European Journal of Cell Biology

Intervertebral disc degeneration is associated with loss of nucleus pulposus (NP) cell phenotype and extracellular matrix, both processes linked to changes in cytoskeletal contractility and cell shape. Here, we tested whether microenvironment-specific modulation of RhoA signaling can restore NP-like morphology and gene expression in NP cells cultured in 2D and in 3D alginate. In 2D monolayer culture, where cells are spread and mechanically activated, pharmacologic inhibition of RhoA with CT04 reduced RhoA activity, decreased actomyosin contractility gene expression, and shifted morphology toward a smaller, more circular phenotype. Bulk RNA sequencing showed that CT04 treatment increased expression of NP phenotypic and matrix-related genes including ACAN, GDF5, CHST3, and MUSTN1 while decreasing expression of catabolic and fibroblast-associated genes including ADAMTS1/9 and COL1, consistent with enrichment of extracellular matrix pathways. In contrast, RhoA activation with CN03 in 2D culture increased actin and phosphorylated myosin light chain intensity but produced limited phenotypic improvement. In 3D alginate, which minimizes integrin-mediated adhesion, baseline actomyosin markers were reduced relative to 2D culture. In alginate, RhoA activation with CN03 increased the amount of actin, phosphorylated myosin light chain, and actomyosin gene expression, yet also promoted a more compact, circular morphology and increased NP markers, including ACAN and KRT19 with repeated dosing. Across culture conditions, increased cell roundness was consistently associated with increased ACAN expression, indicating strong coupling between cytoskeletal state, morphology, and NP matrix programs. Together, these findings demonstrate that RhoA pathway perturbation can promote NP phenotypic gene expression in both 2D and 3D culture, but the direction of optimal modulation depends on the microenvironment, supporting RhoA signaling as a context-dependent therapeutic target for disc regeneration.

The extracellular matrix (ECM) is a meshwork of proteins that orchestrates a broad range of cellular phenotypes, including proliferation, adhesion, migration, and differentiation. SNED1 is a newly characterized ECM glycoprotein that promotes cell adhesion and is essential for embryonic development. Its upregulation is also associated with breast cancer metastasis and poor prognosis for breast cancer patients. We recently showed that SNED1 assembles into fibrillar structures, but the mechanisms guiding its incorporation into the ECM scaffold remain unknown. Combining biochemical assays and confocal immunofluorescence imaging, we found that SNED1 assembly in the ECM occurs early in the process of ECM building and is concomitant and overlaps with the deposition of fibronectin and collagen I, two major ECM proteins. By knocking down fibronectin or destabilizing collagen I fibers, we further demonstrate that SNED1 requires the presence of these proteins for its assembly. Last, using biolayer interferometry, we identify collagen I as the first direct binding partner of SNED1. Altogether, our results lay the foundation for future studies aimed at determining the mechanisms by which SNED1 fibers contribute to SNED1 pathophysiological functions. SUMMARY STATEMENTThe novel protein SNED1 requires the presence of fibronectin and collagen I to assemble into fibrillar structures in the extracellular matrix scaffold.

Cartilage is characterized by a highly specialized extracellular matrix (ECM) secreted by chondrocytes and limited self-regenerative capacity. In vivo investigations of chondrogenesis are limited by difficult and traumatic access, especially in humans. While it is known for decades that disturbances of chondrocyte differentiation and changed cartilage ECM composition cause severe skeletal phenotypes in vertebrates, a detailed molecular understanding of chondrogenesis and cartilage ECM formation is still missing, especially in the context of human genetic skeletal diseases. ATDC5 cells, derived from AT805 mouse teratocarcinoma cells, have been used in the past to model chondrogenic differentiation, however, most studies have investigated few major cellular differentiation markers only so that the composition of the secreted ECM as well as effects on the ATDC5 transcriptome upon differentiation are still unclear. Here, we performed time-resolved transcriptomic and ECM proteomic analyses of differentiating ATDC5 cells. Both datasets confirmed the formation of a cartilage-like matrix with increasing expression of key chondrocyte genes over the course of differentiation. ECM proteomics further revealed a number of ECM components not previously reported in ATDC5 cells or the secreted ECM, encompassing collagens, proteoglycans, glycoproteins and other secreted factors. Overall, our findings provide a more detailed molecular characterization of ATDC5 chondrogenesis and highlight the potential of this model system for ECM-focused studies.

