European Journal of Cell Biology

Cell migration depends on coordinating cell shape changes with force generation, yet how these processes are integrated remains unclear. Here, we combine live-cell imaging with traction force microscopy and computational analysis to quantify cell morphology, motility and force generation in migrating fibroblasts. We find that traction force magnitudes display a multimodal distribution, suggesting discrete migratory regimes. Using a Hidden Markov Model, we identify distinct force states that exhibit differences in shape and motion metrics, and show that individual cells transition between force states over time. To test the role of cytoskeletal organization in establishing the identified states, we analyzed cells lacking Arpc2, which disrupts branched actin assembly. Despite reduced forces and altered morphology, these cells also exhibit three migratory states. State transitions occur more frequently in cells lacking Arpc2 and unlike normal cells their protrusion geometry is force dependent. Together, our findings show that cell migration is organized into discrete mechanical states that couple morphology, motility and force generation. SUMMARY STATEMENTFibroblast motility involves distinct migratory states. These states exist independent of branched actin. However, state transition frequencies, traction force magnitudes and protrusion geometry are branched actin dependent.

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.

Cofilin is a key regulator of actin dynamics that, along with a myriad of other actin-binding proteins, controls the balance of F- and G-actin in numerous cell types. While prior structural studies of the cofilin-actin binding interface have delineated many critical interactions between cofilin and actin, the roles of some residues within the cofilin-actin binding interface remain poorly defined. In this study, we investigate the role of cofilin S119 in the cofilin-actin interaction. Despite its unique position within the cofilin-actin interface and its putative role as a phosphorylation site, relatively little direct evidence exists to define whether it plays an important role in cofilin-actin dynamics. Using site-directed mutagenesis, we demonstrate that mutation of S119 to aromatic amino acids (W, F, Y) results in cofilins with strong actin bundling activity in living cells. This activity can be countered by the incorporation of mutants that disfavor actin rod forming activity (R21Q). Mutation of S119 to phospho-mimic (E) and non-phosphorylated (A) residues either strongly inhibits (E) or modestly increases (A) actin bundling activity. Expression of the S119W mutant in neurons reveals its impacts on spine length and size, while FRAP studies show that its mobile fraction is intermediate between that of LifeAct and WT cofilin. Finally, it is shown that the strong actin bundling phenotype associated with S119W inhibits the progression of optogenetically induced apoptosis.

Epigenetic regulation, particularly histone acetylation, plays a critical role in skeletal muscle differentiation by modulating gene expression programs without altering DNA sequence. Histone deacetylases (HDACs) tightly regulate myogenesis by controlling the timing of differentiation. Pharmacological inhibition of HDACs has shown context-dependent effects on muscle cells. We investigated the effects of 1 {micro}M SAHA (suberoylanilide hydroxamic acid) on C2C12 and L6 myoblasts during differentiation using morphological, immunofluorescence, transcriptomic, and proteomic analyses. SAHA delayed early differentiation, reducing myotube formation with partial recovery at later stages. Transcriptomic analysis revealed time-dependent changes in pathways related to cytoskeleton, cell cycle, and chromatin regulation. Proteomics showed increased mitochondrial metabolism and reduced cytoskeletal components in C2C12 cells, while L6 cells displayed alterations in muscle structural and extracellular matrix proteins. SAHA induces stage- and model-dependent reprogramming of myogenesis, highlighting the importance of timing and cellular context in HDAC-targeted therapies.

We performed a new set of experiments to confirm findings reported in our previous publication regarding the role of Sulf-1 and Sulf-2 in regulating BMP-7 and FGF-2 signaling in human articular chondrocytes. Using primary chondrocytes from five independent human donors, we examined the effects of Sulf knockdown on Smad1/5 and Erk1/2 phosphorylation. Sulf-1 and Sulf-2 knockdown consistently reduced BMP-7-induced Smad1/5 phosphorylation and enhanced FGF-2-induced Erk1/2 phosphorylation. Although the magnitude of Erk1/2 activation was somewhat lower than originally reported, the direction and statistical significance of the effects were preserved. These results confirm the original conclusions and support the role of Sulfs as dual regulators of BMP-7 and FGF-2 signaling pathways in human chondrocytes.

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.

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.

