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Long-read RNA sequencing enables isoform-resolved transcriptomics, but library preparation introduces systematic biases that shape biological interpretation. We benchmarked Oxford Nanopores two protocols--PCR-cDNA and direct RNA--using SKMM2 myeloma cells stimulated with interleukin-6 (IL-6) and ERCC synthetic spike-ins. Direct RNA produced longer, higher-quality reads and more high-confidence isoforms, but showed pronounced 5' coverage loss. PCR-cDNA yielded shorter fragments with 3' underrepresentation, detecting more low-abundance transcripts at reduced confidence. Protocol-specific biases had major consequences: differential expression analysis revealed limited overlap in IL-6-responsive genes, and pathway enrichment was broader in direct RNA. At the isoform level, differential transcript usage was almost entirely protocol-specific, with case studies (e.g. RPL22L1, GRB2, RNF220) illustrating concordance and divergence. ERCC controls confirmed these biases as technical rather than biological. Together, our results show that while both methods provide accurate gene-level quantification, transcript-level conclusions depend critically on protocol choice, highlighting the need for careful selection in long-read transcriptomics.

The level of cellular organization bridging the mesoscale and whole-cell scale is coming into focus as a new frontier in cell biology. Great progress has been made in unraveling the complex physical and functional interconnectivity of organelles, but how the entire organelle network is spatially arranged within the cytoplasm is only beginning to be explored. Drawing on cross-disciplinary research synthesis methods, we systematically curated the whole-cell volumetric imaging literature, resulting in a corpus consisting of 89 studies and 118 image datasets. We describe the trajectory and current state of the field between 2004 and 2024. A broad characterization, or "scoping review", of bibliometrics, study design, and reporting practices shows accelerating technological development and research output. We find high variability in study design and reporting practices, including imaging modality, model organism, cellular contexts, organelles imaged, and analyses. Due to the laborious, low-throughput nature of most volumetric imaging methods, we find trends toward small sample sizes (<10 cells) and small cell types. We describe common quantitative analyses across studies, including volumetric ratios of organelles and inter-organelle contact analyses. This work establishes the initial iteration of a growing dataset of whole-cell imaging literature and data, and motivates a call for standardized whole-cell imaging study design, reporting, and data sharing practices in the context of an emerging sub-field of cell biology. Our curated dataset now provides the basis for a plethora of future aggregate and comparative analyses to reveal larger patterns and generalized hypotheses about the systems behavior and regulation of whole-cell organelle networks. More broadly, we showcase the potential of new rigorous secondary research methods to strengthen cell biologys literature review and reproducibility toolkit, create new avenues for discovery, and promote open research practices that support secondary data-reuse and integration.

