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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2017

The Habc Domain of Syntaxin 3 is a Ubiquitin Binding Domain with Specificity for K63-Linked Poly-Ubiquitin Chains

Syntaxins are a family of membrane-anchored SNARE proteins that are essential components required for membrane fusion in all eukaryotic intracellular membrane trafficking pathways. Syntaxins contain an N-terminal regulatory domain, termed the Habc domain, that is not highly conserved at the primary sequence level but folds into a three-helix bundle that is structurally conserved among family members. The syntaxin Habc domain has previously been found to be structurally very similar to the GAT domain present in GGA family members and related proteins that are otherwise completely unrelated to syntaxins. Because the GAT domain has been found to be a ubiquitin binding domain we hypothesized that the Habc domain of syntaxins may also bind to ubiquitin. Here, we report that the Habc domain of syntaxin 3 (Stx3) indeed binds to monomeric ubiquitin with low affinity. This domain binds efficiently to K63-linked poly-ubiquitin chains within a narrow range of chain lengths but not to K48-linked poly-ubiquitin chains. Other syntaxin family members also bind to K63-linked polyubiquitin chains but with different chain length specificities. Molecular modeling suggests that residues of the GGA3-GAT domain known to be important for ionic and hydrophobic interactions with ubiquitin have equivalent, conserved residues within the Habc domain of Stx3. We conclude that the syntaxin Habc domain and the GAT domain are both structurally and functionally related, and likely share a common ancestry despite sequence divergence. Binding of K63-linked poly-ubiquitin chains to the Habc domain may regulate the function of syntaxins in membrane fusion or may suggest additional functions of this protein family.

cell biology↗

bioRxiv2017

Assessment of the aging of the brown adipose tissue by 18F-FDG PET/CT imaging in the progeria mouse model LmnaG609G/G609G

Brown adipose tissue (BAT) is an important energy metabolic organ that is closely related to obesity, type 2 diabetes, and atherosclerosis. Aging is one of the most important determinants of BAT activity. In this study, we used 18F-FDG PET/CT imaging to assess the aging of the BAT in LmnaG609G/G609G mice. To evaluate the BAT activity, LmnaG609G/G609G and wild-type (WT) mice were injected with 18F-FDG, and PET/CT imaging was performed. The maximum standardized uptake value (SUVMax) of the BAT was measured and the target/nontarget (T/NT) values of BAT were calculated. The transcription and the protein expression levels of the uncoupling protein 1 (UCP1), beta3-adrenergic receptor ({beta}3-AR), and the PRdomain-containing16 (PRDM16), were measured by quantitative real-time polymerase chain reaction (RT-PCR) and Western blotting or immunohistochemical analysis. Apoptosis and cell senescence of the BAT, in WT and LmnaG609G/G609G mice, was detected by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and by CDKN2A/p16INK4a immunohistochemical staining, respectively. At 14 weeks of age, the BAT SUVMax and the expression levels of UCP1, {beta}3-AR and PRDM16 in LmnaG609G/G609G mice was significantly lower than that in WT mice. At the same time, the number of p16INK4a and TUNEL positively stained cells (%) increased in LmnaG609G/G609G mice. LmnaG609G/G609G mice are an ideal model for studying BAT aging. The aging characteristics and the aging mechanism of BAT in LmnaG609G/G609G mice can mimic normal BAT aging.

cell biology↗

bioRxiv2017

Actomyosin contractility modulates Wnt signaling through adherens junction stability

Mechanical forces can influence the canonical Wnt signaling pathway in processes like mesoderm differentiation and tissue stiffness during tumorigenesis, but a molecular mechanism involving both in a developing epithelium and its homeostasis is lacking. We identified that increased non-muscle myosin II activation and cellular contraction inhibited Wnt target gene transcription in developing Drosophila. Genetic interactions studies identified this effect was due to myosin-induced accumulation of cortical F-actin resulting in clustering and accumulation of E-cadherin to the adherens junctions. E-cadherin titrates any available {beta}-catenin, the Wnt pathway transcriptional co-activator, to the adherens junctions in order to maintain cell-cell adhesion under contraction. We show that decreased levels of cytoplasmic {beta}-catenin result in insufficient nuclear translocation for full Wnt target gene transcription. Our work elucidates a mechanism in which the dynamic activation of actomyosin contractility refines patterning of Wnt transcription during development and maintenance of epithelial tissue in organisms.

