EMBO reports

The master kinases of the DNA damage response (DDR), ATR, ATM and DNA-PK, become active in response to DNA damage and orchestrate a downstream wave of phosphorylations contributing to DNA damage repair and preservation of cellular homeostasis. Of them, we recently demonstrated that ATM binds the pool of the lipid phosphatidyl-inositol-4-phosphate (PI4P) situated at the Golgi membrane. Depending on PI4P availability at Golgi membranes, ATM is more or less titrated away from the nucleus, which translates into responses to nuclear DNA damage of matching intensity. Building on this knowledge, in this work we asked if, beyond the Golgi merely serving as a docking platform that retains ATM away from the nucleus, ATM does exert any role important for Golgi biology. We found that ATM maintains Golgi morphology by counteracting its excessive deployment. This occurs both by its mere presence (likely antagonizing the Golgi-stretching action of the protein GOLPH3) and by phosphorylating Golgi-resident substrates. Of relevance, we also report that the morphological alterations caused to the Golgi without ATM affect the biology of a model Golgi cargo. Our findings nourish the growing evidence that kinases of ATMs family display functional interactions with membranes and highlights an underappreciated crosstalk between the Golgi and the nucleus.

The intestinal epithelial lining is highly dynamic, with size and cellular composition adapting to nutrient status. This requires regulation of intestinal stem cell (ISC) proliferation and enterocyte (EC) size. How the absorptive area of the intestine matches physiological nutrient conditions remains unclear. Here, we show that the Nuclear Factor Y (NF-Y) transcription factor plays a role in this process. NF-Y loss-of-function (LOF) in ISCs led to high proliferation and cell growth, a phenotype influenced by dietary nutrients. NF-Y LOF also increased nutrient metabolism, shown by more mitochondria and larger lipid droplets in progenitors. Mechanistically, NF-Y restrains mTOR complex 1 (mTORC1) activity in ISC by controlling transcription of mTORC1 signaling components such as Iml1 and Sestrin. Overall, our results demonstrate that NF-Y limits excessive intestinal epithelial growth under nutrient-rich conditions. HighlightsO_LINF-Y is a cell-autonomous regulator of ISC activity C_LIO_LINF-Y restricts ISC proliferation and epithelial growth under nutrient-rich condition C_LIO_LINF-Y regulates mitochondrial biogenesis and lipid storage in progenitors C_LIO_LINF-Y limits mTORC1 activity in progenitors C_LI

The biogenesis of integral membrane proteins is complex, as revealed by an ever-growing number of cellular components shown to be dedicated to the insertion, folding, surveillance, rectification, or quality control of specific client membrane proteins. The zinc metalloprotease ZMPSTE24 and its yeast homolog Ste24 have well-established roles in the proteolytic maturation of the nuclear scaffold protein lamin A and yeast a-factor, respectively. Additionally, Ste24 has been implicated through yeast genetic screens in a variety of membrane processes, including ER- associated degradation (ERAD), Sec61 translocon "unclogging," the unfolded protein response (UPR), and potentially as a membrane protein topology determinant. Recently, an interaction was demonstrated between ZMPSTE24 and the antiviral interferon induced transmembrane protein IFITM3, although the functional significance of this interaction is not well-understood. IFITM3 is a tail-anchored protein with a cytoplasmic N-terminus, a single transmembrane span, and a lumenal/exocellular C-terminus. Here, we show that a catalytic-dead version of ZMPSTE24, ZMPSTE24E336A, exhibits enhanced binding to IFITM3, and this bound species of IFITM3 is hypo-palmitoylated. Using a split fluorescence topology reporter, we demonstrate that ZMPSTE24E336A "traps" and stabilizes a subpopulation of IFITM3 molecules with an atypical membrane topology, whose C-terminus is cytosolic instead of lumenal. Such inverted forms of IFITM3 are also detected in the presence of ERAD inhibitors when ZMPSTE24E336A is absent. We hypothesize the ZMPSTE24E336A trap mutant reveals a normally transient isoform of IFITM3 whose transmembrane span is inverted and that ZMPSTE24 is involved in the quality control of IFITM3 topology, either inverting, correcting or assisting in removal of aberrant IFITM3 molecules.

