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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.11.511646v1?rss=1 Authors: Novy, B., Adoff, H., De Maria, M., Kampmann, M., Tsvetanova, N., Von Zastrow, M., Lobingier, B. Abstract: G protein-coupled receptors (GPCRs) are the largest family of membrane-bound signaling molecules. Activity of these receptors is critically regulated by their trafficking through the endo-lysosomal pathway. Identifying the genes involved in GPCR trafficking is challenging due the complexity of sorting operations and low affinity protein-protein interactions. Here we present a chemical biology fluorescence-based technique to interrogate GPCR trafficking. We show that the engineered enzyme APEX2 is a highly sensitive biosensor for GPCR trafficking to the lysosome, and this trafficking can be monitored through APEX-based activation of fluorogenic substrates such as Amplex UltraRed (AUR). We used this approach to perform a genome-wide CRISPR interference screen focused on the delta type opioid receptor (DOR), a GPCR which modulates anxiety, depression, and pain. The screen identified 492 genes including known- and novel-regulators of DOR expression and trafficking. We demonstrate that one of the novel genes, RME-8, localizes to early endosomes and plays a critical role in regulating DOR trafficking to the lysosome. Together, our data demonstrate that GPCR-APEX2/AUR is a flexible and highly sensitive chemical biology platform for genetic interrogation of receptor trafficking. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.11.511735v1?rss=1 Authors: Elcocks, H., Brazel, A. J., McCarron, K. R., Kaulich, M., Husnjak, K., Mortiboys, H. J., Clague, M. J., Urbe, S. Abstract: The selective autophagy of mitochondria is linked to mitochondrial quality control and is critical to a healthy organism. We have conducted a CRISPR/Cas9 screen of human E3 ubiquitin ligases for influence on mitophagy under both basal cell culture conditions and following acute mitochondrial depolarisation. We identify two Cullin RING ligases, VHL and FBXL4 as the most profound negative regulators of basal mitophagy. We show that these converge through control of the mitophagy adaptors BNIP3 and BNIP3L/NIX through different mechanisms. FBXL4 suppression of BNIP3 and NIX levels is mediated via direct interaction and protein destabilisation rather than suppression of HIF1-mediated transcription. Depletion of NIX but not BNIP3 is sufficient to restore mitophagy levels. Our study enables a full understanding of the aetiology of early onset mitochondrial encephalomyopathy that is supported by analysis of a disease associated mutation. We further show that the compound MLN4924, which globally interferes with Cullin RING ligase activity, is a strong inducer of mitophagy providing a research tool in this context and a candidate therapeutic agent for conditions linked to mitochondrial dysfunction. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.12.511914v1?rss=1 Authors: Di, A., Huang, L. S., Zhou, B., Toth, P. T., Krishnan, Y., Malik, A. B. Abstract: Potassium efflux via the two pore K+ channel TWIK2 is a requisite step for the activation of the NLRP3 inflammasome, however it is unclear how the efflux is activated in response to cues. Here we report that during homeostasis, TWIK2 resides in endosomal compartments. TWIK2 is transported by endosomal fusion to the plasmalemma in response to increased extracellular ATP resulting in extrusion of K+ ATP-induced endosomal TWIK2 plasmalemma translocation is regulated by Rab11a. Deleting Rab11a or ATP ligated purinergic receptor P2X7 prevented endosomal fusion with the plasmalemma and K+ efflux and NLRP3 inflammasome activation in macrophages. Adoptive transfer of Rab11a-deleted macrophages into mouse lungs prevented NLRP3 inflammasome activation and inflammatory lung injury. Rab11a-mediated endosomal trafficking in macrophages thus regulates TWIK2 abundance and activity on the cell surface and downstream activation of the NLRP3 inflammasome. Endosomal trafficking of TWIK2 to the plasmalemma is therefore a potential therapy target in acute or chronic inflammatory states. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.30.514401v1?rss=1 Authors: An, Y., Wang, C., Fan, B., Li, Y., Kong, F., Zhou, C., Cao, Z., Liu, J., Wang, M., Sun, H., Zhao, S., Gong, Y. Abstract: Lipolysis-stimulated lipoprotein receptor (LSR) is a multi-functional protein that is best known for its roles in assembly of epithelial tricellular tight junctions and hepatic clearance of lipoproteins. Here, we investigate the function of LSR in intestine biology. By using multiple conditional deletion mouse models and ex vivo cultured organoids, we find that LSR elimination in intestinal stem cells results in disappearance of Paneth cell without affecting the differentiation of other cell lineages. Mechanistic studies reveal that LSR deficiency increase abundance and nuclear localization of YAP by modulating its phosphorylation and proteasomal degradation. Intestinal LSR-deficient mice are susceptible to development of necrotizing enterocolitis. In addition, LSR can sense and interpret fatty acid signals derived from dietary lipids and transduce into inactivation of YAP. Thus, this study identifies LSR as an upstream negative regulator of YAP activity and part of the mechanism mediating connection between fat diet and YAP signaling in intestine. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534685v1?rss=1 Authors: Alsehli, H. S., Roy, E., Williams, T., Kuziola, A., Guo, Y., Green, J. B., Gentleman, E., Danovi, D. Abstract: In early human development, gastrulation is tightly associated with lineage specification. The interplay between mechanical forces and biochemical signals during these processes is poorly understood. Here, we dissect the effects of biochemical cues and physical confinement on a 3D in vitro model of gastrulation that uses spheroids formed from human induced pluripotent stem cells (hiPSCs). First, we compare self-renewing versus differentiating media conditions in free-floating cultures, and observe the emergence of organised tri-germ layers. In these unconfined cultures, BMP4 exposure induces polarised expression of SOX17 in conjunction with spheroid elongation. We then physically confine spheroids using PEG-peptide hydrogels and observe dramatically reduced SOX17 expression, albeit rescued if gels that soften over time are used instead. Our study combines high-content imaging, synthetic hydrogels and hiPSCs-derived models of early development to define the drivers causing changes in shape and emergence of germ layers. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.28.534513v1?rss=1 Authors: pan, x., ye, f., Ning, P., Zhang, Z., Zhang, B., Chen, G., Gao, W., Qiu, C., Wu, Z., Gong, K., Li, J., Xia, J., Du, Y. Abstract: Hydroxycarboxylic acid receptor 2 (HCAR2) belongs to the family of class A G-protein-coupled receptors with key roles in regulating lipolysis and free fatty acid formation in humans. It is deeply involved in many pathophysiological processes and serves as an attractive target for the treatment of neoplastic, autoimmune, neurodegenerative, inflammatory, and metabolic diseases. Here, we report four cryo-EM structures of human HCAR2-Gi1 complexes with or without agonists, including the drugs niacin and acipimox, and the highly subtype-specific agonist MK-6892. Combined with molecular docking and functional analysis, we have revealed the recognition mechanism of HCAR2 for different agonists and summarized the general pharmacophore features of HCAR2 agonists, which are based on three key residues R1113.36, S17945.52, and Y2847.43. Notably, the MK-6892-HCAR2 structure shows an extended binding pocket relative to other agonist-bound HCAR2 complexes. In addition, the key residues that determine the ligand selectivity between the HCAR2 and HCAR3 are also illuminated. Our findings provide structural insights into the ligand recognition, selectivity, activation, and G protein coupling mechanism of HCAR2, which sheds light on the design of new HCAR2-targeting drugs for greater efficacy, higher selectivity, and fewer or no side effects. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.23.532505v1?rss=1 Authors: Ge, X., Lu, L., Bai, W., Wang, M., Han, C., Du, H., Wang, N., Gao, M., Li, D., Dong, F. Abstract: Exosomes are small vesicles released from cells and present in various mammal biological fluids, such as bovine milk, which worked for skin care for many years besides dairy. In addition, Exosomes were regarded as a vehicle for intercellular communication. Therefore, we aimed to investigate the novel roles of bovine milk-derived exosomes (MK-Exo) on human skin anti-aging. Purified MK-Exo can be directly uptake by the keratinocytes and fibroblast in vitro and upregulate the expression of the natural factors related to skin