Cells are mechanosensitive, responding to external mechanical stimulation. Nanovibrational stimulation has been shown to enhance cell contractility and actin stress fibre formation. These changes in morphology occur quickly, alongside associated mechanical changes. Here, the relationship between acute morphological and mechanical changes in NIH 3T3 fibroblastic cells in response to nanovibrational stimulation is presented. A 1 kHz, 30 nm vibration is applied continuously for 72 hours. Atomic force microscopy (AFM) quantifies mechanical properties of the nucleus and cytoplasm at multiple timepoints, while immunofluorescence tracks morphological changes. Within 3 hours of stimulation, both nuclear and cytoplasmic stiffness increase significantly, accompanied by a decrease in the cellular fluid exponent, suggesting a shift of the cell towards more solid-like behaviour. These changes correlate with increased nuclear area. Actin polymerisation also increases within 24 hours, although variably. To understand the role of the cytoskeleton, actin polymerisation and contraction are inhibited using cytochalasin D and blebbistatin. Results show that inhibition prevents stiffness increases and results in a higher fluid exponent, indicating a more fluid-like state. These findings demonstrate that actin-myosin dynamics mediate cell stiffening under nanovibrational stimulation. Interestingly, prolonged stimulation appears to reverse this effect, suggesting that temporal optimisation of stimulation may enhance long-term mechanotransducive responses.

Mechanosensing involves proteins detecting mechanical changes in the cytoskeleton or at cell adhesion sites. These interactions initiate signaling cascades that produce biochemical effects such as post-translational modifications or cytoskeletal rearrangements. Filamin is a ubiquitous mechanosensing protein that binds actin filaments and senses pulling forces within the cytoskeleton. Drosophila Filamin (Cheerio) is structurally similar to mammalian Filamin, with roles in egg chamber development, embryo cellularization, and integrity of muscle attachment sites and Z discs in Drosophila indirect flight muscles (IFMs). Here we report a potential novel binding partner of Drosophila Filamins: the death-associated protein kinase Drak that functions as a myosin light chain kinase. We found that Drak biochemically bound to an open mutant of Filamin that resembles the mechanically activated form partially bound to wild type Filamin and did not bind to closed mutant of Filamin. The interaction site was mapped to the intrinsically unfolded C-terminal region of Drak. To study the functional role of Drak-Filamin interaction, we studied two developmental events where Drak has been earlier shown to be expressed and where Filamin also functions: early embryonic cellularization and indirect flight muscle development at pupal stages. We found partial colocalization between Drak-GFP and Filamin-mCherry during the initiation of cellularization furrow, and at the time of myotube attachment site maturation in tendon cells. However, functionally we could not show direct correlation between Filamin and Drak. Our studies reveal interesting new expression patterns of Drak during Drosophila development and provide detailed information about Filamin localization during IFM development.

Missense mutations in the TPM2 gene encoding skeletal muscle tropomyosin Tpm2.2 cause congenital myopathies associated with hyper- and hypocontractile phenotypes. Mutation-dependent defects in thin filament stability and length maintenance may contribute to sarcomere dysfunction. To address this possibility, four disease-associated substitutions in Tpm2.2 were analyzed: hypercontractile D20H and E181K, and hypocontractile E41K and N202K. Recombinant proteins were examined in vitro for their effects on actin filament polymerization, stability, and cofilin-2-dependent filament length regulation in the absence and presence of troponin (+Ca2+). Wild-type Tpm2.2 inhibited spontaneous actin polymerization and reduced polymerization cooperativity in the presence of cofilin-2. Hypercontractile substitutions D20H and E181K further decreased the polymerization rate, whereas hypocontractile variants had little effect. Under ATP-driven actomyosin interactions, E41K and N202K stabilized filaments, resulting in increased filament length, but this effect was abolished by troponin. All variants slightly decreased cofilin-2 affinity for F-actin without affecting cooperativity. Troponin prevented displacement of Tpm2.2 from the filament at increasing cofilin-2 occupancy, indicating concomitant binding of all proteins to the thin filament, consistent with a structural model based on high-resolution F-actin-Tpm-Tn and cofilactin structures.Tpm2.2-N202K inhibited cofilin-2-dependent depolymerization, whereas Tpm2.2-E181K increased susceptibility to depolymerization. Although cofilin-2 induced filament severing in all cases, the Tpm2.2-Tn complex protected filaments from disassembly. These findings support a model in which the Tpm2.2-Tn complex forms a cooperative regulatory strand that constrains filament dynamics and transmits structural perturbations along the filament. Disease-causing substitutions differentially alter filament length and stability, potentially contributing to the pathogenesis of myopathies.