Initial symptoms of COVID-19 infection depend on viral replication, while hyperinflammation is a hallmark of critical illness and may drive severe pneumonia and death. Among the mechanisms potentially involved in the hyperinflammatory state, we focused on the unfolded protein response, because the IRE1-XBP1 branch can be activated as result of the endoplasmic reticulum stress produced by the overwhelming synthesis of viral components and synergizes with Toll-like receptor signaling to induce cytokine expression. Viral RNA may trigger the IRE1-XBP1 branch via TLR7/8 activation and like TLR2 and TLR4 may underpin cytokine expression trough XBP1 splicing (sXBP1). The expression of IL1B, IL6, and TNF mRNA in bronchoalveolar aspirates (BAAs) were higher in COVID-19 patients under mechanical ventilation and intubation who showed sXBP1. The scrutiny of monocytic/macrophagic markers during active infection showed a reduction of those involved in antigen presentation and survival, as well as the IFN stimulated gene MX1. These changes reverted after infection tests turned negative. In contrast, the expression of the mRNA of the serine protease TMPRSS2 involved in S protein priming showed a high expression during active infection. TLR8 mRNA showed an overwhelming expression as compared to TLR7 mRNA, which suggests the presence of monocyte-derived dendritic cells (MDDCs). In vitro experiments in MDDCs activated with ssRNA40, a positive-sense, single-stranded RNA (+ssRNA) like SARS-CoV-2 RNA, induced sXBP1 and the expression of IL-1{beta}, IL-6, and TNF at mRNA and protein levels. These responses were blunted by the IRE1 ribonuclease inhibitor MKC8866. Given the analogies between the results observed in BAAs and the effects induced by +ssRNA in MDDCs, IRE1 ribonuclease inhibition might be a druggable target in severe COVID-19 disease. O_FIG O_LINKSMALLFIG WIDTH=180 HEIGHT=200 SRC="FIGDIR/small/22269752v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@13b04b3org.highwire.dtl.DTLVardef@1b1af7corg.highwire.dtl.DTLVardef@780104org.highwire.dtl.DTLVardef@8ad0ba_HPS_FORMAT_FIGEXP M_FIG C_FIG Author summaryCOVID-19 pandemics put an unprecedented pressure on health systems. The need of new therapies urged research on the mechanisms triggered by the interaction of SARS-CoV-2 virus with host cells and the ensuing pathophysiology driving pneumonia and multiorgan failure. Hyperinflammation soon appeared as a mechanism involved in mortality that could even proceed after viral infection comes to an end. Hyperinflammation is supported by an inappropriate production of cytokines, and this explains the use of the term cytokine storm to refer to this phase of the disease. Given that insight into the molecular mechanisms driving cytokine storm should focus on the interaction of viral components with immune cells, experiments addressing the effect of viral components on its cognate receptors were carried out. It was observed that viral RNA induces a cytokine pattern like the one observed in bronchoalveolar aspirates of COVID-19 patients with critical disease. Overall, the study revealed that both cell organelle overload and receptors involved in the recognition of viral RNA may team up to induce proinflammatory cytokines. This mechanism can be exploited to develop new treatments for COVID-19 disease.

Withdrawal StatementThe authors have withdrawn this manuscript because following a careful internal review conducted in the course of manuscript revision, the authors identified that parts of the presented data could not be reliably traced back to the corresponding original datasets in their current form. While the general biological interpretation is not necessarily invalidated by this finding, the integrity of the present data representation no longer meets the standard required for publication. For this reason, the authors have decided to withdraw this preprint and will refrain from pursuing publication until the underlying data situation has been fully clarified and, if possible, reconstructed. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author. The authors regret any inconvenience caused to the scientific community.

