Cellular and Molecular Life Sciences

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.

The most severe phenotype of alpha-1-antitrypsin deficiency (AATD) is caused by the Z-mutation within the SERPINA1 gene. The Glu342Lys substitution causes misfolding and polymerisation of the alpha-1-antitrypsin (AAT) protein, its accumulation in the ER and increases the susceptibility of hepatocytes towards ER-stress. Here, we present an induced pluripotent stem cell (iPSC)-based hepatic model to study AATD. We demonstrate that iPSCs from AATD patients differentiate equally well to hepatocyte-like cells (HLCs) as control iPSCs. We detected ZAAT polymers in patient-derived HLCs which could be reduced by SAHA or CBZ treatment. Transcriptome analyses revealed major differences in metabolism and signalling between control and AATD HLCs and indicated increased stress levels affecting intracellular organelles. Importantly, the transcriptomes of control and patient-derived cells separated into distinct clusters with respect to the expression of Heat-shock protein (HSP) encoding genes. SAHA treatment increased expression of various HSPs which might contribute towards reduced ZAAT polymers.

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.

In our society, ageing, longevity, and neurodegenerative diseases are major concerns of public health. Recently, in Drosophila, we have identified a new cluster of three snoRNAs, including jouvence, and showed that each of them affect longevity and neurodegeneration. As these snoRNAs are required in the epithelium of the gut, these results point-out a causal relationship between the epithelium of the gut and the neurodegenerative lesions through the metabolic parameters, indicating a gut-brain axis. Here, we demonstrate that each snoRNA pseudouridylates a specific site on ribosomal-RNA, which consequently affects the amount of ribosomes as well as the translational efficacy. Moreover, using TRAP experiment assay, we also show that these lacks of pseudouridylations modify the translation of specific genes involved in lipid metabolism. Consequently, these lead to a chronic deregulation of trigycerides and sterols levels, whose correlate to an increase of neurogenerative lesions in old flies, as well as to a modification of longevity.

Ciliogenesis associated kinase 1 (CILK1) deficiency in human and mice results in kidney developmental defects including cystogenesis. However, the biology of CILK1 in autosomal dominant polycystic kidney disease (ADPKD), the most common inherited kidney disease, remains to be investigated. Here, we show that CILK1 is overexpressed in dedifferentiated cells of renal tissue from ADPKD human patients in comparison to normal control tissue samples. We demonstrate that CILK1 overexpression results in protein accumulation in a non-phosphorylated inactive form. Using mouse polycystic kidney disease models, we reveal that inactive CILK1 accumulation is progressive over the course of disease progression. We show that genetic inactivation of the Polycystic Kidney Disease 1 (PKD1) gene is sufficient to trigger CILK1 accumulation. Altogether, these findings demonstrate that CILK1 regulation is altered in ADPKD and it represents a hallmark of disease progression.

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.