ObjectiveAnterior cruciate ligament (ACL) tears increase the risk for developing posttraumatic osteoarthritis (PTOA). Females have greater risk for both. However, studies defining sex-specific protein responses in human cartilage after ACL injury are lacking. We hypothesize that articular cartilages response to an injurious environment differs depending on sex. DesignWe compared the proteomic profiles of normal cartilage with injured cartilage harvested from the intercondylar area during ACL surgery. Sex-specific injury effects were estimated through contrasts between Injured Male and Normal Male and between Injured Female and Normal Female. Pathway enrichment analysis was done using gene ontology (GO) and compared against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Extracellular matrix (ECM) proteins were further analyzed using the Matrisome AnalyzeR. ResultsFrom the 2,188 proteins identified, males and females shared 1,121 upregulated and 23 downregulated proteins in injured compared to normal cartilage. Analysis of ECM proteins and enriched pathways revealed mostly similar male and female responses to an injurious environment, with evidence of early cartilage remodeling in both sexes. Nevertheless, more than 240 proteins were affected specifically by sex, and significant sex differences were found in inflammation, ECM-related, and metabolic pathways. Males were enriched mostly in "ECM-receptor interaction", while females were enriched in "Citrate cycle (TCA cycle)", "Fatty acid degradation", and "Fatty acid metabolism" pathways. ConclusionArticular cartilage shows signs of remodeling soon after ACL injury, even when only exposed to an injurious environment rather than being physically impacted. Sex differences were observed in inflammation, metabolic pathways, and ECM synthesis.

A hallmark of damaged skeletal muscle fibers is displaced myonuclei that are no longer peripherally positioned. Displaced myonuclei are dogmatically thought to be derived exclusively from muscle stem cell (satellite cell) fusion. Using a surgical resection muscle injury model and in vivo recombination-independent resident myonuclear labeling, we detail the prevalence, time course, and origin of displaced myonuclei in response to a non-chemically-mediated muscle trauma. We found that: 1) non-satellite cell-derived (resident) displaced myonuclei emerge seven days after surgical injury in similar proportion to exogenous (satellite cell-derived) displaced myonuclei in intact muscle fibers, with a biased prevalence in myosin heavy chain IIB muscle fibers, 2) muscle fibers with multiple ([≥]2) displaced resident myonuclei was an unexpected but noteworthy feature of muscle fibers 7 days after injury, 3) embryonic myosin-expressing fibers at seven days post-surgery expectedly contain predominantly satellite-cell derived displaced myonuclei, but a subset have displaced resident myonuclei, and 4) satellite cell numbers in intact muscle do not increase until 7 days post-surgery. These data may help inform whether to target satellite cell-initiated processes, myonuclear-initiated processes, or both to facilitate muscle fiber injury repair. This information could lead to more effective therapeutic strategies for treating muscle trauma.

