muscle atrophy preferentially affecting fast glycolytic fibers, but the mechanisms involved in this fiber selectivity is unclear. REDD1 is a glucocorticoid-induced stress protein that limits muscle protein synthesis inducing atrophy. However, it remains unknown whether REDD1 exerts myofiber type-specific effects and through which precise mechanisms it regulates protein synthesis. We investigated the role of myofiber REDD1 expression in dexamethasone (DEX)-induced muscle atrophy, with a particular focus on its involvement in mitochondria-ER contact sites (MERCS), protein synthesis, and neuromuscular junction (NMJ) integrity. Methods: We generated tamoxifen-inducible, muscle-specific REDD1 knockout mice (REDD1fl/flHSA-CreERT2) and compared them with floxed littermates (WT) in a 2x2 design (WT/KO x PBS/DEX, 7 days). We combined single-nucleus RNA sequencing, RNAscope, immunofluorescence, transmission electron microscopy, proximity ligation assay, SUnSET puromycin labelling, western blot and RT-qPCR, and AdenoFATE1-mediated MERCS disruption in C2C12 myotubes. Results: Glucocorticoid receptor and REDD1 transcripts were co-enriched in fast glycolytic fibers mostly atrophied by DEX (~20%). REDD1 deletion in myofiber drove to lower basal muscle mass and fast fiber volume but protected them from DEX-induced atrophy. DEX inhibited protein synthesis (~70%) in WT mice with no matching change in Akt/mTOR-pathway activity. In REDD1 KO mice, protein synthesis was already low and was not affected by DEX. DEX-induced REDD1 expression remodelled mitochondrial network and MERCS in a subcellular compartment-specific manner. The intermyofibrillar MERCS minimum distance shortened in both genotypes reaching pathological distances only in WT mice (WT ~28 --> ~5 nm; KO ~25 --> ~15 nm). Perinuclear MERCS and mitochondria-nuclei distances increased in WT mice only (~18 --> ~45 nm and ~130 --> ~460 nm). In WT mice only, DEX-induced alteration of the perinuclear mitochondrial network was associated with a loss of myonuclei accumulating mt-RNA and exhibiting an anabolic transcriptomic signature notably enriched in sarcomeric transcripts. These findings suggest that REDD1-dependent MERCS remodelling may regulate muscle anabolism beyond the control of mRNA translation, by shaping the myonuclear transcriptome. Finally, REDD1 localised to the NMJ and reduced endplate area during DEX treatment. Interestingly, MERCS were denser in NMJ than in myofiber body and we showed in vitro that FATE1-mediated MERCS disruption was sufficient to reduce protein synthesis and agrin-induced acetylcholine-receptor clustering demonstrating that REDD1 and MERCS are important for NMJ stabilization. Conclusions: Muscle REDD1 links the glucocorticoid response to compartment-specific mitochondrial network remodelling, protein synthesis as well as NMJ stability in fast glycolytic fibers. Our results also show that REDD1 is important for maintaining basal mitochondrial network and protein synthesis homeostasis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by progressive motor neuron degeneration. Mutations in the SOD1 gene represent the second most common genetic cause of ALS (ALS), and distinct SOD1 missense variants present with markedly different clinical profiles. A4V leads to an aggressive form of the disease (median survival [~]1y), H46R confers a mild, slowly progressive course and I113T exhibits an intermediate phenotype. The molecular basis by which these mutations produce divergent clinical outcomes remains poorly understood. We performed extensive classical molecular dynamics simulations of wild-type SOD1 and the three ALS-associated variants in the apo monomeric state to attempt to investigate the mechanisms behind such phenotypic differences. Structural stability, global compactness, and conformational flexibility, as well as analysis of collective motions between residues and estimation of free energy, were assessed. The H46R, A4V, and I113T variants exhibited distinct dynamic behaviours, highlighting differences in structural stability, local flexibility, and intramolecular interactions. These findings suggest that specific structural regions may contribute differently to protein dysfunction and could represent key elements for understanding the relationship between molecular dynamic properties and the differing clinical severity associated with these variants. Most strikingly, H46R exhibited exceptional structural stability across every analytical level, the lowest global deviation, most attenuated local flexibility, strongest internal dynamic coordination, and the deepest, most confined free energy basins of any system examined. This convergent multi-layered evidence of structural restraint provides a compelling mechanistic basis for the mild and slowly progressive clinical course of H46R ALS, suggesting that enhanced conformational rigidity, rather than bulk destabilisation, is the defining biophysical feature of this variant, and that its pathogenic mechanism operates through a route fundamentally decoupled from the aggregation-driven toxicity that characterises the more aggressive SOD1-ALS mutations.