The Arp2/3 complex has long been considered to only assemble branched actin structures in the cell (lamellipodia, endocytic patches, comet tails, and many more). We show for the first time by single-molecule tracking (SMT) that the Arp2/3 complex and SPIN90, which activates Arp2/3 complex to nucleate unbranched filaments, bind to and move in the basal cortex with stress fibers and focal adhesions (FA) that, unlike known sites of Arp2/3 enrichment, employ linear actin bundles. SPIN90 knockout in U2OS cells significantly increases the rate of collective cell migration while decreasing cellular traction (myosin-II and actin speeds) and adhesion (FA size and maturation markers). SPIN90's SH3 domain, similar to its adapter protein Nck1, shows enrichment in FAs, suggesting a possible location for SPIN90-Arp2/3 complex activity. Together, our findings indicate that SPIN90-Arp2/3 nucleated filaments also function in stress fibers where they help define the mechanics of traction and adhesion to regulate cell motility.

The surface layer of articular cartilage provides for low-friction joint movement and protects the tissue from mechanical wear. The superficial zone chondrocytes (SZCs) of the surface layer produce proteoglycan-4 (PRG4), which is a lubricant that is necessary to reduce friction. Articular cartilage has limited capacity for self-repair and cell-based therapies, such as autologous chondrocyte implantation (ACI), is used to stimulate repair. However, in ACI, cells are expanded on tissue culture polystyrene where SZC poorly attach, proliferate slowly and dedifferentiate. Consequently, expanded SZC produce fibrocartilage tissue with insufficient PRG4. We previously demonstrated that culturing SZC on chondrocyte-derived decellularized extracellular matrix (CM) enhances SZC attachment and preserves phenotype. Since fibronectin (FN) was identified as the most abundant matrix protein within CM, here we tested the hypothesis that FN-coated culture surfaces would partially reproduce the beneficial effects of CM. We found that, similar to CM, SZC on FN-coated polystyrene increased SZC attachment and proliferation. However, unlike CM, SZCs expanded on FN-coated polystyrene remained more dedifferentiated as indicated by spread cells, elevated fibroblastic and contractile mRNA levels, and increased formation of SMA positive stress fibers. Consistent with the dedifferentiated phenotype, SZC on FN-coated polystyrene displayed extensive stress fibers, and higher nuclear myocardin-related-transcription-factor-a (MRTF-A). In contrast, CM reduced stress fiber formation and diminished nuclear MRTF-A in SZC. CM provides matrix cues beyond FN that suppress dedifferentiation and preserve the SZC phenotype. Identifying the matrix cues necessary to improve SZC expansion could lead to the generation of a superior surface in ACI repair tissue.

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.

Neural progenitor cell differentiation is a complex process requiring the proper integration of instructive and permissive factors. Instructive cues including signaling molecules and transcription factor networks have been well studied in this context, but permissive factors such as cell homeostasis have not. Cell homeostasis is critical to support the health and stability of a cell and enable the cell to act on instructive differentiation cues. Our study investigates a homeostasis protein, FAF2, and its function in neural progenitor cells. FAF2 is an adaptor protein involved in endoplasmic reticulum (ER) associated degradation to remove misfolded proteins and restore ER homeostasis. Here we show that knocking out Faf2 in neural progenitor cells results in increased ER stress signature at the protein and transcription level, indicating a conserved functional role in neural progenitor cells. Induced neural differentiation of FAF2 deletion cells shows a failure of neurite development but RNA-seq indicates genes that support neural differentiation are induced. Reducing ER stress in FAF2 knockout cells with a small molecule inhibitor can rescue neural differentiation, providing evidence that excess ER stress contributes to the inhibited differentiation. Taken together, these results reveal that FAF2 is a critical protein in neural progenitor cells for the maintenance of ER homeostasis and execution of neural differentiation. Highlights- FAF2 is required to regulate ER homeostasis in neural progenitor cells - FAF2 knockout blocks differentiation of neural progenitor cells to neurons at the cell morphological level, but does not inhibit the mounting of transcriptional programs associated with neural differentiation. - Excess ER stress due to FAF2 knockout contributes to blocked neural differentiation.