BackgroundPhysical loading mediates postnatal growth, homeostasis, and healing of the tendon and its attachment to bone, which is critical for rotator cuff functional integrity. Our prior studies have highlighted the mechano-sensing role of primary cilia; However, the mechanisms through which cilia convert mechanical stimuli into structural functional adaptation under altered loading conditions remain unanswered. MethodsPublicly available scRNA-seq datasets of mechanically loaded human patellar tendon cells were re-analyzed to identify cilia-related transcriptional changes. Tendon-specific cilia knockout mice (ScxCre;Ift88fl/fl) and wild-type controls (Ift88fl/fl) underwent mechanical unloading induced by botulinum toxin A injection, followed by micro-computed tomography, biomechanical testing, histology, qPCR, and immunohistochemistry to evaluate structural, mechanical, and Hedgehog (Hh) signaling responses. Primary tendon fibroblasts from wild-type and cilia-deletion mice were treated with Hh agonist or antagonist to assess Hh signaling responsiveness in vitro. Students t-test for two groups and two-way ANOVA for two groups with two treatments were performed for our statistical analysis. ResultsHere, we find that mechanical force causes changes in cilia- and hedgehog (Hh)-related gene expression in human tendon fibroblasts. Cilia ablation in the enthesis blunts force-driven remodeling of tissue structure and mechanical strength. Cilia deletion also leads to impaired Hh signaling in tendon cells and decreased responsiveness to activation and inactivation of hedgehog signaling. ConclusionsOur results demonstrate loading-regulated ciliary Hh signaling during postnatal growth of the tendon and enthesis and provide proof-of-concept for developing new cilia-targeted mechanical and biological therapies for enthesis repair.

Muscle satellite cells (MuSCs) are muscle-resident stem cells that are responsible for myofiber regeneration. Although the importance of calcium ions (Ca2+) in muscle physiology has been well established, the mechanism by which Ca2+ mobilization governs MuSC function remains poorly understood. In this study, we aimed to systematically characterize Ca2+ dynamics in MuSCs and to define the mechanisms regulating these signals during muscle regeneration. By employing modified protocols for mouse MuSC isolation and Ca2+ measurement, we observed spontaneous Ca2+ fluctuations in MuSCs isolated from regenerating muscle after cardiotoxin-induced myofiber injury. Our detailed analysis using chemical Ca2+ indicators and a genetically encoded Ca2+ indicator revealed that the frequency and amplitude of Ca2+ fluctuations increased significantly during the activated and proliferative stages of MuSCs in muscle regeneration. This effect was more pronounced in MuSCs isolated from dystrophic and aged mice. Mechanistically, these Ca2+ fluctuations were at least partially mediated by mechanosensitive ion channels, including PIEZO1 and TRPM7, which promote MuSC migration. Collectively, our findings demonstrate that Ca2+ fluctuations through mechanosensitive ion channels act as a key regulator of MuSC activation during muscle regeneration and may provide new insights into the role of Ca2+ influx in muscle biology and the pathogenesis of muscle diseases.

Accurate chromosome segregation relies on proper centromere and kinetochore formation and phospho-regulation. We previously demonstrated that a pluripotent state confers a low fidelity of chromosome segregation, however it is unknown how a pluripotent state impacts centromere and kinetochore function. Here, we demonstrate that both centromere and kinetochore structural organization and phosphorylation in mitosis are developmentally regulated. CENP-A, CENP-C, and HEC1 protein abundance is reduced at mitotic centromeres and kinetochores of human pluripotent stem cells (hPSCs) compared to isogenic somatic cells; however, elevating their levels does not improve chromosome segregation fidelity. Rather, we find that reduced phosphorylation of kinetochores is responsible for their low fidelity. HEC1 is hypophosphorylated at kinetochores of hPSCs compared to isogenic somatic cells at Cyclin B/Cdk1 and Aurora kinase phospho-sites. Inhibiting PP2A phosphatase activity or differentiation increases HEC1 phosphorylation at hPSC kinetochores decreasing chromosome segregation errors. Thus, mitotic fidelity in non-transformed human cells depends on the developmental regulation of the kinase and phosphatase networks controlling kinetochore phosphorylation. SummaryGalaviz Sarmiento et al show that the developmental regulation of kinetochore phosphorylation governs mitotic fidelity. HEC1 is hypophosphorylated at kinetochores of hPSCs during mitosis contributing to their high rate of chromosome segregation errors. While differentiation increases HEC1 phosphorylation improving chromosome segregation fidelity.