As part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Bhargava et al., 2016) that described how we intended to replicate selected experiments from the paper "RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth" (Hatzivassiliou et al., 2010). Here we report the results. We found two unrelated RAF inhibitors, PLX4720 or GDC-0879, selectively inhibited BRAF(V600E) cell proliferation, while the MEK inhibitor, PD0325901, inhibited BRAF(V600E), wild-type RAF/RAS, and mutant RAS cancer cell proliferation, similar to the original study (Figure 1A; Hatzivassiliou et al., 2010). We found knockdown of CRAF, but not BRAF, in mutant RAS cells attenuated the phospho-MEK induction observed after PLX4720 treatment, similar to the original study (Figure 2B; Hatzivassiliou et al., 2010). The original study reported analogous results with GDC-0879, which was not observed in this replication, although unexpected control results confound the interpretation. We also attempted a replication of an assay with recombinant proteins to test the differential effect of RAF inhibitors on BRAF-CRAF heterodimerization (Figure 4A; Hatzivassiliou et al., 2010). Although we were unable to conduct the experiment as planned, we observed differential binding of BRAF by RAF inhibitors; however, it was between BRAF and beads, independent of CRAF. While these data were unable to address whether, under the conditions of the original study, the same observations could be observed, we discuss key differences between the original study and this replication that are important to consider for further experiments. Finally, where possible, we report meta-analyses for each result. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/470372v2_fig1.gif" ALT="Figure 1"> View larger version (7K): org.highwire.dtl.DTLVardef@1f77d0eorg.highwire.dtl.DTLVardef@1333494org.highwire.dtl.DTLVardef@c10824org.highwire.dtl.DTLVardef@fda30f_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Cellular dose response curves for RAF and MEK inhibitors in BRAF(V600E), mutant RAS, and wild-type RAF/RAS cell lines Cell viability assays were performed for RAF inhibitors (PLX4720 and GDC-0879) and MEK inhibitor (PD0325901) against A375, (BRAF(V600E)), HCT116 (mutant RAS), and MeWo (wild-type RAF/RAS) cells. Absolute half-maximum effective concentration (EC50) values ({micro}M) were determined for each biological repeat. EC50 values unable to be accurately estimated are reported as either >20 {micro}M or <0.078 {micro}M, which are the largest and smallest doses tested, respectively. Absolute EC50 values for each biological repeat for A375, HCT116, and MeWo cells treated with (A) PLX4720, (B) GDC-0879, or (C) PD0325901 for this replication attempt are plotted with the EC50 value reported in Hatzivassiliou et al. (2010) displayed as a single point (red triangle) for comparison. Where possible the mean and 95% confidence interval of the replication data are shown. Additional details for this experiment can be found at https://osf.io/52zp9/. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/470372v2_fig2.gif" ALT="Figure 2"> View larger version (34K): org.highwire.dtl.DTLVardef@1dc3058org.highwire.dtl.DTLVardef@e9f5d9org.highwire.dtl.DTLVardef@baf5eorg.highwire.dtl.DTLVardef@126717a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 2.C_FLOATNO Cellular dose response curves for each biological repeat This is the same experiment as in Figure 1. The dose response curve of each biological repeat [n=4] for A375, HCT116, and MeWo cells treated with (A) PLX4720, (B) GDC-0879, or (C) PD0325901 for this replication attempt are plotted. Additional details for this experiment can be found at https://osf.io/52zp9/. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/470372v2_fig4.gif" ALT="Figure 4"> View larger version (33K): org.highwire.dtl.DTLVardef@e4357eorg.highwire.dtl.DTLVardef@73e9ceorg.highwire.dtl.DTLVardef@1fb01c6org.highwire.dtl.DTLVardef@31a799_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 4.C_FLOATNO Testing of BRAF and CRAF antibodies used in original study The BRAF and CRAF antibodies reported in the original study (Hatzivassiliou et al., 2010) were tested for suitability by Western blot. (A) Representative Western blots and Ponceau-stained membrane (experiment performed independently 3 times) of 0.5 {micro}g of purified recombinant BRAF kinase domain (KD). Membranes were probed with the BRAF antibody used in the original study (RRID:AB_1840330) or with a different BRAF antibody used in this replication attempt (RRID:AB_2721113). (B) Representative Western blots and Ponceau-stained membrane (experiment performed independently 3 times) of 0.5 {micro}g of purified recombinant GST tagged CRAF KD. Membranes were probed with the CRAF antibody used in the original study (RRID:AB_397552) or with a different CRAF antibody used in this replication attempt (RRID:AB_10989977). (C) Representative Western blots (experiment performed once) of 16 {micro}g of whole cell lysates from the indicated human cell lines. Membranes were probed with the BRAF (RRID:AB_1840330) or CRAF (RRID:AB_397552) antibodies used in the original study. (D) Screenshot from the Constraint-based Multiple Alignment Tool (COBALT) (Papadopoulos and Agarwala, 2007) showing the alignment of full-length BRAF (NCBI Reference Sequence: NP_004324.2), the region of BRAF used as the immunogenic peptide used to generate the BRAF antibody used in the original study (RRID:AB_1840330), the BRAF KD used in this replication attempt, and the BRAF KD used in the original study. (E) Screenshot from COBALT showing the alignment of full-length CRAF (NCBI Reference Sequence: NP_002871.1), the region of CRAF used as the immunogenic peptide used to generate the CRAF antibody used in the original study (RRID:AB_397552), and the CRAF KD used in the original study and this replication attempt. Additional details for this experiment can be found at https://osf.io/6f3sk/. C_FIG