Centrosome positioning is governed by mechanical interactions between the actin and microtubule cytoskeletons, yet the contribution of different actin architectures and their attachment to the cell membrane remains unclear. We use numerical simulations to study a centrosomal array of microtubules interacting with cortical, bulk, and centrosome-associated actin networks, including passive cross-linkers, motor proteins, and ERM-mediated coupling of actin to the cell membrane. We find that bulk and centrosome-associated actin alone are insufficient for robust centrosome centering, whereas sufficiently dense cortical actin promotes centering through steric interactions with growing microtubules. ERM-mediated attachment of cortical actin to the cell membrane enhances this effect, allowing stable centering at lower actin densities. In a two-centrosome system, microtubule interactions generate a spindle-like structure, while the organization of actin and its membrane attachment through ERM regulate centrosome separation. Antagonistic microtubule motors further limit centrosome separation and establish a finite spindle length. These results demonstrate that ERM-mediated coupling of actin to the cell membrane provides an important mechanical link between the actin cortex and microtubule-driven centrosome positioning, influencing both centrosome centering and spindle geometry.

Injuries to individual cells happen frequently as a result of physiological and environmental stresses during their normal daily functions that can lead to a ruptured cell cortex (plasma membrane and underlying cortical cytoskeleton). The capacity of cells to rapidly repair general daily injuries, as well as ones resulting from trauma, infection, or diseases/cancer, is essential for their survival. While we know the general cell biological outline of the highly-conserved physiological events taking place during cell wound repair, our knowledge of the molecular mechanisms governing the repair process is still fairly limited, due in large part to the lack of information regarding the molecules, machineries, and pathways involved. Here, we performed a genetic screen of 1322 fluorescent-tagged proteins to identify cell wound repair components that are recruited upon laser wounding or whose expression is lost and/or altered upon laser wounding. We identified 129 proteins that are recruited to wounds during the cell repair process through high resolution spatio-temporal expression analyses of these gene fusions in conjunction with a fluorescent actin reporter. Strikingly, we find that many members of the Rab family GTPases are recruited to wounds where, in addition to their well-known roles in intracellular membrane trafficking, they are affecting actin cytoskeletal organization and dynamics during the repair process. These studies are allowing us to define the earliest acting proteins, as well as those required at specific steps in the repair process based on their recruitment patterns and the precise timing of their recruitment to wounds. Thus, our imaging-based screen is providing us with a global view of the repair processes, as well as a large number of genes/gene families that provide new entry points for examining specific steps in the cell wound repair process. Author SummaryCells in our bodies get injured every day from normal activity, environmental stress, infection, or disease. To survive, they must quickly repair these injuries and restore normal function. While some molecules have been identified as key players of cell wound repair, many of the molecules involved and their roles remain unknown. In this study, we identified new molecules that are involved in different steps of cell wound repair. Using laser-induced injury in the Drosophila model, we examined 1322 proteins and observed their spatial and temporal dynamics in a cell after injury. From the 1322 proteins examined, we identified 129 proteins recruited to distinct regions around the damage site during cell wound repair, suggesting roles in specific steps of the repair process. Interestingly, a subset of these proteins are Rab family GTPase members, highlighting new roles for these proteins in regulating actin dynamics. By identifying new candidate repair molecules, we provide a foundation for understanding how cells maintain their integrity and how repair processes may be influenced by factors such as wound size, infection, aging, and disease.

Endothelial-to-mesenchymal transition (EndMT) has become a central mechanism in developmental biology, fibrosis, vascular disease, and cancer. We previously reported on an integrated signaling model in which TGF-{beta}2 induces EndMT through coordinated activation of Smad-dependent and Smad-independent signaling pathways converging on Snail, while GSK-3{beta} regulates Snail activity. We performed a systematic figure-by-figure reproducibility analysis of the original publication. Independent studies published between 2011 and 2026 were identified and curated according to predefined inclusion criteria. Each original experimental conclusion was evaluated for independent confirmation. In parallel, selected biochemical experiments were independently reproduced using newly acquired reagents and contemporary Western blot methodologies. Independent publications consistently reproduced each major mechanistic conclusion of the original study, including activation of Smad, ERK, PI3K/AKT, and p38 MAPK signaling, regulation of Snail expression, EndMT-associated marker switching, and GSK-3{beta}-dependent control of Snail activity. Independent laboratory experiments reproduced the principal biochemical findings using contemporary reagents and experimental workflows. The combined literature analysis and independent laboratory replication demonstrate that the mechanistic framework in our previous study has remained reproducible across multiple laboratories, endothelial cell types, disease models, and fifteen years of investigation. This work illustrates a complementary framework for assessing reproducibility that integrates direct experimental replication with cumulative independent validation.