cell biology↗

bioRxiv2017

Phospho-signal flow from a pole-localized microdomain spatially patterns transcription factor activity

Selective recruitment and concentration of signaling proteins within membrane-less compartments is a ubiquitous mechanism for subcellular organization. However, little is known about how such dynamic recruitment patterns intracellular signaling and cellular development. Here, we combined transcriptional profiling, reaction-diffusion modeling, and single-molecule tracking to study signal exchange in and out of a microdomain at the cell pole of the asymmetrically dividing bacterium Caulobacter crescentus. Our study revealed that the microdomain is selectively permeable, and that each protein in the signaling pathway that activates the cell fate transcription factor CtrA is sequestered and uniformly concentrated within the microdomain or its proximal membrane. Restricted rates of entry into and escape from the microdomain enhance phospho-signaling, leading to a sublinear gradient of CtrA~P along the long axis of the cell. The spatial patterning of CtrA~P creates a gradient of transcriptional activation that serves to prime asymmetric development of the two daughter cells.

cell biology↗

bioRxiv2017

A FRET based biosensor for measuring Gα13 activation in single cells

Forster Resonance Energy Transfer (FRET) provides a way to directly observe the activation of heterotrimeric G-proteins by G-protein coupled receptors (GPCRs). To this end, FRET based biosensors are made, employing heterotrimeric G-protein subunits tagged with fluorescent proteins. These FRET based biosensors complement existing, indirect, ways to observe GPCR activation. Here we report on the insertion of mTurquoise2 at several sites in the human G13 subunit. Three variants were found to be functional based on i) plasma membrane localization and ii) ability to recruit p115-RhoGEF upon activation of the LPA2 receptor. The tagged G13 subunits were used as FRET donor and combined with cp173Venus fused to the G{gamma}2 subunit as the acceptor. We constructed G13 biosensors by generating a single plasmid that produces G13-mTurquoise2, G{beta}1 and cp173Venus-G{gamma}2. The G13 activation biosensors showed a rapid and robust response when used in primary human endothelial cells that were treated with thrombin, triggering endogenous protease activated receptors (PARs). This response was efficiently inhibited by the RGS domain of p115-RhoGEF and from the biosensor data we inferred that this is due to GAP activity. Finally, we demonstrated that the G13 sensor could be used to dissect heterotrimeric G-protein coupling efficiency in single living cells. We conclude that the G13 biosensor is a valuable tool for live-cell measurements that probe G13 activation.

cell biology↗

bioRxiv2017

Stopping Transformed Growth with Cytoskeletal Proteins: Turning a Devil into an Angel

The major hallmark of cancer cells is uncontrollable growth on soft matrices (transformed growth), which indicates that they have lost the ability to properly sense the rigidity of their surroundings. Recent studies of fibroblasts show that local contractions by cytoskeletal rigidity sensor units block growth on soft surfaces and their depletion causes transformed growth. The contractile system involves many cytoskeletal proteins that must be correctly assembled for proper rigidity sensing. We tested the hypothesis that cancer cells lack rigidity sensing due to their inability to assemble contractile units because of altered cytoskeletal protein levels. In four widely different cancers, there were over ten-fold fewer rigidity-sensing contractions compared with normal fibroblasts. Restoring normal levels of cytoskeletal proteins restored rigidity sensing and rigidity-dependent growth in transformed cells. Most commonly, this involved restoring balanced levels of the tropomyosins 2.1 (often depleted by miR-21) and 3 (often overexpressed). Restored cells could be transformed again by depleting other cytoskeletal proteins including myosin IIA. Thus, the depletion of rigidity sensing modules enables growth on soft surfaces and many different perturbations of cytoskeletal proteins can disrupt rigidity sensing thereby causing transformed growth of cancer cells.

cell biology↗

bioRxiv2017

LEM-3 is a midbody-tethered DNA nuclease that resolves chromatin bridges during cytokinesis

Faithful chromosome segregation and genome maintenance requires the removal of all DNA bridges that physically link chromosomes before cells divide. Using C. elegans embryos we show that the LEM-3/Ankle1 nuclease defines a new genome integrity mechanism by processing DNA bridges right before cells divide. LEM-3 acts at the midbody, the structure where abscission occurs at the end of cytokinesis. LEM-3 localization depends on factors needed for midbody assembly, and LEM-3 accumulation is increased and prolonged when chromatin bridges are trapped at the cleavage plane. LEM-3 locally processes chromatin bridges that arise from incomplete DNA replication, unresolved recombination intermediates or the perturbance of chromosome structure. Proper LEM-3 midbody localization and function is regulated by AIR-2/Aurora B kinase. Strikingly, LEM-3 act cooperatively with the BRC-1/BRCA1 homologous recombination factor to promote genome integrity. These findings provide a molecular basis for the suspected role of the LEM-3 orthologue Ankle1 in human breast cancer.