In Drosophila, Arginine kinase 1 (Argk1) is involved in maintaining ATP homeostasis during bursts of activity in tissues with high and variable rates of energy turnover such as muscle. However, its role beyond stress conditions is less understood. Here, we show that Argk1 maintains energy homeostasis during flight muscle development and is required for animal viability and proper muscle function. The knockdown of Argk1 causes defects in both early and late stages of myogenesis. In the proliferating myoblasts associated with the wing disc, Argk1 depletion results in a reduction in cell size without changes in cell cycle progression. Single cell RNA-sequencing revealed that the overall composition of differentiating and undifferentiating myoblasts is not altered. Nonetheless, Argk1 knockdown causes broad alterations in the expression of genes involved in various metabolic pathways. This correlates with low levels in both ATP content and NAD+/NADH ratio. Later in muscle development, Argk1-depleted muscles completely lack spontaneous muscle contractions that are essential in myofibrillogenesis. Accordingly, Argk1 knockdown results in severe defects in sarcomere structure, while the mitochondrial network is highly fragmented. Furthermore, muscle growth is severely reduced. Thus, our data reveal an essential role for Argk1 in maintaining energy homeostasis throughout muscle development, which is required to meet the demand to support myofibrillogenesis, muscle growth and proper muscle function.

Guanylate-binding proteins (GBPs) are part of a family of large interferon gamma (IFN{psi})-inducible GTPases, with ascribed roles in infection control and induction of programmed cell death. While pathogen-specific functions of GBPs have been studied in detail, their broader regulation of frontline immune defences remain unexplored. Here, we analysed the global contribution of human GBP1-5 to cellular metabolism in IFN{psi}-stimulated macrophages. We found a robust role of GBP2 and GBP5 in macrophage glycolysis. Only GBP5, and not GBP2 deficiency impaired surface expression and cytokine production of classically IFN{psi}/LPS-activated macrophages. The GTPase activity of GBP5 was required for the regulation of glycolysis and cytokine production. We found that GBP5 deficiency impaired cellular glucose uptake and lactate production specifically. Isotopic tracing with [U-13C6]-Glucose confirmed a decrease in several glycolytic intermediates, including glucose 6-phosphate, pyruvate, and lactate, but stable levels of traced tricarboxylic acid cycle (TCA) intermediates. Elevated ribose-5-phosphate and glycerol levels suggest an altered cytosolic redox balance and enhanced breakdown of fatty acids. GBP5 localised predominantly to the cis-Golgi and in the absence of GBP5 we observed increased Golgi fragmentation, however the total Golgi size remained unchanged. Our results underscore the fundamental role of GBP5 in glycolytic fluxes and Golgi integrity in IFN{psi}-stimulated macrophages, highlighting its significance in immune function in general and immunometabolism specifically.

The PIWI-interacting RNA (piRNA) biosynthesis pathway is best studied for its role suppressing Drosophila germline transposable elements. Piwi, the founding member of the pathway, is involved in adult intestinal stem cell (ISC) homeostasis. Whether a broader role of the PIWI pathway exists in the intestine, remains unknown. Here, we characterise a role of the PIWI family protein Aubergine (Aub) in ISCs. While dispensable for basal ISC self-renewal, upregulation of Aub by damage-induced reactive oxygen species drives regenerative ISC proliferation through increased protein synthesis, including translation of ISC factors Myc and Sox21a. Unexpectedly, such roles of Aub in ISCs appear uncoupled from its piRNA regulatory function. Additionally, Aub and mammalian PIWIL1, mediate tumorigenic intestinal growth in Drosophila and human organoids, respectively. Our results reveal regulated protein translation as a fundamental aspect of regenerative ISC function and discover a central role of Aub in such process.

Photoreceptor integrity depends on the precise coordination of membrane trafficking and signal transmission. Despite their well-known roles in germline biology, the functions of PIWI family proteins in post-mitotic neuronal cells remain unclear. We investigated the role of HIWI2 in photoreceptor-derived 661W cells. Silencing of HIWI2 resulted in a significant decrease in the early endosomal marker, Rab5, and its effector EEA1, and reduced expression of the recycling endosome marker Rab11, indicating poor endosomal sorting and receptor recycling. In contrast, the marker for late endosomes, Rab7, was significantly upregulated, suggesting a shift toward degradative trafficking pathways, in line with increased receptor breakdown. These trafficking shifts led to the degradation of EphA2 and EphB2 receptors, as confirmed by a phospho-proteome receptor tyrosine kinase array and further supported by immunoblotting, and were accompanied by a compensatory increase in Akt phosphorylation. Furthermore, HIWI2 deficiency impaired cell motility in wound-healing assays. These results propose HIWI2 as a critical regulator of endosomal sorting and Eph receptor stability, providing a novel link between the PIWI pathway and photoreceptor integrity.