Nicotinic acetylcholine receptors (nAChRs) belong to the pentameric ligand-gated ion channel superfamily (pLGICs). Among them, the neuronal homomeric 7 nAChR is highly permeable to calcium and plays critical roles in synaptic transmission, cell signaling, and inflammation modulation. The biogenesis of 7 nAChRs is enhanced by the chaperone proteins RIC-3 and NACHO. Previously, we reported a motif in the 5-HT3A receptor, another pLGIC, involved in RIC-3 modulation. Residues in this motif are conserved and also found within the L1-MX segment of the 7 nACh subunit. We therefore explored the regulatory roles of these conserved residues in the biogenesis of 7 nAChRs using multiple approaches, including heterologous expression in Xenopus laevis oocytes, mutagenesis, pull-down assays, cell-surface labeling, and two-electrode voltage-clamp (TEVC) recordings. We find that synthetic 7 L1-MX peptide interacts with both RIC-3 and NACHO. In particular, conserved residues W330, R332, and L336 in the L1-MX positively regulates the assembly of 7 oligomers and the biogenesis of 7nAChR. In presence of residues W330, R332, and L336, NACHO promotes an assembly of an 7 pentamer which is resistant to strong denaturing conditions. NACHO-promoted 7 pentamer is also resistant to Endo H enzyme. Sensitivity of the pentamer to moderate temperatures (37 {degrees}C, 45 {degrees}C, and 50 {degrees}C) suggests that NACHO stabilizes the pentamer via non-covalent interactions. In contrast, Ala replacements at these residues disrupt the biogenesis and abolish 7 current. NACHO and RIC-3 co-expression yields partial rescue of functional expression for some Ala replacement constructs. SUMMARYThis work identifies regulatory roles of conserved residues W330, R332, and L336 in the biogenesis of 7 nAChR. This discovery positions MX subdomain as a promising target for future drug development that can minimize adverse effects.

The intracellular transport system is pivotal for cellular function and integrity, facilitated by cytoskeletal motor proteins such as dynein, which traverse along microtubules (MTs). The heterogeneity of the tubulin isotypes composing MTs introduces functional diversity, potentially affecting cytoskeletal motor proteins interactions with the MT. This in silico study investigated the influence of amino acid sequence variations in the C-terminal tails (CTTs) of six different Homo sapiens tubulin isotypes, TUBB2A, TUBB2B, TUBB2C, TUBB3, TUBB4A, and TUBB5, highly expressed in human brain tumors, and assessed the isotypes effect on the binding of motor protein dynein to MT. Among these isotypes, TUBB2A, TUBB2B, and TUBB2C were found to affect conformational motions of the dyneins microtubule-binding domain (MTBD) and stalk domain. The investigation highlighted the novel role of isotype-specific variations in lateral interactions between tubulin protofilaments (PFs) in determining the proximity of the {beta}-CTT of the adjacent PF to the MTBD, potentially affecting dyneins motility and suggesting how changes in isotype expression directly influence dyneins velocity and processivity and contribute to transport defects associated with neurological disorders and cancers. Isolating specific tubulin isotypes experimentally is challenging due to their high sequence similarity and complex interactions with other microtubule-associated proteins. This makes it challenging to distinguish between different tubulin isotypes and their effects, particularly in tissues where multiple isotypes are co-expressed. Additionally, these isotypes are heavily modified in vivo by post-translational modifications, which further complicate the isolation of a single, unmodified tubulin isotype. As a result, computational approaches have been necessary in this study for exploring these effects in a controlled, isotype-specific manner.

As human spaceflight becomes increasingly relevant, understanding how microgravity affects the human brain is an important but largely unexplored question, particularly in the context of neuronal function and vulnerability to neurodegeneration. Direct investigation of these processes in humans is not feasible, necessitating the use of physiologically relevant in vitro model systems. Three-dimensional human brain organoids recapitulate key aspects of brain development and organization and provide an experimentally accessible platform to study neuronal responses under controlled conditions. Here, within the framework of the student competition "Uberflieger 2", we investigated the effects of long-term microgravity on human midbrain organoids cultured for 40 days aboard the International Space Station (ISS). Midbrain organoids reproduce essential features of dopaminergic neuron development and are widely used to model Parkinsons disease and related neurodegenerative processes. To enable spaceflight experiments, we developed and implemented an autonomous culture system adapted to the constraints of the ISS environment. During the mission, a hardware malfunction impaired scheduled medium exchange, introducing an additional metabolic stress condition. Despite these limitations, ISS-cultured organoids remained viable and showed robust neurite outgrowth. Molecular and imaging analyses revealed that exposure to microgravity in combination with nutrient limitation induced a coordinated response involving cytoskeletal remodeling, neuronal plasticity, and selective vulnerability of dopaminergic neurons. These findings demonstrate that human midbrain organoids can maintain key structural and functional properties under prolonged spaceflight-associated stress while activating adaptive response programs. This work highlights the potential of organoid-based systems to investigate neurobiological effects of microgravity and provides a foundation for future studies addressing mechanisms relevant to neurodegenerative disease.