Collective cell migration is coordinated by adherens junctions (AJs) which serve both as structural links between the actin cytoskeleton of adjacent cells, as well as mechano-transductory structures allowing cells to transmit mechanical signals. Vinculin contributes to AJ maturation in multiple ways, binding to actin and the cadherin-catenin complex, bundling actin filaments, antagonising branched actin polymerisation and recruiting late AJ proteins. Here we have analysed the effect of vinculin on junctional actin organisation and its role in collective migration during unjamming in the human epithelial cell line MCF10A. At the apical surface of MCF10A monolayers, we found transcellular actin fibres (TAFs) that are directionally coordinated across long ranges, up to 10 cells. These TAFs are contractile and "cross" cell-cell contacts at AJs. Analysis of a vinculin knockout cell line revealed that this protein is essential for the coordination of TAFs across multiple cells. Arp2/3 activity must be tightly regulated to establish a long-range network of TAFs, since its downregulation by CK666 treatment, as well as its upregulation by the expression of an activated Rac1 mutant or a mutation that prevents the vinculin-Arp2/3 interaction, all impair TAF formation. During hypotonic unjamming of monolayers, we found that MCF10A cells with long-range TAFs migrate more collectively than vinculin KO cells in which TAFs only connect adjacent cells. Similarly, space-induced unjamming of MCF10A and vinculin knockout monolayers showed that cells connected by the long-range TAFs can collectively coordinate the direction in which they extend their lamellipodia. Thus, we show that vinculin plays a novel role in organising long-range actin networks across multiple cells and coordinating collective migration within cell monolayers.

The endoplasmic reticulum (ER) and its muscle-specialized form, the sarcoplasmic reticulum (SR), are crucial organelles in muscle cells, involved notably in protein synthesis, calcium regulation and muscle contraction. A well-known process involved in ER remodeling and homeostasis is ER-phagy, also called reticulophagy, a selective form of autophagic process in which ER-phagy receptors mediate the delivery of ER portions to lysosomes for degradation. SH3KBP1 is an adaptor protein involved in membrane trafficking. Recently, it was shown to control ER morphology and SR formation in striated skeletal muscle. In this study, we demonstrate that SH3KBP1 can bind to LC3B and CKAP4 proteins, bridging ER to autophagosome membranes, and is degraded by autophagy, in developing muscle fibers. Moreover, SH3KBP1 down-regulation impacts basal autophagy efficiency and ER-phagy stimulation; it also impairs the turnover of numerous ER-resident proteins. Our work highlights a new role for SH3KBP1 as a soluble ER-phagy receptor in striated skeletal muscle.

BackgroundMYORG (myogenesis-regulating glycosidase) and STRADB (STE20-related kinase adapter protein beta) were previously identified as activity-mediated skeletal muscle genes with potential roles in frailty and sarcopenia. We hypothesized that, if these genes are sustained by neuromuscular contractile activity, their expression should be consistently downregulated in muscular dystrophies, conditions defined by progressive muscle degeneration and secondary functional disuse. MethodsWe performed a systematic cross-dataset transcriptomic analysis of five publicly available GEO microarray datasets of human skeletal muscle. Discovery analysis was conducted in GSE3307 (Affymetrix HG-U133A/B; samples spanning DMD, LGMD2A/B/I, BMD, FSHD, JDM, ALS, AQM versus healthy controls). Independent external validation was performed in GSE38417 (HG-U133 Plus 2.0, DMD; n=16/6), GSE11681 (HG-U133A/B, LGMD2A; n=8-10/9-10), GSE465 (HG-U95Av2/B/C, multi-disease), and GSE1007 (HG-U95B/C/E, DMD; n=10-11/11). Raw CEL files underwent array-level quality assessment using NUSE and RLE diagnostics prior to normalization. Seven poor-quality arrays were excluded (none from Control, DMD, or LGMD groups). Remaining arrays were processed by robust multi-array average (RMA) normalization, and differential expression was assessed by limma with Benjamini-Hochberg FDR correction. ResultsMYORG was significantly downregulated in DMD (log2 fold-change [logFC] = -0.93, adj.P<0.001), LGMD2A (logFC = -0.82, adj.P<0.01), LGMD2B (logFC = -1.01, adj.P<0.01), and LGMD2I (logFC = -1.03, adj.P<0.01) in GSE3307. STRADB was significantly reduced in DMD (logFC = -0.33, adj.P<0.05) and showed a near-significant trend in LGMD2I (logFC = - 0.42, adj.P = 0.061). MYORG downregulation in DMD was independently replicated in GSE38417 (logFC = -1.40, adj.P<0.001) and GSE1007 (logFC = -0.80, adj.P<0.001). STRADB was also significantly downregulated in GSE38417 DMD (logFC = -0.45, adj.P<0.001). Deoxygalactonojirimycin, an iminosugar and an FDA/EMA-approved pharmacological chaperone (migalastat/Galafold) for Fabry disease, has been reported to be a specific molecular interactor that stabilizes MYORG protein in skeletal muscle. ConclusionsThis multi-dataset study further supports the role of MYORG and STRADB as activity-sensitive muscle genes that are robustly downregulated in DMD and LGMD. The pharmacological interaction between migalastat and MYORG provides a mechanistically grounded rationale for investigating this approved agent as an adjunct therapy in muscular dystrophies, in combination with the existing standard of care. This also supports active investigation of iminosugar analogs to target MYORG as potential therapeutics for improving skeletal muscle function in dystrophies, frailty, and sarcopenia.