The MYC associated factor X (MAX) is the heterodimeric partner of the MYC paralogs (MYC, MYCN and MYCL). When deregulated, high level of the MYC paralogs contribute to all aspects of tumorigenesis and tumor growth. MAX can also heterodimerize with the MXD proteins, MNT and MGA. Heterodimerization and sequence specific DNA binding to the E-Box sequences at gene promoters is controlled by their heterodimerization with the MAX b-HLH-LZ. As a heterodimer with MAX, MYC proteins activate genes involved in cell metabolism, growth and proliferation whereas MXD proteins, MNT and MGA repress them. MAX can also bind to the E-Bos sequence as a homodimer. Being devoid of a transactivation domain it can act as an antagonist of the MYC/MAX heterodimers. Variants of MAX have been reported to be linked to cancer. These variants are either not expressed, inactivated or lead to missense mutations. This has led to the notion that MAX may have a tumor suppressor role. Here, we characterize three of those variants with missense mutations in the basic region, i.e. E32K, R35P and R35C. We analyzed their heterodimerization with the b-HLH-LZ of MYC and their DNA binding properties as homo-and heterodimers. The R35C variant b-HLH-LZ was found to have a markedly increased affinity for the b-HLH-LZ of MYC. We also observed that all three b-HLH-LZ variants have a lower affinity as homodimers for the E-Box than the WT. This was shown to lead to a preferential binding of all the heterodimeric b-LHLH-LZ to the E-Box. This effect is exacerbated in the case of the R35C variant. We argue that this preferential binding of MYC as heterodimers with these variants to E-Box sequences could contribute to tumorigenesis. Hence, our results suggest that, mechanistically, the MAX homodimer bound to the E-Box could act as a tumor suppressor. MATERIALS AND METHODSO_ST_ABSMolecular modelingC_ST_ABSThe open source version 1.7.6.0 of Pymol was used for modeling and molecular rendering [1]. The crystal structure of the MAX homodimer bound to the E-Box (1HLO [2]) was used as a template for the generation of the models. The variants were generated using the mutagenesis function in the wizard. The conformation of the K32 side chain was manually set in order to avoid introducing steric clashes with DNA. Protein expression and purificationThe cDNA, coding for the MAX b-HLH-LZ (Max* hereafter, residues 22-103, UniProt entry P61244-1) to which are added the GSGC residues in c-terminal, inserted in the pET3a vector was already available in the laboratory [3] and was used as a template to generate the plasmids with inserts coding for each of the mutants (E32K, R35C and R35P) through quick-change PCR with Q5 DNA polymerase and DpnI from New England Biolabs. The primers used were purchased from IDT DNA, their sequences are listed in Table S1. Sequence for each construct was confirmed by Sanger sequencing at the Plateforme de sequencage SANGER - Centre de recherche du CHU de Quebec - Universite Laval. The primary structure for the basic region of each construct is given in Fig. 2A. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/715400v1_fig2.gif" ALT="Figure 2"> View larger version (41K): org.highwire.dtl.DTLVardef@1b05d5eorg.highwire.dtl.DTLVardef@1c1d692org.highwire.dtl.DTLVardef@ee469dorg.highwire.dtl.DTLVardef@15e0ba4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 2.C_FLOATNO Structure schematics, specific and non-specific interactions dictating specificity and stability of binding of the basic region of MAX to the canonical (CACGTG) E-Box. A. Primary structure for the basic region of MAX and each of the variants. Positions making the most important contacts with the E-box are indicated by black arrows. Positions for the variants studied here are colored according to the Zappo colour scheme, following their physico-chemical properties: red for negative, blue for positive, magenta for proline and yellow for cysteine. B. The side chain (carboxylate) of E32 receives H-Bonds from the CA nucleobases in the leading strand (white carbon atoms). R35 and R36 make a salt bridges with phosphate groups while and the guanidino moiety of R36 makes a specific H-Bond with the nucleobase of the G in the strand of the reverse complement (cyan carbon atoms). C. The R35C mutation removes one non-specific salt-bridge at the interface of the complex. D. The aliphatic portion of the K side chain in the E32K variant is unable to accept the H-Bonds from the CA nucleobases and leads to the stabilisation of the complex and the helical structure of the basic region. E. In addition to removing a salt-bride, the Pro residue in the R35P kinks the path of the basic region, prevents the establishment of the specific H-Bonds mandatory for recognition of the E-Box and leads to unfolding of the helical state. C_FIG The MYC b-HLH-LZ (Myc*), the Max*WT b-HLH-LZ and its variants were expressed and purified as previously described [3,4] After lyophilisation, the b-HLH-LZs were kept at -20{degrees}C and solubilised in Myc buffer (50 mM NaCl, 50 mM NaH2PO4 pH 5.5) for Myc* or PBS for Max* at a final concentration of 1 mM before use. Circular dichroismAll circular dichroism (CD) measurements were performed on a Jasco J-810 spectropolarimeter equipped with a Peltier-type thermostat. The instrument was routinely calibrated using an aqueous solution of d-10-(+)-camphorsulfonic acid at 290.5 nm. Samples were prepared as follows: Max* (either WT or a variant) was diluted in 100 {micro}l 2X CD buffer (40 mM KCl, 11.4 mM K2HPO4, 28.6 mM KH2PO4, pH 6.8) and the volume adjusted to 106 {micro}l with PBS. 10 {micro}l TCEP 16 mM were added, and the volume further adjusted to 192 {micro}l with ddH2O before samples were incubated overnight at room temperature. After reduction, Myc* was added and the volume adjusted to 198 {micro}l with Myc buffer (Na2HPO4 0.95 mM, NaH2PO4 49.05 mM, 50 mM NaCl, pH 5.5). The DNA complexes were prepared as follows. After a 10 minutes incubation of the protein samples at room temperature, 0, 1 or 2 {micro}l of 2 mM of specific or non-specific DNA duplexes in 10 mM Tris pH 8.0 were added and the volume adjusted to 200 {micro}l with 10 mM Tris pH 8.0. The strands of the specific probe were: 5-ATT ACC CAC GTG TCC T*AC-3 and 5-GTA GGA CAC GTG GGT* AAT-3 (with the E-box sequence underlined) and the non-specific probe: 5-ATT ACC TCC GGA TCC T*AC-3 and 5-GTA GGA TCC GGA GGT* AAT-3 (Integrated DNA Technologies). Samples were further incubated for 10 minutes at room temperature and transferred to a 1 mm path length quartz cuvette. All spectra were recorded from 250 to 195 nm at 0.1 nm intervals by accumulating 10 spectra at 25 {degrees}C. Thermal denaturations were recorded at 222 nm from 5 to 95 {degrees}C at a heating rate of 1 {degrees}C/min. CD signal for spectra and thermal denaturations was corrected by substracting the signal from corresponding spectra or thermal denaturation either for buffer alone or the appropriate DNA duplex. CD signal was then converted to mean residue ellipticity using the following formula [5]: [{theta}] = {delta} {middle dot} MRW/(10{middle dot}c l) where [{theta}] is the mean residue ellipticity in deg {middle dot} cm2 dmol-1, {delta} is the CD signal in millidegrees, MRW is the mean residue weight, c is the concentration in mg/ml and l is the pathlength in mm. For the heterodimers, the concentration used was the sum of Max* and Myc* and the MRW was determined using a weighted average.