Na+/H+ exchangers are found in all cells to regulate intracellular pH, sodium levels and cell volume. Na+/H+ exchangers are physiological homodimers that operate by an elevator alternating-access mechanism. While the structure of the core ion translocation domain is fairly conserved, the scaffold domain and oligomerization show larger structural variation. The Na+/H+ exchanger NhaA from E. coli has a weak oligomerization interface mediated by a {beta}-hairpin domain and homodimerization was shown to be dependent of the lipid cardiolipin. Organellar Na+/H+ exchangers NHE6, NHE7 and NHE9 are likewise predicted to contain {beta}-hairpin domains and a recent analysis of horse NHE9 indicated that the lipid PIP2 binds at the dimerization interface. Despite predicted lipid-mediated oligomerization, their structural validation has been lacking. Here, we report cryo-EM structures of E. coli NhaA and horse NHE9 with the coordination of cardiolipin and PI(3,5)P2 binding at the dimer interface, respectively. Cell based assays confirms that NHE9 is inactive at the plasma membrane and thermal-shift assays, solid-supported membrane (SSM) electrophysiology and MD simulations, corroborates that NHE9 specifically binds the endosomal PI(3,5)P2 lipid, which stabilizes the homodimer and enhances activity. Taken together, we propose specific lipids regulate Na+/H+ exchange activity by stabilizing oligomerization and stimulating Na+ binding under lipid-specific cues.

Interleukin (IL)-1 is a pro-inflammatory member of the IL-1 cytokine superfamily and is important for inflammatory responses to infection and injury. Unlike pro-IL-1{beta}, pro-IL-1 is mainly localised to the nucleus upon expression. This is mediated by a nuclear localisation sequence (NLS) responsible for its importin-dependent transport into the nucleus. This nuclear localisation and the presence of histone acetyl transferase (HAT)-binding domains within the pro-domain suggest a role of this cytokine in gene transcription regulation. In addition, nuclear trafficking of pro-IL-1 is proposed to regulate its secretion. To-date, studies on the nuclear role of pro-IL-1 have used overexpression systems. Here, we generated a mouse where the endogenous Il1a gene was edited with CRISPR to disrupt the NLS (mNLS). Using an in vitro approach with murine macrophages we found that this NLS mutation did not affect pro-IL-1 RNA expression levels in response to LPS but increased its protein expression levels. Moreover, we found that the transcriptional signature induced by LPS was not altered between WT and mNLS macrophages. Release of IL-1 in response to different stimuli such as ionomycin was not negatively impacted by disrupted nuclear localisation, although higher levels of IL-1 release were detected, potentially due to increased levels of pro-IL-1. Inflammatory responses in an in vivo model of peritonitis and an influenza infection model were comparable between WT and mNLS mice. Thus, we have established a mouse model in which pro-IL-1 nuclear localisation is disrupted, although future research is required to reveal the importance of this nuclear localisation for IL-1 function.

Lysosomal proteases such as the cathepsin family and the asparaginyl endopeptidase, legumain, govern vital processes to maintain cellular proteostasis, and their dysregulation contributes to diverse pathologies. Recent studies have reported extra-lysosomal localisation of these proteases, especially in the nucleus, cytoplasm, and extracellularly, yet their function is not completely understood. To examine the relationship between legumain and cathepsins, we assessed the activity and expression of cathepsins in wild-type and legumain-deficient (LGMN-/-) cells using chemical activity-based probes and immunoblots. Processing of cathepsins (CTS) L, V, B, and D from the single-chain to the two-chain form was abrogated in the absence of legumain, with some cell type- and species-specific variation observed. This processing was dependent on legumain activity, although the mechanism remains unclear since recombinant legumain does not appear to directly cleave cathepsins in vitro. In cell types where CTSL exists in the nucleus preferentially in its double chain form, loss of legumain led to a reduction in nuclear CTSL levels. To understand the potential role of these lysosomal proteases in the nucleus, we applied our newly refined chemical N-terminomics pipeline, No-enrichment Identification of Cleavage Events (NICE). This analysis revealed widespread changes in both protein abundance and proteolysis, including putative nuclear substrates of CTSL and legumain, that primarily suggest roles in cell proliferation, cell cycle regulation, inflammation, and ribosomal biogenesis. Overall, this study builds on our understanding of the relationship between legumain and cathepsins and provides the first systematic characterisation of lysosomal protease substrates in the nucleus. Our results offer valuable insight into the potential extra-lysosomal roles of these critical proteases.