moisturizing, including Filaggrin (FLG), Aquaporin 3 (AQP3), CD44 in the keratinocytes and hyaluronidase (HAS2) in the fibroblast, and MK-Exo promoted the cell migration of the fibroblast, while rescue its expression of type I collagen (Col I), type III collagen (Col III) after ultraviolet radiation. Furthermore, the phototoxicity test, photoallergy test, repeated skin irritation test, skin allergy test, and patch test confirm the safety of MK-Exo on the skin. Finally, the roles of MK-Exo in preserving moisture and anti-wrinkle were also identified in humans. Then, MK-Exo was smeared on the facial skin of 31 female volunteers twice a day for 28 days, and the functions were evaluated following the safety assessment in vivo. These studies reveal the novel roles of bovine milk-derived exosomes in human skin aging, which opens a new way of skin care. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.27.534456v1?rss=1 Authors: Li, J., Jin, C., Gustafsson, S., Rao, A., Wabitsch, M., Park, C. Y., Quertermous, T., Bielczyk-Maczynska, E., Knowles, J. W. Abstract: Adipogenesis is a process in which fat-specific progenitor cells (preadipocytes) differentiate into adipocytes that carry out the key metabolic functions of the adipose tissue, including glucose uptake, energy storage, and adipokine secretion. Several cell lines are routinely used to study the molecular regulation of adipogenesis, in particular the immortalized mouse 3T3-L1 line and the primary human Simpson-Golabi-Behmel syndrome (SGBS) line. However, the cell-to-cell variability of transcriptional changes prior to and during adipogenesis in these models is not well understood. Here, we present a single-cell RNA-Sequencing (scRNA-Seq) dataset collected before and during adipogenic differentiation of 3T3-L1 and SGBS cells. To minimize the effects of experimental variation, we mixed 3T3-L1 and SGBS cells and used computational analysis to demultiplex transcriptomes of mouse and human cells. In both models, adipogenesis results in the appearance of three cell clusters, corresponding to preadipocytes, early and mature adipocytes. These data provide a groundwork for comparative studies on human and mouse adipogenesis, as well as on cell-to-cell variability in gene expression during this process. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.28.534499v1?rss=1 Authors: Rehl, K. M., Selvakumar, J., Hoang, D., Arumugam, K., Gorfe, A., Cho, K.-J. Abstract: Ras proteins are membrane-bound GTPases that regulate essential cellular processes at the plasma membrane (PM). Constitutively active mutations of K-Ras, one of the three Ras isoforms in mammalian cells, are frequently found in human cancers. Ferrocene derivatives, which elevate cellular reactive oxygen species (ROS), have shown to block the growth of non-small cell lung cancers (NSCLCs) harboring oncogenic mutant K-Ras. Here, we developed and tested a novel ferrocene derivative on the growth of human pancreatic ductal adenocarcinoma (PDAC) and NSCLC. Our compound inhibited the growth of K-Ras-dependent PDAC and NSCLC and abrogated the PM binding and signaling of K-Ras, but not other Ras isoforms. These effects were reversed upon antioxidant supplementation, suggesting a ROS-mediated mechanism. We further identified K-Ras His95 residue in the G-domain as being involved in the ferrocene-induced K-Ras PM dissociation via oxidative modification. Together, our studies demonstrate that the redox system directly regulates K-Ras PM binding and signaling via oxidative modification at the His95, and proposes a role of oncogenic mutant K-Ras in the recently described antioxidant-induced metastasis in K-Ras-driven lung cancers. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.28.534608v1?rss=1 Authors: Asrul, A. Abstract: The use of biological agents as a biocontrol against plant pathogens was often ineffective because it worked slowly. The objective of this research was to examine the potential of local isolates of Bacillus spp. as a biocontrol agent in suppressing Fusarium wilt disease (Fusarium oxysporum f. sp. cepae) on wakegi onions. The research was designed using a completely randomized design with the treatment of rhizosphere bacterial isolates. The treatments consisted of control (without isolate application), isolates KP17, KP5, DB9, DB12, DB18, DG4, and DG11 so that the number of treatments was eight. Each treatment was repeated 5 times and each replication consisted of 10 wakegi onion plants. This