Astroglia can counteract the harmful effects of -synuclein (-SYN) protofibrils and reverse premature cellular senescence by promoting tunneling nanotubes (TNTs). However, the mechanism behind this recovery is unknown. This study is the first to examine TNT-mediated mechanical stability in senescent astroglial recovery. We demonstrate that disruption of Lamin A/C in -SYN-protofibrils-treated senescent cells reduces actin-cytoskeleton stress, as measured by nucleus flatness index and isometric scale factor from quantitative microscopy. ROCK (Rho-associated kinase) inhibition, which is crucial for reducing actin-cytoskeleton tension, promotes TNTs. Small molecules like Cytochalasin-D, Nocodazole, and Jasplakinolide, which inhibit TNTs by altering actin tension other than ROCK pathway, cannot reverse senescence. RNA-sequence heatmaps reveal changes in senescence-, integrin-, and ROCK-pathway genes; STRING links these to the Hippo pathway. Experimental results show that cytosolic YAP translocation, a key regulator of Hippo pathway, is vital for TNT formation and actin-based stability in U87-MG astrocytoma and primary astrocytes. Interestingly, TNTs form between two cells with different actin tensions: one exhibits low actin tension with Hippo signaling on, while the other has higher actin tension with Hippo signaling off. The most notable observation is the high abundance of YAP inside the TNTs, along with actin. The study shows that TNTs maintain mechanical stability through Lamin A/C integrity and actin tension in -SYN-induced senescent astroglia, thereby protecting the cells, reversing senescence. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/711517v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@192307dorg.highwire.dtl.DTLVardef@ad8b75org.highwire.dtl.DTLVardef@19ece78org.highwire.dtl.DTLVardef@1056395_HPS_FORMAT_FIGEXP M_FIG C_FIG

Alternative pathways of antigen presentation are crucial in different immunological contexts such as autoimmunity and anti-microbial defense. Among these pathways, autophagy has a central role in delivering cytosolic substrates to the MHC class II compartment. However, its contribution to endogenous MHC class II presentation was only demonstrated for a few antigens. Here we focused our study on the contribution of autophagy to the peptidome of one major allele of the HLA-DR shared epitope, HLA DRB1*04:01 conferring the greatest risk factor for the development of rheumatoid arthritis (RA). We provide an extensive qualitative and quantitative mass spectrometry analysis of the autophagy related MHC class II peptide repertoire of the human DRB1*04:01 allele. A fraction of peptides representing 30% of the repertoire differ profoundly between autophagy sufficient and deficient cells. Our analysis demonstrates that autophagy contributes to MHC class II presentation of peptides from seven described RA autoantigens, the majority of them being related to the ER folding and stress response (calreticulin, calnexin, the 78 kDa glucose-regulated protein (GRP78)-also known as binding immunoglobulin protein (BiP) and several protein from the heat-shock-protein 70 family). Our results correlate with an increased activation of autophagy, in situ, in synovial biopsies and synovial fibroblast (SF) of RA patients. We could further show that SF upregulate MHC class II and present peptides from autophagy related auto-antigens to CD4 T cells from RA patients. Our finding identifies autophagy as a potential process that could contribute to the break of peripheral tolerance during RA.