We show that chronic impairment of mitochondrial respiration is associated with marked accumulation of cytochrome c (Cytc) protein. Using SCO2-deficient HCT116 cells lacking functional cytochrome c oxidase and wild-type cells exposed to sustained hypoxia, we found that substantial mitochondrial Cytc accumulation parallels reduced electron flux through Cytc. SCO2-deficient cells exhibited equally elevated Cytc levels under normoxia (19% O2) and hypoxia (0.1-3% O2). Wild-type cells under sustained hypoxia accumulated Cytc, reaching levels comparable to those in SCO2-deficient cells. This effect was reversible upon reoxygenation. Increased Cytc protein levels were also observed in other cell models, including primary cortical neurons cultured under chronic hypoxia and in cerebral cortex tissue from hypoxia-exposed mice. Cytc accumulation occurred independently of CYCS transcription, mRNA translation, HIF activation, ROS production and changes in mitochondrial network. Pharmacological inhibition of complex III was likewise accompanied by increased Cytc levels, whereas mitochondrial uncoupling had no effect, suggesting that impaired electron transfer rather than membrane depolarisation per se underlies this association. Raman spectroscopy revealed enrichment of reduced Cytc and an increased Cytc-to-cytochrome b ratio in respiration-deficient cells. Further supporting a stabilisation-based mechanism, the fraction of membrane-unbound ferro-Cytc was decreased in SCO2-deficient cells, consistent with moderate cardiolipin enrichment, which is known to enhance retention of Cytc at the inner mitochondrial membrane. Despite elevated mitochondrial Cytc content, SCO2-deficient cells were less susceptible to apoptosis induced by intermittent hypoxia or dichloroacetate. Together, these findings indicate that reduced electron flux through complex IV is associated with Cytc accumulation through increased protein stability and membrane retention without enhancing apoptotic sensitivity.

Microtubules are cytoskeletal polymers that play essential roles in eukaryotic cells, including structural support, cell division, and intracellular transport. During intracellular transport, kinesin motor proteins move cargo along microtubule tracks via their processive stepping. Recent studies have shown that the kinesin-1 KIF5C can damage the microtubule lattice while stepping. Microtubule damage can be repaired through incorporation of new tubulin subunits, however, excessive lattice damage results in microtubule breakage and disassembly. To identify cellular factors involved in microtubule repair, we performed an siRNA screen targeting microtubule-associated proteins (MAPs) known to regulate microtubule dynamics and stability. Based on the results, we investigated whether the end binding protein EB1 and cytoplasmic linker-associated protein 2 (CLASP2) contribute to repair of microtubule damage. To test this, we used a microtubule destruction assay in which damage was induced in microtubules gliding over surfaces coated with wild-type or mutant KIF5C proteins. Our findings suggest that CLASP2 directly facilitates microtubule repair, whereas EB1 does not. We further examined CLASP function using a microtubule repair assay and found that CLASP2 promotes repair by enhancing tubulin incorporation and reducing microtubule breakage. Together, these findings demonstrate that CLASP proteins play an important role in repairing and protecting against lattice damage caused by kinesin-1 motor activity. Our results further suggest that MAPs can directly regulate microtubule lattice integrity under mechanical stress generated by motor protein-driven intracellular transport.