Oxidative phosphorylation is the most efficient way of generating ATP in respiring cells. As high energy electrons are the major source of reactive oxygen species their production needs to be carefully calibrated. In most organisms, NADH dehydrogenase serves as the primary source and gateway of electrons. This complex is responsible for oxidizing NADH to NAD+, which liberates two electrons that are then fed into the respiratory chain. In the Gram-positive model bacterium, Bacillus subtilis, a transcription factor (Rex) is utilized to monitor the rise in NADH level and subsequently increase the production of the NADH dehydrogenase Ndh. Thus, the generation of electrons through this pathway is tightly regulated. In this report, we reveal the presence of another independent mechanism to moderate Ndh activity involving a previously uncharacterized protein, YfhS. Additionally, we present the first experimental evidence showing that the functional NADH dehydrogenase is a two-protein complex comprised of a membrane-associated YjlC and the enzyme Ndh. We find that absence of YfhS leads to cell morphology and growth defects that are corrected by spontaneous mutations in ndh. We note that increased production of NADH dehydrogenase complex proteins by itself is not detrimental. However, strikingly, it is lethal in a strain lacking yfhS. These results reveal that YfhS is an important moderator of NADH dehydrogenase activity. We also demonstrate that YfhS and YjlC are interaction partners. A model developed based on our data indicates that YfhS is an important regulator of intracellular NADH concentration. Compounds that target specific microbial (Type II) NADH dehydrogenase, which is absent in human mitochondria, are considered promising drug candidates to help address the threat posed by antibiotic-resistant bacteria. Overall, our data unveiling the importance of YfhS and YjlC in controlling Ndh activity could be harnessed for the development of new therapeutics.

Odorants stimulate olfactory sensory neurons (OSNs) to create a bilateral sensory map defined by a set of glomeruli present in the left and right olfactory bulbs. Using Xenopus tropicalis tadpoles we challenged the notion that glomerular activation is exclusively determined ipsilaterally. Glomerular responses evoked by unilateral stimulation were potentiated following transection of the contralateral olfactory nerve. The gain of function was observed as early as 2 hours after injury and faded away with a time constant of 4 days. Potentiation was mediated by the presence of larger and faster calcium transients driving glutamate release from OSN axon terminals. The cause was the reduction of the tonic presynaptic inhibition exerted by dopamine D2 receptors. Inflammatory mediators generated by injury were not involved. These findings reveal the presence of a bilateral modulation of glomerular output driven by dopamine that compensates for imbalances in the number of operative OSNs present in the two olfactory epithelia. Considering that the constant turnover of OSNs is an evolutionary conserved feature of the olfactory system and determines the innervation of glomeruli, the compensatory mechanism here described may represent a general property of the vertebrate olfactory system to establish an odor map.

The different types of muscle fibres respond in a specific way to hypertrophy or atrophy. The mechanisms underlying these heterogeneous adaptations remain poorly understood. Using single-nucleus RNA sequencing, we propose that fast glycolytic fibres show genetic limitations to hypertrophy induced by mechanical overload. We show that a prior fibre transition, achieved by reducing SIX1 protein expression (hypomorphism), enhances and accelerates overload-induced hypertrophy, bypassing the genetic limitations of fast glycolytic fibres. In contrast and unexpectedly, Six1 knockout in myofibers abolished overload-induced hypertrophy and instead caused atrophy of IIb/IIx fibers, despite the induction of a strong slow oxidative phenotype. In particular, Six1 deletion leads to metabolic defects caused by inhibition of glycolysis, AMPK and mitochondrial biogenesis. Our findings highlight the critical role of SIX1/AMPK/glycolysis-dependent aerobic metabolism in muscle growth and suggest that fibre type transitions, coupled with preserved metabolic function, may optimise hypertrophic responses.