cell biology↗

bioRxiv2017

Identification and characterisation of the cryptic Golgi Apparatus in Naegleria gruberi

Although the Golgi apparatus has a conserved morphology of flattened stacked cisternae in the vast majority of eukaryotes, the organelle has lost the stacked organization in several eukaryotic lineages raising the question of what range of morphologies is possible for the Golgi. In order to understand this range of organellar diversity, it is necessary to characterise the Golgi in many different lineages. Here we identify the Golgi apparatus in Naegleria, the first description of an unstacked Golgi organelle in a non-parasitic eukaryote, other than fungi. We provide a comprehensive list of Golgi-associated membrane trafficking genes encoded in two separate species of Naegleria and transcriptomic support to show that nearly all are expressed in mouse-passaged N. fowleri cells. We then study distribution of the Golgi marker NgCOPB by fluorescence, identifying membranous structures that can be disrupted by Brefeldin A treatment consistent with Golgi localisation. Confocal and immuno-electron microscopy revealed that NgCOPB is localized to membranous structures consistent with tubules. Our data not only identify the Golgi organelle for the first time in this major eukaryotic lineage, but also provide the rare example of a tubular form of the organelle representing an important sampling point for the comparative understanding of Golgi organellar diversity.

cell biology↗

bioRxiv2017

Allele-specific control of replication timing and genome organization during development

DNA replication occurs in a defined temporal order known as the replication-timing (RT) program. RT is regulated during development in discrete chromosomal units, coordinated with transcriptional activity and 3D genome organization. Here, we derived distinct cell types from F1 hybrid musculus X castaneus mouse crosses and exploited the high single nucleotide polymorphism (SNP) density to characterize allelic differences in RT (Repli-seq), genome organization (Hi-C and promoter-capture Hi-C), gene expression (nuclear RNA-seq) and chromatin accessibility (ATAC-seq). We also present HARP: a new computational tool for sorting SNPs in phased genomes to efficiently measure allele-specific genome-wide data. Analysis of 6 different hybrid mESC clones with different genomes (C57BL/6, 129/sv and CAST/Ei), parental configurations and gender revealed significant RT asynchrony between alleles across ~12 % of the autosomal genome linked to sub-species genomes but not to parental origin, growth conditions or gender. RT asynchrony in mESCs strongly correlated with changes in Hi-C compartments between alleles but not SNP density, gene expression, imprinting or chromatin accessibility. We then tracked mESC RT asynchronous regions during development by analyzing differentiated cell types including extraembryonic endoderm stem (XEN) cells, 4 male and female primary mouse embryonic fibroblasts (MEFs) and neural precursors (NPCs) differentiated in vitro from mESCs with opposite parental configurations. Surprisingly, we found that RT asynchrony and allelic discordance in Hi-C compartments seen in mESCs was largely lost in all differentiated cell types, coordinated with a more uniform Hi-C compartment arrangement, suggesting that genome organization of homologues converges to similar folding patterns during cell fate commitment.

cell biology↗

bioRxiv2017

Topography and refractometry of sperm cells using SLIM

Characterization of spermatozoon viability is a common test in treating infertility. Recently, it has been shown that label-free, phase-sensitive imaging can provide a valuable alternative for this type of assay. Here, we employ spatial light interference microscopy (SLIM) to decouple the thickness and refractive index information of individual cells. This procedure was enabled by quantitative phase imaging cells on media of two different refractive indices and using a numerical tool to remove the curvature from the cell tails. This way, we achieved ensemble averaging of topography and refractometry of 100 cells in each of the two groups. The results show that the thickness profile of the cell tail goes down to 150 nm and the refractive index can reach values of 1.6 close to the head.