C. burnetii is a Gram-negative, obligate intracellular bacterium and the causative agent of Q fever. The disease is either asymptomatic or manifests as a mild flu-like illness, but pneumonia or hepatitis might also occur. In most cases, the infection is self-limiting and the pathogen is cleared. In a small percentage of patients, the host immune system fails to eliminate the pathogen, potentially allowing the development of chronic Q fever months or even years after primary infection. The elimination of the bacteria, and thereby prevention of disease onset, would require an inflammatory response. Inflammasomes are multimeric protein complexes that induce a pro-inflammatory response to combat pathogens. Here we show that C. burnetii fails to induce a strong activation of the non-canonical inflammasome, independently of its type IVB secretion system. However, the pathogen is unable to prevent external activation of the non-canonical inflammasome, which subsequently results in a reduction of the bacterial burden. Importantly, the acylation pattern of lipid A was identified to be involved in avoiding the activation of the non-canonical inflammasome. C. burnetii harbors a tetra-acylated lipid A. Modification of the C. burnetii lipid A to penta-/hexa-acylation resulted in increased secretion of IL1{beta} and reduced bacterial load. Together, these results suggest that the acylation pattern of lipid A constitutes an important immune evasion strategy of C. burnetii by failing to activate the non-canonical inflammasome. In addition, evidence was provided that oxygen limitation arrests activation of the NLRP3 inflammasome in murine BMDM, which might prevent efficient elimination of bacteria under hypoxic conditions, such as in granulomas or in inflamed tissue. AUTHOR SUMMARYSeveral pathogens have evolved mechanisms to persist in the human host, which allows reoccurring or late onset of infection. The human innate immune system has therefore established several pathways, including the inflammasome, to prevent bacterial survival. Here we show that the obligate intracellular pathogen Coxiella burnetii, the causative agent of Q fever, prevents detection by the non-canonical NLRP3 inflammasome. This is mediated by the acylation pattern of its lipid A. Altering this acylation pattern allows activation of the inflammasome and, consequently, improved clearance of the pathogen. This information opens new avenues to target the immune response to C. burnetii infection with the goal to eliminate the bacteria and thereby prevent disease.

The primary cilium has been implicated in multiple developmental processes, such as cell migration and asymmetric cell division of stem- and progenitor cells. While most in vitro model systems examine ciliogenesis induced by serum starvation, it is not fully understood how de- and re-ciliation are regulated in proliferating stem- and progenitor cells. Here we employ the hierarchically organized C2C12 skeletal muscle cell line to examine how K-Ras4B participates in de- and re-ciliation processes of ciliated stem- and progenitor cells. We show that MAPK-pathway activation supports ciliogenesis through phosphorylation of centrosomal protein CEP55, which can then no longer stabilize the master regulator of de-ciliation Aurora kinase A. K-Ras4B localizes to the primary cilium aided by the ciliary trafficking chaperone PDE6D, which promotes ciliation. In line with this, depletion of components of the PDE6D machinery, RPGR and RPGRIP1L, decreases ciliation. Activation of the ciliary AMPK-PKG2-pathway increases S181-phosphorylation of K-Ras4B, which negatively regulates its binding to PDE6D, its ciliary abundance and promotes differentiation. Our work integrates a major mediator of mitogenic signaling into the regulation of ciliogenesis of proliferating muscle stem- and progenitor cells.

The carotid body (CB) is the key peripheral oxygen sensor. CB mitochondria are hypothesised to be uniquely adapted with unusually low intrinsic oxygen affinity which, in association with nitric oxide (NO) and reactive oxygen species signalling, enables acute responsiveness to hypoxia. However, CB mitochondrial physiology or intrinsic oxygen affinity have never been measured directly. We sought to address this key gap by isolating sheep CB mitochondria and comprehensively characterising their phenotype and contrasting them to a non-oxygen sensing tissue, left ventricular myocardium (LV). High resolution respirometry, liquid chromatography mass spectrometry, enzymatic assays and in silico modelling were used to characterise mitochondrial content, aerobic capacity, oxygen affinity, complex subunit abundance and activity, H2O2 production and NO sensitivity in ovine CB and LV. Mitochondrial oxygen affinity (P50 = 0.089 mmHg) was lower in the CB than the LV (P50 = 0.058 mmHg; p = 0.005). Whilst mitochondrial content was lower in the CB, CB mitochondria had higher respiratory rates and enzymatic activity than LV. H2O2 production and NO sensitivity were similar in the two tissues. While intrinsic mitochondrial oxygen affinity is slightly lower in the oxygen sensing CB than in the non-oxygen sensing LV, this difference is small. Hence, any role of mitochondria in CB oxygen sensing is not due to an intrinsic difference in the O2 affinity of cytochrome oxidase due to differential expression of its subunits. Instead, this work suggests that differences in O2 affinity in vivo are secondary to other factors, perhaps including NO, that alter mitochondrial O2 affinity.

Generative diffusion models have demonstrated an ability to produce novel images sampled from the learned underlying data distribution. These models are able to infer system characteristics for parameter combinations that were not seen during training. We investigate the ability of these models to infer trends in biological data from limited samples. Specifically, we consider the response of system scale behaviors such as cortical flow in a simulated actomyosin system as we tune filament turnover rates. We train a diffusion model on coarse grained actin curvature and density heatmap images, and are able to generate images from conditioning variables not seen during training. These images are predictive of nonlinear trends in the system. We also consider characteristics of the system that allows this level of inference, such as the strong linear relationship between average density and filament turnover in the system, and by exploring minimal underlying dynamics with a motor binding model.