The authors have withdrawn their manuscript because many of the experiments described in this paper have not been reproducible, or at least are not robust, in the hands of other members of the Kaelin Laboratory who were not initially involved in this work. While we do see apparent secretion of histone H3 under some conditions, it is usually accompanied by secretion of histone H4. In this regard, the Halo tagged-histone H3 and Halo-tagged H4 constructs used for the single molecule imaging studies we reported, which seemingly confirmed specific secretion and transfer of histone H3, were purported to be sequence validated. Upon resequencing these constructs we discovered a non-synonymous mutation in the Halo tag of the H4 construct. We then redid the imaging experiments with the corrected Halo-H4 together with Halo-H3 and, in contrast to our earlier study, unfused Halo. These experiments were difficult to interpret because of the background signal seen with the unfused Halo but did not support specific secretion and transfer of histones (let alone specific secretion and transfer of histone H3). We have, in some experiments, observed transfer of H3-Cre into reporter cells ex vivo and in vivo, but 1) the transfer is not specific for H3-Cre versus H4-Cre, 2) the transfer efficiency is highly variable, and 3) the transfer efficiency is typically much less than reported in our original paper. We do not yet know whether this lack of reproducibility and robustness reflects technical and biological variables that we do not yet understand and hence were not captured in our experimental protocols. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

Cell migration frequently involves the formation of lamellipodia induced by Rac GTPases mediating activation of WAVE Regulatory Complex (WRC) driving Arp2/3 complex-dependent actin assembly. Previous genome editing studies solidified the view of an essential, linear pathway employing aforementioned components. Using disruption of the WRC subunit Nap1 and its paralogue Hem1 followed by serum and growth factor stimulation or expression of active GTPases now revealed a pathway to formation of Arp2/3 complex-dependent, lamellipodia-like structures (LLS) that require both Rac and Cdc42, but not WRC. These observations were independent of WRC subunit eliminated and coincided with the lack of recruitment of Ena/VASP family actin polymerases. Moreover, aside from the latter, induced LLS contained all common lamellipodial regulators tested, including cortactin, the Ena/VASP ligand lamellipodin or FMNL subfamily formins. Our studies thus establish the existence of a signaling axis to Arp2/3 complex-dependent actin remodeling at the cell periphery operating without WRC and Ena/VASP.

The Plasminogen-Apple-Nematode (PAN) domain, with a core of four to six cysteine residues, is found in > 28,000 proteins across 959 genera but its role in protein function is not fully understood. The PAN domain was initially characterized to be present in numerous proteins including hepatocyte growth factor (HGF). Dysregulation of HGF-mediated signaling results in numerous deadly cancers. All biological impacts of HGF in cell proliferation are triggered by binding of HGF to its cell surface receptor, cellular mesenchymal-epidermal transition (c-MET). Here, we show that four PAN domain cysteine residues are essential for HGF/c-MET signaling. Mutating these residues resulted in retardation of perinuclear localization, cellular internalization of HGF and its receptor, c-MET, and c-MET ubiquitination. Our observations indicate that the PAN domain of HGF is required for the c-MET binding and subsequent c-MET autophosphorylation and phosphorylation of its downstream targets, protein kinase B (AKT), extracellular signal-regulated kinase (ERK), and signal transducer and activator of transcription 3 (STAT3). Furthermore, transcriptional activation of HGF/c-MET signaling-related genes including matrix metalloproteinase-9 (MMP9), ETS translocation variant 1, 4, and 5 (ETV1, ETV4, ETV5), and early growth response 1 (EGR1) was impaired and cell proliferation was attenuated. These results suggest that core cysteine residues in the PAN domain are critical for HGF/c-MET interaction, c-MET mediated signal transduction, and cell survival. Thus, targeting the PAN domain of HGF may represent a mechanism for selectively regulating the binding and activation of the c-MET pathway. SignificanceHGF/c-MET signaling induces multifunctional cellular responses. Dysregulation of HGF/c-MET signaling cascade can lead to tumorigenesis by transforming normal cells to tumor cells. This work defines the importance of core cysteine residues in the PAN domain of HGF in downstream activation of HGF/c-MET signaling. To understand the role of cysteines in the PAN domain, PAN mutants of HGF were used to stimulate c-MET signaling in cells and the impact was delineated by determining phosphorylation and transcription of downstream targets. Mutations in core cysteines in the HGF-PAN domain completely blocked downstream phosphorylation and perinuclear accumulation of c-MET. These results suggest an indispensable role for the cysteine-rich PAN domain in HGF/c-MET interaction and could set the stage for future therapies that selectively disrupt the MET signaling cascade with limited off-target effects in tumors overexpressing HGF/c-MET.