cell biology↗

bioRxiv2017

Growth inhibition by amino acids in Saccharomyces cerevisiae

Amino acids are essential metabolites but can also be toxic when present at high levels intracellularly. Substrate-induced down-regulation of amino acid transporters in Saccharomyces cerevisiae is thought to be a mechanism to avoid this toxicity. It has been shown that unregulated uptake by the general amino acid permease Gap1 causes cells to become sensitive to amino acids. Here, we show that overexpression of eight other amino acid transporters (Agp1, Bap2, Can1, Dip5, Gnp1, Lyp1, Put4 or Tat2) also induces a growth defect when specific single amino acids are present at concentrations of 0.5-5 mM. We can now state that all proteinogenic amino acids, as well as the important metabolite ornithine, are growth inhibitory to S. cerevisiae when transported into the cell at high enough levels. Measurements of initial transport rates and cytosolic pH show that toxicity is due to amino acid accumulation and not to the influx of co-transported protons. The amino acid sensitivity phenotype is a useful tool that reports on the in vivo activity of transporters and has allowed us to identify new transporter-specific substrates.

cell biology↗

bioRxiv2017

BMPR2 inhibits activin- and BMP-signaling via wild type ALK2

Activin A is a member of the TGF-{beta} superfamily and activates the transcription factors SMAD2/3 through the ALK4 type 1 receptor. Activin A has also been shown to activate SMAD1/5/8 through mutated variants of the type 1 receptor ALK2. Interestingly, we here show that both activin A and activin B could activate SMAD1/5/8 through endogenous wild type ALK2 in multiple myeloma cells. Knockdown of the type 2 receptor BMPR2 strongly potentiated activin A- and activin B-induced SMAD1/5/8 activation and subsequent cell death. Furthermore, activity of BMP6, BMP7 or BMP9, which also signal via ALK2, was potentiated by BMPR2 knockdown. Similar results were seen in HepG2 liver carcinoma cells. We propose that BMPR2 inhibited ALK2-mediated signaling by preventing ALK2 from oligomerizing with the type 2 receptors ACVR2A and ACVR2B, necessary for ALK2 activation by activins and several BMPs in these cells. In conclusion, BMPR2 could be explored as a possible target for therapy in patients with multiple myeloma.\n\nSummary StatementThe activation of SMAD1/5/8 via endogenous wild type ALK2 by activin A, activin B, and certain BMPs was enhanced when BMPR2 levels were knocked down.

cell biology↗

bioRxiv2017

Higher eukaryote-specific APC/C composition is a determinant of spindle assembly checkpoint importance

The multisubunit ubiquitin ligase APC/C (anaphase promoting complex/cyclosome) is essential for mitosis by promoting timely degradation of cyclin B1. Proper timing of APC/C activation is regulated by the spindle assembly checkpoint (SAC), which is initiated by the kinase MPS1 and implemented by MAD2-dependent inhibition of the APC/C. Here we analysed the contribution of the higher eukaryote-specific APC/C subunits APC7 and APC16 to APC/C composition, function and regulation. APC16 is required for APC7 assembly into the APC/C, while APC16 assembles independently of APC7. {Delta}APC7 and {Delta}APC16 cells display no major defects in mitotic progression, cyclin B1 degradation or SAC response. Strikingly, however, deletion of either APC7 or APC16 is sufficient to provide synthetic viability to MAD2 deletion. {Delta}APC7{Delta}MAD2 cells display an accelerated mitosis and require SAC-independent MPS1 function for maintaining their genome stability. Overall, these results show how human APC/C composition critically influences the cellular fate upon loss of SAC activity.

cell biology↗

bioRxiv2017

Cytoplasmic import and processing of mRNA amplify transcriptional bursts accounting for the majority of cellular noise

Transcription is an episodic process characterized by probabilistic bursts; but how these bursts are modulated by cellular physiology remains unclear and has implications for how selection may act on these fluctuations. Using simulations and single-molecule RNA counting, we examined how cellular processes influence cell-to-cell variability (noise). The results show that RNA noise is amplified in the cytoplasm compared to the nucleus in ~85% of genes across diverse promoters, genomic loci, and cell types (human and mouse). Surprisingly, measurements show further amplification of RNA noise in the cytoplasm, fitting a model of biphasic mRNA conversion between translation- and degradation-competent states. The multi-state translation-degradation of mRNA also causes substantial noise amplification in protein levels, ultimately accounting for ~74% of intrinsic protein variability in cell populations. Overall, the results demonstrate how transcriptional bursts are intrinsically amplified by mRNA processing and indicate mechanisms through which noise could act as a substrate for evolutionary selection.