Triggering receptor expressed on myeloid cells 2 (TREM2) plays a crucial role in regulating various microglial functions, including phagocytosis, inflammation, chemotaxis, and proliferation. Recent studies have demonstrated that TREM2 cooperates with DAP12 to mediate intracellular signaling essential for these processes. Despite the importance of the TREM2-DAP12 complex in microglial physiology, the mechanisms controlling its expression and activity remain poorly understood. In this study, we report that the soluble ectodomain of TREM2 (sTREM2) regulates microglial phagocytic activity by attenuating the surface expression of DAP12. We found that stimulation of the microglial cell line BV2 with recombinant sTREM2 reduces the membrane expression of DAP12, but not that of TREM2. In addition, sTREM2 binds to full-length TREM2, leading to the uncoupling of TREM2 from DAP12. Furthermore, pre-treatment of BV2 cells with sTREM2 significantly inhibited amyloid-{beta} incorporation. These findings suggest that sTREM2 negatively regulates TREM2 signaling through the destabilization of the TREM2-DAP12 complex, and act as a novel bioactive molecule that modulates TREM2 signaling under physiological and pathological conditions.

The extracellular matrix (ECM) is a complex scaffold of proteins that supports multicellular structures. Interactions between cells and the ECM via receptors, like integrins, govern cellular phenotypes (e.g., proliferation, adhesion), but also contribute to ECM assembly. Understanding how ECM-receptor interactions regulate matrix assembly is critical to uncover how alterations of the ECM cause or accompany congenital diseases, cancer, or fibrosis. SNED1 is a novel ECM protein with roles in development and metastasis. However, the mechanisms governing its assembly and signaling functions remain largely unknown. SNED1 contains two integrin-binding motifs, RGD and LDV, and we recently showed that its interaction with RGD-integrins mediates cell adhesion. Here, we investigated the role of SNED1/integrin interactions in SNED1 ECM assembly. While SNED1/integrin interactions were not necessary for its initial incorporation in the ECM, interaction with LDV-, but not RGD-, integrins, was required for ECM build-up and the patterning of SNED1 and the fibrillar proteins fibronectin and collagen I. Moreover, SNED1/LDV-integrin interaction promoted ECM alignment, cell alignment, and cell proliferation, processes essential to SNED1-driven neural crest cell migration during craniofacial development and breast cancer invasion. SUMMARY STATEMENTInteraction of SNED1 with LDV-binding integrins, but not RGD-binding integrins, mediates ECM remodeling and controls cytoskeletal rearrangement and cell proliferation.