Meiotic prophase-I chromosomes are organized into linear arrays of chromatin loops anchored to proteinaceous axes that define the interaction interfaces for the pairing and synapsis of homologous chromosomes. Chromatin loop size and axial chromosome length are inversely correlated and vary widely both between and within species, including between the sexes. The molecular basis of this variation remains unclear. Here, we provide evidence that the small ubiquitin-like modifier, SUMO, regulates loop-axis organization in mouse meiosis. Our analysis shows that the longer axes of oocyte chromosomes contain more SUMO per unit length than the shorter axes of spermatocyte chromosomes. In mouse models, the loss of SUMO1 results in shorter axes and longer chromatin loops. Conversely, increased SUMO1 conjugation, caused by mutation of the SENP1 isopeptidase, produces longer axes with shorter loops. Axis length positively correlates with meiotic recombination. Accordingly, Sumo1 and Senp1 mutations respectively decrease and increase crossover frequency. These findings identify SUMO as a key regulator of meiotic chromosome architecture and suggest a molecular basis for the physiological variation in chromosome length and recombination rates seen among species, sexes, individuals, and individual meiocytes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/710713v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@145c465org.highwire.dtl.DTLVardef@160c8aborg.highwire.dtl.DTLVardef@1165b76org.highwire.dtl.DTLVardef@ced5e0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Viruses manipulate the host cell membrane of infected cells for evasion of antiviral immunity, prevention of superinfection and optimization of viral replication and spread. The Ebola virus glycoprotein (EBOV GP) mediates virus entry, but is also known as important factor for subversion of the hosts antiviral immune response. We characterized the dysregulation of cell surface-residing proteins by EBOV GP and found that among several membrane proteins GP interferes with the tetraspanins CD81, CD63 and CD9. This was a conserved function of several filoviral GPs and not observable for viral glycoproteins of other virus families. While CD63 and CD9 were largely dispensable for EBOV replication, CD81 suppressed virus-like particle entry and replication at multiple steps. This phenotype might be explainable by sustained suppression of NF{kappa}B by CD81, that is otherwise activated by VP40 and EBOV trVLP replication. We further demonstrate that not only GP but also VP40 interferes with CD81 functionality and that antibody-mediated clustering of CD81 suppresses EBOV infection. Altogether, the tetraspanin CD81 emerges as druggable NF{kappa}B and EBOV-inhibiting factor, supporting an important role of NF{kappa}B in EBOV replication and potentially virus-induced immunopathogenesis. HighlightsO_LIEBOV GP and VP40 interfere with CD81 cell surface expression C_LIO_LICD81 suppresses NF{kappa}B signaling which is activated by VP40 C_LIO_LICD81 restricts EBOV VLP uptake and replication C_LIO_LITargeting CD81 by a crosslinking antibody inhibits EBOV infection C_LI

The metabolic needs of a cell are tightly linked to its proliferative state and increasing evidence indicate an extensive bidirectional crosstalk between metabolic pathways and cell cycle regulators. In cancer cells, metabolism is reprogrammed to couple energetic needs and relentless proliferation. The cyclin/CDK inhibitor p27Kip1 (p27) is frequently inactivated in cancers. p27 is also involved in multiple cellular processes, including transcriptional regulation or autophagy induction. Herein, we investigated the effect of p27 loss on cell metabolism. The knockout of p27 in immortalized mouse fibroblasts increases glucose uptake and glycolysis, while decreasing mitochondrial ATP production, consistent with induction of a Warburg effect, and this was accompanied by an increased glutamine dependency to feed the TCA cycle. Our data suggest that p27 loss causes this phenotype through extensive transcriptional remodeling of metabolic gene expression. Importantly, p27 silencing in human retinal RPE1-hTERT cells was sufficient to induce a Warburg effect. Together, these results reveal a new function of p27 in regulating energy metabolism and that loss of p27 expression is sufficient to induce metabolic reprogramming and a Warburg effect, suggesting that p27 inactivation in cancer cells not only results in the loss of cell cycle inhibition but also enables the metabolic rewiring needed for increased proliferation.