cell biology↗

bioRxiv2017

Asymmetrical localization of components of the Nup107-160 subcomplex within the nuclear pore complex in fission yeast

The nuclear pore complex (NPC) forms a gateway for nucleocytoplasmic transport. The outer ring protein complex of the NPC (the Nup107-160 subcomplex in humans) is a key component for building the NPC. Nup107-160 subcomplexes are believed to be symmetrically localized on the nuclear and cytoplasmic sides of the NPC. However, in S. pombe immunoelectron and fluorescence microscopic analyses revealed that the homologous components of the human Nup107-160 subcomplex had an asymmetrical localization: constituent proteins spNup132 and spNup107 were present only on the nuclear side (designated the spNup132 subcomplex), while spNup131, spNup120, spNup85, spNup96, spNup37, spEly5 and spSeh1 were localized only on the cytoplasmic side (designated the spNup120 subcomplex), suggesting the complex was split into two pieces at the interface between spNup96 and spNup107. This contrasts with the symmetrical localization reported in other organisms. Fusion of spNup96 (cytoplasmic localization) with spNup107 (nuclear localization) caused cytoplasmic relocalization of spNup107. In this strain, half of the spNup132 proteins, which interact with spNup107, changed their localization to the cytoplasmic side of the NPC, leading to defects in mitotic and meiotic progression similar to an spNup132 deletion strain. These observations suggest the asymmetrical localization of the outer ring spNup132 and spNup120 subcomplexes of the NPC is necessary for normal cell cycle progression in fission yeast. Author summaryThe nuclear pore complexes (NPCs) form gateways to transport intracellular molecules between the nucleus and the cytoplasm across the nuclear envelope. The Nup107-160 subcomplex, that forms nuclear and cytoplasmic outer rings, is a key complex responsible for building the NPC by symmetrical localization on the nuclear and cytoplasmic sides of the nuclear pore. This structural characteristic was found in various organisms including humans and budding yeasts, and therefore believed to be common among "all" eukaryotes. However, in this paper, we revealed an asymmetrical localization of the homologous components of the human Nup107-160 subcomplex in fission yeast by immunoelectron and fluorescence microscopic analyses: in this organism, the Nup107-160 subcomplex is split into two pieces, and each of the split pieces is differentially distributed to the nuclear and cytoplasmic side of the NPC: one piece is only in the nuclear side while the other piece is only in the cytoplasmic side. This contrasts with the symmetrical localization reported in other organisms. In addition, we confirmed that the asymmetrical configuration of the outer ring structure is necessary for cell cycle progression in fission yeast. This study provides notions of diverse structures and functions of NPCs evolved in eukaryotes.

cell biology↗

bioRxiv2017

The conserved LEM-3/Ankle1 nuclease is involved in the combinatorial regulation of meiotic recombination repair and chromosome segregation in Caenorhabditis elegans

Homologous recombination is essential for crossover (CO) formation and accurate chromosome segregation during meiosis. It is of considerable importance to work out how recombination intermediates are processed leading to CO and non-crossover (NCO) outcome. Genetic analysis in budding yeast and Caenorhabditis elegans indicates that the processing of meiotic recombination intermediates involves a combination of nucleases and DNA repair enzymes. We previously reported that in C. elegans meiotic Holiday junction resolution is mediated by two redundant pathways, conferred by the SLX-1 and MUS-81 nucleases, and by the HIM-6 Blooms helicase in conjunction with the XPF-1 endonucleases, respectively. Both pathways require the scaffold protein SLX-4. However, in the absence of all these enzymes residual processing of meiotic recombination intermediates still occurs and CO formation is reduced but not abolished. Here we show that the LEM-3 nuclease, mutation of which by itself does not have an overt meiotic phenotype, genetically interacts with slx-1 and mus-81 mutants, the respective double mutants leading to 100% embryonic lethality. LEM-3 and MUS-81 act redundantly, their combined loss leading to a reduced number of early meiotic recombination intermediates, to a delayed disassembly of foci associated with CO designated sites, and to the formation of univalents linked by SPO-11 dependent chromatin bridges (dissociated bivalents). However, LEM-3 foci do not co-localize with ZHP-3 a marker that congresses into CO designated sites. In addition, neither CO frequency nor distribution is altered in lem-3 single mutants or in combination with mus-81 or slx-4 mutations, indicating that LEM-3 drives NCO outcome. Finally, we found persistent chromatin bridges during meiotic divisions in lem-3; slx-4 double mutants. Supported by the localization of LEM-3 between dividing meiotic nuclei, this data suggests that LEM-3 is able to process erroneous recombination intermediates that persist into the second meiotic divisions.\n\nAuthor SummaryMeiotic recombination is required for genetic diversity and for the proper chromosome segregation. Recombination intermediates, such as Holliday junctions (HJs), are generated and eventually resolved to produce crossover (CO) and non-crossover (NCO). While an excess of meiotic double-strand breaks is generated, most breaks are repaired without leading to a CO outcome and usually only one break for each chromosome pair matures into a CO-designated site in Caenorhabditis elegans. Resolution of meiotic recombination intermediates and CO formation have been reported to be highly regulated by several structure-specific endonucleases and the Bloom helicase. However, little is known about enzymes involved in the NCO recombination intermediate resolution. In this study, we found that a conserved nuclease LEM-3/Ankle1 acts in parallel to the SLX-1/MUS-81 pathway to process meiotic recombination intermediates. Mutation of lem-3 has no effect on CO frequency and distribution, indicating LEM-3 functions as a nuclease promoting NCO outcome. Interestingly, a prominent localization of LEM-3 is found between dividing meiotic nuclei. We provide evidence that LEM-3 is also involved in processing remaining, erroneous recombination intermediates during meiotic divisions.