Eya3 and Eya4 are two Eya genes expressed in adult myogenic stem cells, where they may act as SIX cofactors. We analyzed muscle regeneration in single and compound Eya3 and satellite cell-specific Eya4 mutant mice. A kinetic analysis of muscle regeneration after Notexin injury of the Tibialis Anterior revealed no major phenotype at 4, 14, and 30 days after injury in terms of PAX7+ cell number and myofiber cross-sectional area in Eya3 mutants, while all parameters were decreased in Eya4 mutants and further worsened in Eya3/Eya4 double mutants, in which we also observed a modification of the myofiber phenotype at 30 days after injury. Satellite cells were cultured ex vivo and Eya4 deletion was induced by Ad-Cre-mediated recombination. While single Eya3 mutant cells showed normal proliferation and differentiation, double mutant cells exhibited normal proliferation but failed to fuse. Analysis of their transcriptome revealed that the expression of Myomixer, Follistatin, and Noggin was severely downregulated specifically in double mutant cells, explaining their fusion deficiency. To gain a better understanding of the involvement of Eya genes during embryonic development and the genesis of PAX7+ myogenic stem cells, we analyzed Eya1 / ;Eya2 / , Eya3 / , Eya4 / , and Eya3 / ;Eya4 / E18.5 mutant fetuses at the limb and craniofacial levels. In Eya1 / ;Eya2 / fetuses, we confirmed the absence of distal limb muscles and observed reduced craniofacial muscles. In Eya3 / ;Eya4 / fetuses, craniofacial myogenesis appeared preserved and PAX7+ myogenic stem cells were present. BackgroundThe Eyes absent (Eya) genes encode transcriptional co-activators and phosphatases that function within the PAX-SIX-EYA-DACH (PSED) regulatory network. In skeletal muscle, EYA proteins cooperate with SIX homeoproteins to control myogenic gene expression during both embryonic development and adult regeneration. While Eya1 and Eya2 are predominantly expressed in embryonic myogenic progenitors and Eya3 and Eya4 are the dominant paralogs in adult satellite cells (SC), the specific and redundant contributions of individual family members to myogenesis remain poorly characterized. MethodsWe analyzed compound Eya mutant mice during adult Tibialis anterior muscle regeneration and during embryogenesis. We complemented this analysis by performing ex vivo myogenic stem cell cultures from compound Eya mutants and examining their fusion capacity. ResultsAnalysis of muscle regeneration following Notexin injury revealed that Eya2 and Eya3 single mutants display no major regenerative deficit. In contrast, satellite cell-specific deletion of Eya4 (Eya4sc/sc) caused a transient impairment of early regeneration, with reduced numbers of smaller regenerating MYH3+ (embryonic myosin heavy chain) myofibers and a transient decrease in SC number at 4 days post-injury (dpi). Compound Eya3-/-;Eya4sc/scdouble mutants showed a more severe and persistent phenotype, with decreased myofiber cross-sectional area, reduced myonuclear accretion, accumulation of PAX7+ cells associated with regenerated myofibers, and altered fiber-type composition at 14 and 30 dpi. Ex vivo analysis of double mutant SCs revealed a specific and complete blockade of myogenic fusion without defects in proliferation or MYOD expression. Transcriptomic analysis identified severe downregulation of Myomixer, Noggin, and Follistatin in differentiating Eya3-/-;Eya4-/- SCs. Open-access SIX1 and SIX4 ChIP-seq publicly available data confirmed direct binding at the Myomixer, Noggin, and Follistatin loci, supporting a direct SIX-EYA transcriptional mechanism. In parallel, embryonic analysis demonstrated that Eya1-/-;Eya2-/-E18.5 fetuses lack distal limb musculature and display severe craniofacial muscle hypoplasia, while in Eya3-/-;Eya4-/-fetuses limb and craniofacial musculature developed with no detectable defects. ConclusionsThese results reveal distinct temporal requirements for EYA proteins in skeletal muscle: EYA1 and EYA2 are essential SIX cofactors for embryonic myogenic fate acquisition in hypaxial and craniofacial progenitors, while EYA3 and EYA4 act redundantly in adult satellite cells to enable myogenic fusion by maintaining BMP antagonist expression and Myomixer activation downstream of the SIX-EYA transcriptional complex.

Microtubules are cytoskeletal structures composed of polymers of /{beta}-tubulin heterodimers. They play a central role in cell division and motility by a stochastic process of alternating polymerization and depolymerization episodes (dynamic instability) that can be modulated by phosphorylation. Protein kinase C and cyclin-dependent kinase 1 are known to phosphorylate Ser165 of -tubulin (:Ser165) and Ser172 of {beta}-tubulin, ({beta}:Ser172), respectively. Using all-atom molecular dynamics simulations of 6-mer {beta}-tubulin systems modeled on the cryo-EM structure of a microtubule (PDB 3J6E), the impact of phosphorylation at each site is explored in terms of secondary structures (:helix H8/loop T7 segment and {beta}:loops T3/T5) that lie at the inter-dimer cleft near the E-site {beta}:GTP. If properly aligned, :Glu254 (helix H8) hydrolyzes {beta}:GTP to GDP thereby triggering the transition from a polymerizing to a depolymerizing microtubule. -Tubulin phosphorylated at :Ser165 displaces helix H8 (:Glu254/:Gln256) and loop T5 towards the {gamma}-phosphate of {beta}:GTP. This movement coincides with a shift of the {beta}:GTP nucleotide by 4.5-5.5 [A], stabilization of the {gamma}P of {beta}:GTP by additional H-bonding and weakened inter-dimer interactions. In a phosphorylated {beta}:Ser172 system, loop T5 is displaced toward {beta}:GTP and coincides with stabilization of inter-dimer interactions. Therefore, phosphorylation of either - or {beta}-tubulin generates a distinct profile of intramolecular rearrangements that remodel the inter-dimer cleft and modulate dynamic instability. These profiles may provide a useful reference for screening mutations identified in tumor genomes.