Epidermal Growth factor (EGF) signaling is associated with (oncogenic) proliferation. Conversely, EGF-family ligands are able to trigger a differentiation program in cultured cells, an effect attributed to ligand affinity and EGFR phosphorylation. How EGF/EGFR driven proliferation-differentiation dynamics underlie tissue self-renewal has not been addressed. We show that culturing mouse small intestinal organoids (mSIOs) without EGF enhanced EGFR expression and base phosphorylation while maintaining a balanced development of proliferative crypts and differentiated villi. Addition of EGF or EREG triggers receptor endocytosis, reducing cell-surface and expression levels. While EGF promoted crypt proliferation, EREG promoted both proliferation and villus differentiation compared to untreated controls. Removal or re-introduction of EGF or EREG proved sufficient to induce development comparable to constant presence of ligands over 96h. Sub-saturating concentrations of EGF led to increased villus differentiation, resembling EREG treatments, suggesting that control over EGFR endocytic cycle ultimately regulates the balance of proliferation and differentiation in mSIOs SummaryExpression and signaling competency at the plasma membrane of EGFR drives crypt proliferation vs villus differentiation by medium ligand-composition, aiding mouse intestinal organoids self-renewal and regeneration.

Circadian rhythms, driven by 24-hour molecular oscillators, or "clocks", widely tune physiology to the daily rhythms of light and dark to enhance organismal fitness. In mammals, the cellular immune response is tightly regulated by these rhythms such that immunometabolic output is coordinated across the day, consolidating macrophage physiology into temporally distinct phases that determine the macrophage response to stimuli. Importantly, key proteins in the macrophage response to viral infection have been found to be under circadian control, and time of day of adjuvant application is known to affect the efficacy of vaccinations, including in the case of the SARS-CoV-2 virus. However, little is known about the molecular changes that underly the temporal response to vaccine application. Therefore, to investigate the circadian response of macrophage physiology to adjuvant exposure, we exposed primary mouse and human macrophages to the SARS-CoV-1 and CoV-2 spike proteins at different times over the circadian day. To further explore the time-of-day effect, we performed a multi-omics analysis and in vitro tissue culture assays examining macrophage responses over circadian time. We found that, conserved across the species, the timing of spike protein exposure dictated two distinct temporal responses which were characterized by hallmarks of immunometabolic suppression and modest immunometabolic activation. Intriguingly, these temporal responses were driven by central metabolic and mitochondrial changes rather than classical immune activation, suggesting immunometabolic control is a primary regulator of the temporal response of immune cells to stimuli.

The suppression of type I interferon (IFN) signalling by the ISG15-USP18-STAT2 inhibitory complex (ISG15 IC) is an established regulatory mechanism of the antiviral response. However, a molecular understanding of how the ISG15 IC forms to suppress IFN signalling is still emerging. Here, we use AlphaFold modelling in conjunction with biochemical and biophysical approaches to elucidate the interactions of this multiprotein assembly. Our analysis identified a unique STAT2 binding loop (SBL) in USP18, which is critical for the USP18-STAT2 association. Further biochemical characterisation through site-directed mutagenesis confirmed the importance of residues within and surrounding the SBL, enabling the design of mutants with both increased and decreased binding affinities. Moreover, several USP18 and STAT2 patient mutations severely disrupted this interaction. Lastly, using influenza B virus (IBV) and Zika virus (ZIKV) proteins, we investigated the influence of these viral effector proteins on these interactions. Taken together, these results provide much-needed insights into a key aspect of IFN signalling control.

Mast cells are emerging players in malignant conditions, but the underlying molecular mechanisms remain poorly defined. Based on previous studies showing that steroids can impact on tumour progression in various settings, we here investigated whether mast cell-derived steroid synthesis can have an impact on tumour metastasis in a melanoma model. To this end, we used mice with mast cell-specific ablation of Cyp11a1, a key enzyme in steroid synthesis. We show that lung colonization of melanoma nodules was markedly diminished in mice with mast cell-specific ablation of Cyp11a1, accompanied by reduced infiltration of mast cells into the lungs. Cyp11a1 gene expression was significantly decreased in lungs of mice with mast cell-specific ablation of Cyp11a1, indicating that mast cells account for a substantial fraction of the total Cyp11a1 expression. Our results also revealed that the mast cell-specific deletion of Cyp11a1 led to an overall increase in CD107a/LAMP1 staining intensity of the lung tissue, suggesting that mast cell-derived steroids can suppress immune cell activation/degranulation. A further dissection of this finding by flow cytometry analysis of individual immune cell populations revealed that CD8+ T cells, NK cells and basophils were activated to a higher extent in lungs from mice with mast cell-specific Cyp11a1 ablation. We also demonstrate that both CD8+ and CD4+ T cells in lungs of mice with mast cell-specific deletion of Cyp11a1 expressed elevated levels of IFN-{gamma} in comparison with controls. Altogether, these findings introduce a hitherto unrecognized role of a mast cell-derived steroid axis in regulating tumour metastasis.