cell biology↗

bioRxiv2017

Evidence for, and Metabolic Consequences of, a Cardiac Mitochondrial KNa1.2 Channel

Controversy surrounds the molecular identity of mitochondrial K+ channels that are important for protection against cardiac ischemia-reperfusion injury. While KNa1.2 (Kcnt2 gene) is necessary for cardioprotection by volatile anesthetics, electrophysiologic evidence for a channel of this type in mitochondria is lacking. The endogenous physiologic role of a potential mito-KNa1.2 channel is also unclear. Herein, single channel patch-clamp of 27 independent cardiac mitochondrial inner membrane (mitoplast) preparations from wild type (WT) mice yielded 6 channels matching the known ion-sensitivity, ion-selectivity, pharmacology and conductance properties of KNa1.2 (slope conductance 138{+/-}1 pS). However, similar experiments on 40 preparations from Kcnt2-/- mice yielded zero such channels. The KNa opener bithionol uncoupled respiration in WT but not Kcnt2-/- cardiomyocytes. Furthermore, when oxidizing only fat as substrate, Kcnt2-/- cardiomyocytes and hearts were less responsive to increases in energetic demand. Kcnt2-/- mice also had elevated body fat, but no baseline differences in the cardiac metabolome. These data support the existence of a cardiac mitochondrial KNa1.2 channel, and a role for cardiac KNa1.2 in regulating metabolism under conditions of high energetic demand.

cell biology↗

bioRxiv2017

Skeletal muscle fibro-adipogenic progenitors of dystrophic mice are insensitive to NOTCH-dependent regulation of adipogenesis

Fibro adipogenic progenitors (FAPs) promote satellite cell differentiation in adult skeletal muscle regeneration. However, in pathological conditions, FAPs are responsible for fibrosis and fat infiltrations. Here we show that the NOTCH pathway negatively modulates FAP differentiation both in vitro and in vivo. However, FAPs isolated from young dystrophin-deficient mdx mice are insensitive to this control mechanism. Nonetheless, factors released by hematopoietic cells restore the sensitivity to NOTCH adipogenic inhibition. An unbiased mass spectrometry-based proteomic analysis of FAPs from muscles of wild type and mdx mice, revealed that the synergistic cooperation between NOTCH and inflammatory signals controls FAP differentiation. These results offer a basis for rationalizing the pathological outcomes of fat infiltrations in skeletal muscle and may suggest new therapeutic strategies to mitigate the detrimental effects of fatty depositions in muscles of dystrophic patients.\n\nHighlightsO_LISingle-cell mass cytometry reveals that wt and mdx FAPs are in different cell states.\nC_LIO_LIActivation of the NOTCH signaling pathway negatively regulates adipogenesis of wt but not mdx FAPs.\nC_LIO_LIDeep proteomics suggests a mechanism explaining the different sensitivity of mdx- FAPs to NOTCH.\nC_LIO_LITNF-a stimulation restores the anti-adipogenic effect of NOTCH in mdx FAPs.\nC_LI

cell biology↗