In cells, the disassembly of actin filaments is tightly controlled by molecular mechanisms where cofilin plays a central role. Cofilin binds to actin filaments cooperatively, forming clusters that lead to the severing of the filaments. Since cofilin binds preferentially filaments that have hydrolysed ATP and released inorganic phosphate (Pi), most studies have focused on the action of cofilin on ADP-actin filaments. Here, we ask how cofilin binds and severs filaments that still contain a large fraction of ADP{middle dot}Pi-actin. To answer this, we use microfluidics and fluorescence microscopy to monitor single filaments exposed to cofilin in controlled conditions, allowing us to disentangle and to quantify the different reactions. We find that the step limiting cluster nucleation is the binding of 2 cofilins on a minimal group of 3-4 contiguous ADP-actin subunits. Cofilin clusters then expand mostly in the direction of the pointed end of the filament, by easily binding to the ADP-actin neighbor of an ADP{middle dot}Pi-actin subunit, whose Pi release it accelerates. Severing at the ADP{middle dot}Pi-actin/cofilin cluster boundary is 30-fold slower than for an ADP-actin boundary. Globally, it seems that cofilin senses and modifies the nucleotide state of the actin subunits that are in its direct vicinity only. Our results provide a more comprehensive understanding of the action of cofilin, taking into account the nucleotide-state complexity of actin filaments.

Adult zebrafish efficiently regenerate skeletal muscle following different types of injury; however, the molecular programs involved in repair after extensive cryoinjury remain to be comprehensively characterized. Here, we explored the transcriptomic response of adult zebrafish skeletal muscle at 7 days post cryoinjury (dpci), a stage marked by ongoing tissue clearance, progenitor expansion, myogenic differentiation, and nascent myofiber formation, and compared it with phase-matched stab wound injury. Cryoinjury induced a broad transcriptional response, with 5,330 differentially expressed genes. Integrated enrichment and protein-protein interaction analyses revealed that, at 7 dpci, zebrafish skeletal muscle functions as an integrated regenerative system in which immune remodeling, progenitor expansion, myogenic differentiation, extracellular matrix reconstruction, mechanotransduction, biosynthetic adaptation, proteostasis, and intracellular trafficking operate simultaneously. In parallel, mature sarcomeric and oxidative metabolic programs were suppressed, consistent with ongoing tissue reconstruction and structural immaturity. Comparison with stab-wounded skeletal muscle revealed substantial transcriptional conservation, as 612 of 717 stab-wound-responsive genes (85%) were also differentially expressed after cryoinjury. Shared upregulated genes formed coherent functional modules related to proliferation, extracellular matrix organization and signaling, immune regulation, muscle differentiation, and protein processing. Thus, distinct injury modalities converge on a common regenerative program, while cryoinjury elicits a quantitatively broader transcriptional response. These findings support a conserved regenerative architecture of adult zebrafish skeletal muscle repair, in which interconnected biological modules act coordinately, with the breadth of transcriptional engagement reflecting regenerative demand.

In epithelial wounds, physically enlarged cells emerge along injury edges where they interact with regular-size cells during collective migration to repair tissue continuity. Although such large cells are often interpreted as migration leaders, recent observations suggest that regular-size cells can reciprocally influence them and that mixed-size cell clusters engage in distinct rotational motion before merging into confluent sheets. How cell size, polarity and adhesive coupling jointly control distinct collective migration modes remains unclear. Because many features of collective epithelial migration, including large and regular-size cell phenotypes, are conserved between in vivo and in vitro systems, here we developed a multi-scale computational model of interacting epithelial cells in two-dimensional culture with dynamic cell-substrate adhesion, cell-cell interactions, protrusion-based polarity, and contact-induced myosin redistribution as polarity regulator. We also explicitly modeled two functionally distinct cell states, featuring regular and large sizes respectively. Simulations showed that symmetric and asymmetric myosin redistribution at cell-cell junctions can produce stable cell contacts and persistent rotational motions by cell doublets. Intriguingly, in mixed-size cell clusters, straight translation can arise both from leader-like large cell and regular-size cell-mediated motion, whereas rotation emerges when large-cell-generated torque overcomes translation while cell-cell adhesion is maintained. Thus, collective migration mode depends on the balance among myosin-driven torque, regular-size cell-mediated translation, substrate coupling, and cell-cell adhesion. These modeling results suggest that collective epithelial migration can emerge in vitro from reciprocal biomechanical interactions between distinct cell states, rather than from leader-like cell behavior alone, and that such interactions can produce a predator-prey-like pursuit-escape mode of collective migration. Our model also provides a framework for investigating the biochemical and biomechanical regulation of collective cellular migration. It can be readily extended from in vitro to in vivo context and can incorporate the effects of substrate topology and soluble signaling factor-driven chemotaxis. Author summaryWhen epithelial cells repair a wound, they move as coordinated groups rather than as isolated individuals. Cells of different sizes may contribute in distinct but connected ways. We developed a computational model to examine how a large cell and neighboring regular-size cells move together. In the model, cells attach to the underlying surface and one another, form protrusions that set their direction, and redistribute the force-generating protein myosin after contact. We found that group motion depends on a balance among surface attachment, cell-cell adhesion, movement of regular-size cells toward the large cell, and myosin-driven turning of the large cell. Depending on this balance, a mixed-size cluster can travel along a nearly straight path or rotate persistently, exhibiting pursuit-and-escape-like interactions between the large cell and surrounding regular-size cells. Rotation occurs when the large cells turning effect outweighs the translational motion driven by regular-size cells, while cell-cell adhesion keeps the cluster together. Our results show that collective migration can emerge from reciprocal mechanical interactions between cells in different states, not only from a "leader" cell acting alone. The model also provides experimentally testable predictions for how cell size, adhesion, and internal myosin distribution shape cell cluster movement.