Cell division is a crucial process that ensures proper development of multicellular organisms. Cell division ends in abscission, a process in which the intercellular bridge between two sister cells is cut. Although abscission usually happens shortly after chromosome segregation, abscission is severely delayed in mouse embryos and embryonic stem cells (mESC). The regulation of the duration of abscission influences cell fate transitions but how cell state and abscission dynamics crosstalk remains unknown. Here, we show that a key pluripotency pathway, the Wnt signalling pathway, controls abscission dynamics. Upon deactivation of Wnt signalling in naive mESCs, abscission becomes faster. Wnt signalling regulates abscission dynamics in mESCs through two mechanisms. First, Wnt signalling keeps the amount of Aurora B high at the intercellular bridge, probably by preventing Aurora B degradation. In turn, high Aurora B activity at the bridge delays bridge abscission. Second, a key component of Wnt signalling, the kinase GSK-3{beta} localizes at the intercellular bridge with microtubules and their associated proteins (MAPs). In pluripotent cells, inactivation of GSK-3{beta} leads to an increase of stable microtubules at the bridge stable which causes delayed abscission. Crucially, inhibition of GSK-3{beta} after cells have exited naive pluripotency accelerates abscission, demonstrating that cell state influences the output of the abscission signalling pathway. The permissive function of canonical Wnt on cell state is thought to be mediated by reinforcement of existing pluripotency network; altogether, our work shows that non-canonical Wnt is also context dependent.

Crossing over during meiosis drives genetic diversity and ensures the accurate segregation of homologous chromosomes. Variation in the rate of crossing over has been linked to evolutionary divergence and environmental adaptability, shaping fitness and responses to selective pressures. Despite its significance, the molecular mechanisms underlying this variation remain poorly understood. Crossover sites are selected from a large pool of potential sites initiated by programmed DNA double-strand breaks. Post-translational modification by SUMO (Small Ubiquitin-like Modifier) has been implicated in this process. Here, we show that crossover rate, chromosome length, and abundance of chromosome-associated SUMO are positively correlated across a range of vertebrate species, including mouse, chicken, pig, cattle, sheep, and goat. Crossover variation between goat breeds across the Indian subcontinent was also positively correlated with chromosomal SUMO level. Furthermore, modulating SUMO levels in cultured goat spermatocytes altered crossover frequency. Cumulatively, these observations point to a central role for SUMO in mediating crossover variation both between and within vertebrate species.

Following their engagement towards differentiation, tissue stem cells often transit through a precursor state that is difficult to define because of its transient nature; similarly, the precise role of lineage precursors in implementation of tissue architecture and function is unknown. In the present work, we used two mouse models of deficient feedback regulation to characterize precursors of the pituitary corticotrope lineage that regulates the stress response. Both the POMC knockout and adrenalectomized mouse models develop glucocorticoid deficiency and compensatory accumulation of corticotrope precursors that have so far eluded characterization. We found that pre-corticotrope differentiation depends on the lineage-specific factor Tpit and is repressed by glucocorticoids. We identified brain-derived neurotrophic factor (BDNF) as the signal that engages pituitary stem cells towards differentiation in these models as well as in normal pituitary development. A glucocorticoid-sensitive BDNF autocrine loop active in pre-corticotropes turns these cells into signaling hubs for maintenance of pituitary-adrenal homeostasis. HighlightsO_LIPituitary lineage precursors expand in conditions of deficient feedback regulation C_LIO_LIBDNF mobilizes pituitary stem cells during establishment of tissue size and architecture C_LIO_LICorticotrope precursors are a signaling hub for tissue homeostasis C_LI