Fibroblasts are functionally heterogeneous cells, capable of promoting and suppressing tumour progression. Across cancer types, the extent and cause of this phenotypic diversity remains unknown. We used single-cell RNA sequencing and multiplexed immunohistochemistry to examine fibroblast heterogeneity in human lung and non-small cell lung cancer (NSCLC) samples. This identified seven fibroblast subpopulations: including inflammatory fibroblasts and myofibroblasts (representing terminal differentiation states), quiescent fibroblasts, proto-myofibroblasts (x2) and proto-inflammatory fibroblasts (x2). Fibroblast subpopulations were variably distributed throughout tissues but accumulated at discrete niches associated with differentiation status. Bioinformatics analyses suggested TGF-{beta}1 and IL-1 as key regulators of myofibroblastic and inflammatory differentiation respectively. However, in vitro analyses showed that whilst TGF-{beta}1 stimulation in combination with increased tissue tension could induce myofibroblast marker expression, it failed to fully re-capitulate ex-vivo phenotypes. Similarly, IL-1{beta} treatment only induced upregulation of a subset of inflammatory fibroblast marker genes. In silico modelling of ligand-receptor signalling identified additional pathways and cell interactions likely to be involved in fibroblast activation, which can be examined using publicly available R shiny applications (at the following links: myofibroblast activation and inflammatory fibroblast activation). This highlighted a potential role for IL-11 and IL-6 (among other ligands) in myofibroblast and inflammatory fibroblast activation respectively. This analysis provides valuable insight into fibroblast subtypes and differentiation mechanisms in NSCLC.

Human ZMPSTE24, an integral membrane zinc metalloprotease, is required for conversion of prelamin A to mature lamin A, a component of the nuclear lamina and failure of this processing causes premature ageing disorders. ZMPSTE24 has also been implicated in both type 2 diabetes mellitus and in viral-host response mechanisms, but to date its only confirmed substrate is the precursor for lamin A. Prelamin A is thought to undergo four C-terminal post-translational modifications in the following order: farnesylation, SIM tripeptide cleavage, carboxymethylation and upstream "SY^LL" cleavage. Here we present evidence that the sequence of events does not follow the accepted dogma. We assessed cleavage of long human prelamin A sequence peptides by purified human ZMPSTE24 combined with FRET and mass spectrometry to detect products. Surprisingly, we found that the "SY^LL" cleavage occurs before and independent of the C-terminal CSIM modifications. We also found that ZMPSTE24 does not perform the predicted C^SIM tripeptide cleavage, but rather it removes an IM dipeptide. ZMPSTE24 can perform a tripeptide cleavage with a canonical CaaX box (C: cysteine; a: aliphatic; X: any residue), but the C-terminus of prelamin A is not a true CaaX sequence. Regardless of the C-terminal modifications of prelamin A, ZMPSTE24 can perform upstream SY^LL cleavage, thus removing the unwanted farnesylated C-terminus. Therefore, it is failure of SY^LL cleavage, not the C-terminal processing that is the likely cause of progeroid disorders.