Cellular senescence is a stable cell-cycle arrest state associated with characteristic phenotypes, including enlarged cell morphology, altered secretory signaling, and pronounced lysosomal remodeling. Senescent cells commonly accumulate increased numbers of enlarged lysosomes with changes in acidity and degradative capacity, creating an opportunity for simple live-cell readouts of senescence-linked organelle remodeling. Here, I describe a live-cell lysosomal profiling protocol that uses LysoTracker Deep Red, an acidotropic fluorescent dye, to label and quantify acidic organelles in individual living cells as an indicator of senescence-associated lysosomal expansion. The method is demonstrated in IMR-90 human lung fibroblasts undergoing replicative senescence across serial passaging. The protocol details cell culture and passage tracking, LysoTracker staining, fluorescence imaging, and straightforward image-based quantification of lysosomal signal intensity and lysosome-enriched area per cell. As an optional validation step, senescence-associated {beta}-galactosidase staining is performed on parallel cultures to confirm senescent cell identity. Representative outcomes show increased LysoTracker signal and expanded lysosome-enriched regions in late-passage cultures compared to early-passage controls, consistent with lysosomal remodeling during senescence. This protocol is designed to be simple to adopt and can be adapted to other cell types or senescence-inducing stresses, providing a practical, quantitative complement to conventional endpoint assays. SUMMARYThis article presents a live-cell imaging protocol using LysoTracker Deep Red to quantify lysosomal remodeling as a marker of cellular senescence in IMR-90 human fibroblasts. We demonstrate quantitative lysosomal readouts derived from fluorescence imaging, including lysosome-enriched area and intensity measurements that can be summarized per cell and, when desired, as stitched-field, per-nucleus normalized metrics. Senescence status can be validated against senescence-associated {beta}-galactosidase (SA-{beta}-Gal) staining performed on parallel cultures. The method can be adapted to other cell types or senescence-inducing stresses and enables quantitative analysis of lysosomal remodeling during senescence.

NRF1 is a key mediator of the proteasome recovery pathway, yet its regulation by ER-resident factors is not fully elucidated. Here, we demonstrate that selenoproteins SELS and SELK are critical regulators for NRF1 protein dynamics. SELS stabilizes NRF1, while SELK induces its insolubilization. Their deficiency leads to a hyper-accumulation and increased nuclear localization of NRF1 under proteasome inhibition condition. This results in an augmented transcriptional response of proteasome subunits. These results indicate that SELS and SELK cooperatively gate NRF1 activity by controlling its retrotranslocation and solubility, highlighting a novel layer of selenoprotein-mediated quality control in the proteostasis network.

Since NO can modulate mesenchymal cell function, we posit that NO can modulate gene expression associated with excitation-contraction coupling. Our study shows that treating asthma-derived HASMCs with a low dose of NO plus sGC stimulator BAY-41, in most cases sensitized smooth muscle sGC towards activation via an elevated sGC heterodimer and in some cases also improved sGC{beta}1, catalase, Cyb5r3 or Trx1 expression (n=24 non-asthma and n=25 asthma). Interestingly we found that majority of asthma HASMCs showed a marked downregulation of G6PD expression inducing a low GSH/GSSG ratio in asthma, and these findings were replicated in murine lungs of allergic asthma (OVA and CFA/HDM). Studies with HEK/COS-7 cells showed G6PD synergizing with hsp90 in enabling sGC heme-maturation. G6PD overexpression in HASMCs enhanced the sGC heterodimerization while silencing of endogenous G6PD abrogated it. Complementation of these cellular results with whole animal models of G6PD deficiency or overexpression provided verification to our findings. Mouse lung tissue from the humanized variant of G6PD deficiency, V68M (G6PD A-deficiency) showed significant downregulation in the sGC heterodimer, with a concomitant reduction in its NO heme-dependent activity, thereby showing that G6PD deficiency lowers sGC heme. Conversely, G6PD overexpressing mouse lung tissue displayed an elevated sGC heterodimer and also showed a robust G6PD-sGC{beta}1 interaction, suggesting G6PD to be involved in the heme-maturation of sGC{beta}1. While G6PD maintains the cell redox by generating NADPH, its new role in regulating sGC maturation links sGC dysfunction in asthma to G6PD deficiency and may potentially uncover new targets for asthma treatment.