Purpose. To determine which proposed keratoconus (KC) mechanisms carry its genetic susceptibility and how it relates to corneal structure, and which describe its corneal tissue. Methods. Six mechanism modules and two attribution sets (transforming growth factor-{beta} [TGF-{beta}] and canonical type-2 atopy) were tested for enrichment in three independent European KC genome-wide association studies (GWAS) and in GWAS of central corneal thickness (CCT) and corneal resistance factor (CRF), with colocalization at matrix loci. Tissue expression was pooled across nine arms from eight studies with control-source meta-regression; activator protein-1 (AP-1)/immediate-early regulatory activity was scored in 107 donors, spatial expression in 21, and serum responses in human and rabbit stromal cells. Results. Extracellular matrix (ECM) was the only module enriched in all three KC sources (P [≤] 5.46 x 10-) and for CCT, CRF, and thickness-independent CRF (P [≤] 2.07 x 10-). COL5A1 shared causal variants with CCT in all three sources (posterior probability 0.985-0.991), and FNDC3B and COL1A1 with CRF or its thickness-independent component (0.961-0.986). Pooled, KC corneas had lower general and corneal-local inflammatory and stress/immediate-early expression (log2 fold-change -0.97, -0.98, -0.82; P [≤] 0.049). In whole-cornea and stromal tissue, ECM expression reversed between postmortem-donor and pathological controls (log2 fold-change +0.36, -2.34), whereas inflammatory and TGF-{beta} programs were lower against both. AP-1/immediate-early regulatory activity was lower in four donor cohorts (Hedges' g -1.50) and stress/immediate-early expression in two spatial cohorts; serum raised immediate-early readouts in human and rabbit cells. Conclusions. KC combines matrix-mechanical genetic susceptibility with a low-inflammatory, low-immediate-early corneal state whose matrix expression direction depends on the control source. Because this susceptibility is genetically shared with corneal thickness and resistance, interventions on the inherited disease are judged by corneal structure and mechanics.

BackgroundLamins are major regulators of the spatial and functional organization of chromatin. Lamins at the nuclear periphery form the lamina that anchors heterochromatin to the nuclear envelope. A subpool of A-type lamins localizes in the nuclear interior, where they also bind to euchromatic genomic regions. A-type lamin properties and chromatin association are regulated by lamin-associated polypeptide 2alpha (LAP2). Here we systematically analyze, how LAP2 depletion affects chromatin organization, accessibility and gene expression on a genome-wide level. ResultsLAP2 depletion in mouse dermal fibroblasts positively and negatively affects chromatin accessibility and gene expression throughout the genome, which correlates with changes in chromatin association of A-type lamins and the nucleosomal remodeler proteins BRG1 and CHD4. In particular, A-type lamins bind to open chromatin regions close to BRG1 and CHD4 binding sites and deregulated genes, but do not directly accumulate on genes and BRG1 and CHD4-enriched sites. Unsupervised clustering of the datasets on LAP2-bound genomic regions confirms spreading of A-type lamins to active chromatin regions containing deregulated genes and an enrichment of chromatin remodelers on a subset of these genomic regions. ConclusionsLAP2 depletion in fibroblasts leads to a gross rearrangement of chromatin. Genome-wide chromatin reorganization is linked to spreading of A-type lamins to active chromatin regions and accompanied by a restriction of chromatin remodelers to a subset of active genomic regions. These changes correlate with changes in chromatin accessibility and gene expression throughout the genome, particularly in regions where lamin binding is gained in LAP2 knockout versus wildtype cells.