Type I interferons (IFNs) play crucial roles in antiviral defence, autoinflammation and cancer immunity. The human genome encodes 17 different type I IFNs that all signal through the same receptor. Non-redundant functions have been reported for some type I IFNs. However, whether different type I IFNs induce different responses remains largely unknown. We stimulated human peripheral blood mononuclear cells (PBMCs) with recombinant type I IFNs to address this question in multiple types of primary cells. We analysed signalling responses by mass cytometry and changes in gene expression by bulk and single-cell RNA sequencing. We found cell-type specific changes in the phosphorylation of STAT transcription factors and in the gene sets induced and repressed upon type I IFN exposure. We further report that the magnitude of these responses varied between different type I IFNs, whilst qualitatively different responses to type I IFN subtypes were not apparent. Taken together, we provide a rich resource mapping signalling responses and IFN-regulated genes in immune cells. HighlightsO_LIMass cytometry and scRNAseq analysis of human PBMCs stimulated with type I IFNs C_LIO_LICell type-specific phosphorylation of STAT proteins and induction of ISGs C_LIO_LIDifferent type I IFNs induce qualitatively similar responses that vary in magnitude C_LIO_LIIdentification of ten core ISGs, up-regulated by all cell types in response to all type I IFNs C_LI In briefRigby et al. provide a single-cell map of signalling and transcriptomic responses to type I IFNs in ex vivo stimulated human PBMCs. Different cell types responded in unique ways but differences between different type I IFNs were only quantitative. These rich datasets are available via an easy-to-use interactive interface (https://rehwinkellab.shinyapps.io/ifnresource/). Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/547491v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@17e1e06org.highwire.dtl.DTLVardef@e53d81org.highwire.dtl.DTLVardef@290069org.highwire.dtl.DTLVardef@1da0c5a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cristae membranes have been recently shown to undergo intramitochondrial merging and splitting events. Yet, the metabolic and bioenergetic factors regulating them are unclear. Here we investigated whether and how cristae membrane remodelling is dependent on oxidative phosphorylation (OXPHOS) complexes, the mitochondrial membrane potential ({Delta}{Psi}m), and the ADP/ATP nucleotide translocator. Advanced live-cell STED nanoscopy combined with in-depth quantification were employed to analyse cristae morphology and dynamics after treatment of mammalian cells with rotenone, antimycin A, oligomycin A and CCCP. This led to formation of enlarged mitochondria along with reduced cristae density but did not change the number of cristae remodelling events. CCCP treatment leading to {Delta}{Psi}m abrogation even enhanced the cristae dynamics showing their {Delta}{Psi}m-independent nature. Inhibition of OXPHOS complexes was accompanied by reduced ATP levels but did not affect cristae dynamics. However, inhibition of ADP/ATP exchange led to aberrant cristae morphology and impaired cristae dynamics in a mitochondrial subset. In sum, we provide quantitative data of cristae membrane remodelling under different conditions supporting an important interplay between OXPHOS, metabolite exchange and cristae membrane dynamics. Summary BlurbCristae morphology and dynamics are intricately connected

Human endosomal Toll-like receptors TLR7 and TLR8 recognize self and non-self RNA ligands, and are important mediators of innate immunity and autoimmune pathogenesis. TLR7 and TLR8 are encoded by the adjacent X-linked genes, TLR7 and TLR8. We previously established that TLR7 evades X chromosome inactivation in female immune cells, and that mononuclear blood cells express more TLR7 protein in women than in men. Using RNA fluorescence in situ hybridization, we now show that TLR8 likewise evades X chromosome inactivation in CD14+ monocytes and CD4+ T lymphocytes, and that cells harboring TLR7 or TLR8 transcript foci are more frequent in women than in men. In parallel, we found TLR7 and TLR8 simultaneous transcription to be disproportionally frequent in female monocytes and T cells, and disproportionally scarce in the male cells, resulting in a 7-fold difference in frequency. These transcriptional biases were again observable when comparing the single X of XY males with the active X of female cells. Among (47,XXY) Klinefelter syndrome males, both TLR7 and TLR8 escape X chromosome inactivation, and co-transcription frequencies on the active X of monocytes were intermediate overall between those for XY males and XX females, and encompassed both male- and female-like individual patterns. These findings indicate that the TLR7 and TLR8 genes form a co-regulated gene cluster, which we have called the X-linked Toll-like receptor locus, with different sex- and sexual karyotype-dependent modes of transcription. Interestingly, TLR8 protein expression was significantly higher in female mononuclear blood cells, including all monocyte subsets, than in the male cells. Thus, co-dependent transcription from the active X chromosome and escape from inactivation could both contribute to higher TLR8 protein abundance in female cells, which may have implications for the response to viruses and bacteria, and the risk of developing inflammatory and autoimmune diseases. HighlightsO_LITLR8, like TLR7, escapes X chromosome inactivation in immune cells from women and 47,XXY Klinefelter syndrome (KS) men. C_LIO_LIThe frequency of cells double-positive for TLR7 and TLR8 primary transcripts is 7-fold higher in women than in men. C_LIO_LITLR7 and TLR8 form a co-regulated gene cluster on the human X chromosome, with sex-specific, divergent transcriptional patterns observable in monocytes and CD4+ T lymphocytes. C_LIO_LICo-dependent transcription of the TLR7 and TLR8 genes on the active X was observed in women and KS men, contrasting with mutually exclusive transcription in euploid men. C_LIO_LIBlood mononuclear cells, including monocyte subsets, expressed higher levels of TLR8 protein in females than in males. C_LI