AAA proteases are hexameric ATP-dependent metallopeptidases that perform crucial proteolytic activities within prokaryotic and eukaryotic membranes. Structurally, protomers are comprised of catalytically active C-terminal domains that are anchored to the membrane by an N-terminal autonomous folding unit. In this study, we determined the fold, stability, and oligomeric state of the N-terminal intermembrane domains of human spastic paraplegia type 7 (SPG7)/ paraplegin protein and its bacterial orthologue FtsH using circular dichroism (CD), small-angle X-ray scattering (SAXS), small-angle neutron scattering (SANS) and X-ray crystallography. Solution-state analysis revealed that the N-terminal domain of paraplegin is a monomer in solution whereas FtsH forms a dimer. Unexpectedly, the N-terminal domain of paraplegin presents as a domain-swapped homodimer in our crystal structure that involves the first helix and first two beta-strands from one monomer and beta-strand 3, helix 2 and beta-strand 4 from another symmetry-related molecule. However, together they form an assembly which is similar to protomers observed for the N-terminal regions of FtsH and AfG3L2. Drawing from our structural data, we postulate that domain-swapping interactions of the N-terminal regions contribute to stability of the AAA protease hexamer containing paraplegin, demonstrating the extensive flexibility of the N-terminal portion of this protein and its role in achieving the appropriate molecular architecture required for function. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/720153v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1f4b9b5org.highwire.dtl.DTLVardef@1cc2242org.highwire.dtl.DTLVardef@dd211borg.highwire.dtl.DTLVardef@1a87722_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFtsH-IMS forms a homo-dimer in solution, whereas paraplegin-IMS presents as a well-folded monomer in solution C_LIO_LIparaplegin-IMS crystallises as a domain-swapped homo-dimer but its domain-swapped monomers are structurally similar to other IMS-regions C_LIO_LIAfG3L2/paraplegin hexamer formation may be supported by domain swapping in paraplegin-IMS C_LIO_LIdomain-swapping in paraplegin could be a Bonafide feature under certain cellular conditions and may be related to disease in spastic paraplegia C_LI

Motile cells can sense and exert forces on the extracellular environment through dynamic actin networks. Increased stress against the polymerizing barbed ends of branched actin networks has been shown to lead to an increase in the density of these networks through a force feedback mechanism, though this phenomenon has not been explored through the examination of real-time responses of endogenous actin networks in cells. Here, we utilize mouse embryonic fibroblast CRISPR knock-in lines with labeled ARP2/3 complex to identify cellular and extracellular conditions that regulate branched actin density and enrichment at the leading edge of lamellipodial protrusions. A common theme shared among all branched actin density-increasing conditions is higher levels of interface stress between the plasma membrane and the barbed ends of the lamellipodial actin network. Among these conditions, we find that ARP2/3 is specifically required for robust spreading and protrusion in response to increased extracellular viscosity. Interestingly, time-lapse traction force microscopy of ARP2/3-dependent viscosity responses show significantly reduced changes in strain energy applied to the substrate when compared to spreading and motility through cell-matrix adhesion. In addition, we find that increased extracellular viscosity can bypass the need for extracellular matrix proteins to support lamellipodial protrusion driven by optogenetic Rac activation. Our studies provide strong support for in vitro models of branched actin force feedback responses and further characterize an essential role for branched actin in mediating dramatic cell shape changes in response to increased extracellular viscosity.