Saul-Wilson syndrome (SWS) is a skeletal dysplasia characterized by primordial dwarfism and progeroid features caused by a recurrent dominant COG4 variant (p.G516R). We previously showed that this mutation accelerates Golgi retrograde trafficking and disrupts glycosylation of the proteoglycan decorin, while zebrafish models revealed defects in chondrocyte elongation and intercalation. We have also shown that the SW1353 chondrosarcoma cells carrying the SWS variant exhibit reduced secretion of extracellular matrix (ECM) components. While these results indicate a critical function of COG4 in Golgi processing, the developmental process leading to skeletal dysplasia in SWS patients remains unknown. Here, we generated patient-derived iPSC cartilage organoids (SWS organoids), modeling early human chondrogenesis. SWS organoids failed to produce cartilage structures and displayed poor expression of chondrogenic markers. Time-course RNA-seq analysis of the chondrogenic process revealed reduced activation of gene networks involved in skeletal development, ECM organization, ossification, and glycosaminoglycan metabolism. Spatial multiomic analysis of protein and glycosylation by CODEX and GLYPH imaging revealed an altered chondrogenic trajectory, persistence of mesenchymal states, global glycosylation changes, and reduced deposition of chondroitin sulfate proteoglycans. These results indicate that the COG4 mutation disrupts ECM glycosylation and chondrogenic commitment, and that SWS organoids model early defects in cartilage formation underlies impaired skeletal growth in SWS. HighlightsO_LIPatient iPSC-derived cartilage organoids model development defects in Saul-Wilson syndrome C_LIO_LISWS organoids show defective extracellular matrix deposition and attenuated chondrogenic gene expression C_LIO_LIGlycan profiling reveals global glycosylation defects and deficient proteoglycan GAG chains C_LIO_LIAn early developmental impairment in chondrogenesis alters skeletal formation in Saul-Wilson syndrome C_LI

The selective autophagy receptors SQSTM1/p62 and NBR1 are evolutionary related and involved in autophagy of different substrates including proteins, protein aggregates and organelles. The two proteins interact via their PB1 domains, and essential for their roles in autophagy is the formation of a specific type of condensates named p62 bodies. The scaffold of these structures is formed by the interaction of polymeric p62 with polyubiquitin, but NBR1 is recruited and essential for their formation. Previous studies have shown that p62 contains nuclear export signal (NES) and nuclear localization signal (NLS) motifs and shuttles between the cytoplasm and the nucleus. Its nuclear roles are not fully understood, but there is evidence that p62 is involved in protein quality control in the nucleus. No previous studies have tested if NBR1 is transported into the nucleus. We show here that NBR1 contains two NES motifs and one NLS motif, and like p62, the protein shuttles between the cytoplasm and the nucleus. NBR1 also accumulates in nuclear p62 bodies and the formation of nuclear p62 bodies depends on NBR1.

The Ube2J1 enzyme that mediates the ubiquitination and proteasomal degradation of misfolded proteins at the ER is phosphorylated at serine S184. Following anisomycin treatment of HEK293T cells, we observed an inverse relationship between phosphorylation and dephosphorylation at this site. This suggested a dynamic interchange between the two forms, and we show that S184 is a target for protein phosphatase 2A. The S184-phosphorylated protein is known to exhibit increased sensitivity to proteasomal degradation, and we found that mutation at K186R increased the ratio of S184-phosphorylated to S184-dephosphorylated protein. Although the K186R mutant retained some sensitivity to proteasomal inhibition, our results show that Ube2J1 steady state expression can be exercised at multiple levels, and can involve dynamic phosphorylation and dephosphorylation at S184.

Naked mole-rats (NMRs, Heterocephalus glaber) display unusual longevity and resistance to age-related decline, and accumulating evidence suggests that their autophagy-lysosome pathway (ALP) is regulated differently from that of conventional mammalian models. However, most studies in NMR cells have relied on static biochemical or ultrastructural readouts, leaving the dynamic organisation of autophagy in living cells poorly defined. Here, we establish a stable tandem fluorescent autophagy reporter in NMR skin fibroblasts using an mCherry-EGFP-LC3NMR construct to enable live-cell, single-cell resolution analysis of ALP dynamics. Under basal conditions, NMR skin fibroblasts exhibit a greater abundance of LC3-positive structures than HeLa cells, together with a mixed population of autophagosomes and autolysosomes, indicating a distinct steady-state organisation of the ALP. Chloroquine (CQ)-induced lysosomal stress caused the expected accumulation of LC3-positive structures but also triggered the formation of large cytoplasmic vacuoles in NMR skin fibroblasts. Importantly, this vacuolation was not associated with acute cytotoxicity and progressively resolved following CQ removal, accompanied by reorganisation of LC3-positive compartments and recovery of lysosomal acidity. Electron microscopy showed that CQ-induced vacuoles are membrane-bound, containing internal material and co-existing with multiple ALP-related vesicular compartments. Primary NMR skin fibroblasts display a similar vacuolation phenotype, indicating that this response is not an artefact of immortalisation or reporter expression. Together, these findings establish a live-cell platform for analysing autophagy in NMR cells and identify a distinctive, reversible vacuolation response to lysosomal stress, consistent with dynamic remodelling of the lysosomal system within NMR skin fibroblasts.