Withdrawal Note: The authors have withdrawn their manuscript due to the discovery of artifacts that call into question key findings from the study. Both artifacts originate from cell-type differences that arose during sample processing, creating the impression that non-SMC subunits of condensin proteins were present at substantially higher levels in thymic T cells compared to bone marrow B cells or erythroblast cells. Based on data collected since the preprint was submitted, we no longer believe this to be true. The experimental artifacts do not affect the data contributed by the Papadopoulos laboratory (Figure 4). The authors wish to make it clear that all data and experimental protocols in the original manuscript were accurately reported, and that the experimental artifacts which affect data interpretation were discovered during internal review, independently of peer review, and are being reported at the authors own instigation. They do not, to our knowledge, affect the interpretation of data from any other published work. Due to the issues outlined above, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

The manuscript has been withdrawn because of lack of full consent from all co-authors. Therefore, the corresponding author does not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

Genome replication requires duplication of the complete set of DNA sequences together with nucleosomes and epigenetic signatures. Notwithstanding profound knowledge on mechanistic details of DNA replication, major problems of genome replication have remained unresolved. In this perspective article, we consider the accessibility of replication machines to all DNA sequences in due course, the maintenance of functionally important positional and structural features of chromatid domains during replication, and the rapid transition of CTs into prophase chromosomes with two chromatids. We illustrate this problem with EdU pulse-labeling (10 min) and chase experiments (80 min) performed with mouse myeloblast cells. Following light optical serial sectioning of nuclei with 3D structured illumination microscopy (SIM), seven DNA intensity classes were distinguished as proxies for increasing DNA compaction. In nuclei of cells fixed immediately after the pulse-label, we observed a relative under-representation of EdU-labeled DNA in low DNA density classes, representing the active nuclear compartment (ANC), and an over-representation in high density classes representing the inactive nuclear compartment (INC). Cells fixed after the chase revealed an even more pronounced shift to high DNA intensity classes. This finding contrasts with previous studies of the transcriptional topography demonstrating an under-representation of epigenetic signatures for active chromatin and RNAPII in high DNA intensity classes and their over-representation in low density classes. We discuss these findings in the light of current models viewing CDs either as structural chromatin frameworks or as phase-separated droplets, as well as methodological limitations that currently prevent an integration of this contrasting evidence for the spatial nuclear topography of replication and transcription into a common framework of the dynamic nuclear architecture.

Mini-G proteins are engineered thermostable variants of G subunits designed to specifically stabilise G protein-coupled receptors (GPCRs) in their active conformation for structural analyses. Due to their smaller size and ease of use, they have become popular tools in recent years to assess specific GPCR behaviours in cells, both as reporters of receptor coupling to each G protein subtype and for in-cell assays designed to quantify compartmentalised receptor signalling from a range of subcellular locations. Here, we describe a previously unappreciated consequence of the co-expression of mini-G proteins with their cognate GPCRs, namely a profound disruption in GPCR trafficking and intracellular signalling caused by the co-expression of the specific mini-G subtype coupled to the affected receptor. We studied the Gs-coupled pancreatic beta cell class B GPCR glucagon-like peptide-1 receptor (GLP-1R) as a model to describe in detail the molecular consequences derived from this effect, including a complete halt in {beta}-arrestin-2 recruitment and receptor internalisation, despite near-normal levels of receptor GRK2 recruitment and lipid nanodomain segregation, as well as the disruption of endosomal GLP-1R signalling by mini-Gs co-expression. We also extend our analysis to a range of other prototypical GPCRs covering the spectrum of G subtype coupling preferences, to unveil a widely conserved phenomenon of GPCR internalisation blockage by specific mini-G proteins coupled to a particular receptor. Our results have important implications for the design of methods to assess intracellular GPCR signalling. We also present an alternative adapted bystander intracellular signalling assay for the GLP-1R in which we substitute the mini-Gs by a nanobody, Nb37, with specificity for active Gs:GPCR complexes and no deleterious effect on the capacity for GLP-1R internalisation.