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.

KMT2D, the histone methyltransferase and core component of the COMPASS/MLL4 complex, has been implicated in developmental diseases such as Kabuki Syndrome, interstitial lung disease, and congenital diaphragmatic hernia, with clear links to pediatric pulmonary disorders. Despite this, the mechanism by which KMT2D governs lung development remains unclear. Knock-in mouse models rendering, KMT2D catalytically deactivated (KMT2DKI) and reducing H3K4 methylation, have demonstrated potential in defining KMT2Ds role in pulmonary development. Our examination of the lungs of KMT2DKI mice revealed increased cellular density and impaired sacculation indicated by reduced airspace chord length, thickening of intersaccular septa, and abnormal alveolar cell differentiation. KMT2DKI mice revealed narrowed Sox2+ conducting airways and epithelial differentiation defects characterized by reduced Cc10+ club cells. Accompanying the alveolar and airway hypoplasia, blood vessel luminal area was reduced. Conversely, KMT2DKI lungs had a significantly higher proportion of proliferating cells accompanied by a dramatic expansion in Pdgfr+ mesenchymal progenitor cells. Our findings therefore suggest that KMT2D-mediated H3K4 methylation is vital to normal lung development, and its impairment results in widespread pulmonary hypoplasia and potentially pulmonary hypertension.

IP3R-Grp75-VDAC1 protein complex at the mitochondria-ER contact sites (MERCS) is involved in response to nutrients and control of glucose and energy metabolism, however, early alterations of the complex and MERCS in response to increased fat intake remain inconclusive. We investigated early effects of high-fat diet (HFD) on IP3R-Grp75-VDAC1 protein expression in correlation with ER-mitochondrial interaction in the liver of mice. Five-week-old mice were fed an HFD or a standard diet (SD) for 2 weeks (2W) or 8 weeks (8W). MERCS fractionation by a gradient ultracentrifugation, Western blot, transmission electron microscopy (TEM), Oroboros high-resolution respirometry were used to analyse liver tissues, while real-time PCR was used to profile genes responsive to HFD. No macroscopic morphological or functional alterations were observed in mice at 2W, while, expectedly, at 8W of HFD mice gained weight and glucose intolerance. Total IP3R protein was reduced at both 2W and 8W points by a post-transcriptional mechanism, while in MERCS, IP3R, VDAC1 and Grp75 were reduced at 8W time-point. TEM analysis revealed a significant reduction of mitochondrial coverage by MERCS, mitochondrial fragmentation and shortening of ER-mitochondria distance already at 2W time-point. Mitochondrial function and metabolism were largely spared. Markers of altered protein homeostasis such as Lmp2, Mecl-1 and Lmp7 showed an early upregulation. In conclusion, HFD induces early alterations in liver MERCS that precede gain of weight and glucose intolerance, suggesting their primary role in obesity and metabolic diseases and as potential therapeutic target.

The cellular response to DNA replication stress (DRS) provoked by anticancer drugs involves activation of the G2/M checkpoint (which promotes transient cell cycle arrest at G2 phase) and DNA repair, followed by induction of apoptosis or senescence. Here, we activated the p53-p21 pathway and ATR using DRS-inducing drugs, and found that that the transition to senescence depends on the duration of the G2 phase. Shortening of G2 duration by G2/M checkpoint inhibitors led not only to a switch in cell fate from senescence to mitotic entry, but also to effective cell death through carry-over of chromosomal aberrations (generated by DRS-inducing drugs) into mitosis and subsequent mitotic progression. Such enhanced cell death was also observed in p53 deficient cells, which do not normally undergo senescence. Thus, we propose that temporal regulation of G2 phase is an approach to enhancing the effects of DRS-inducing drugs in a manner that is independent of p53 status.