research was divided into 2 stages, namely laboratory research which included isolation, characterization of colony morphology of rhizosphere bacterial isolates, and in vitro testing of the inhibitory power of biocontrol agents against pathogens. The test in the greenhouse was in the form of a disease case suppression test. The results obtained seven candidate isolates of biocontrol from 46 isolates obtained from the rhizosphere of the wakegi onion. These isolates had similarities with Bacillus spp. based on colony morphology, physiology, and biochemistry characteristics. Among the isolates found, the DB12 isolate had the potential to be developed as a biocontrol agent compared to other isolates. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.28.534628v1?rss=1 Authors: Fontcuberta-Rigo, M., Nakamura, M., Puigbo, P. Abstract: The bone extracellular matrix (ECM) contains minerals deposited on highly cross-linked collagen fibrils, and hundreds of non-collagenous proteins. Some of these proteins are determinant to regulate bone formation and regeneration via signaling pathways, and play important regulatory and structural roles. However, the complete list of bone extracellular matrix proteins, their roles, and the extent of individual and cross-species variation are not yet well understood in both humans and model organisms. Here, we introduce the most comprehensive resource of bone bone extracellular matrix (ECM) proteins that can be used in future studies in research fields such as bone regeneration, osteoporosis and mechanobiology. The Phylobone database (available at https://phylobone.com) includes 255 proteins potentially expressed in the bone extracellular matrix (ECM) of humans and 30 species of vertebrates. A bioinformatics pipeline has been utilized to identify evolutionary relationships of bone ECM proteins. This analysis facilitates the identification of potential model organisms to study the molecular mechanisms of bone regeneration. A network analysis shows the high connectivity of bone ECM proteins. A total of 214 functional protein domains have been identified, including collagen and domains involved in bone formation and resorption. Information from public drug repositories is utilized to identify potential repurposing of existing drugs. The Phylobone database provides a platform to study bone regeneration and osteoporosis in the light of (biological) evolution, and will substantially contribute to the identification of molecular mechanisms and drug targets. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534583v1?rss=1 Authors: Montealegre, S., de Calbiac, H., Straube, M., Debruge, H., Chentout, L., Ciura, S., Imbard, A., Le Guillou, E., Marian, A., Goudin, N., Caccavelli, L., Fabrega, S., Hubas, A., van Endert, P., Dupont, N., Diana, J., Kabashi, E., de Lonlay, P. Abstract: Patients with pathogenic variants in the TANGO2 gene suffer from severe and recurrent rhabdomyolysis (RM) episodes precipitated by fasting. Since starvation promotes autophagy induction, we wondered whether TANGO2-related muscle symptoms result from autophagy insufficiency to meet cellular demands in stress conditions. Autophagy functioning was analyzed in vitro, in primary skeletal muscle cells from TANGO2 patients in basal and fasting conditions. In addition, wce developed a tango2 morphant zebrafish model to assess the effect of tango2 knockdown (KD) on locomotor function and autophagy efficiency in vivo. We report that TANGO2 mutations are associated with decreased LC3-II levels upon starvation in primary muscle cells, but not in fibroblasts. In zebrafish larvae, tango2 knockdown induces locomotor defects characterized by reduced evoked movements which are exacerbated by exposure to atorvastatin, a compound known to cause RM. Importantly, RM features of tango2 KD are also associated with autophagy defects in zebrafish. Calpeptin treatment, a known activator of autophagy, is sufficient to rescue the locomotor function and improves autophagy in zebrafish. LC3-II levels of primary muscle cells of TANGO2 patients are also ameliorated by calpeptin treatment. Overall, we demonstrate that TANGO2 plays an important role in autophagy, and that autophagy efficiency is critical to prevent RM, thus giving rise to new therapeutic perspectives in the prevention of these life-threatening episodes in the context of TANGO2 pathology. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.28.534295v1?rss=1 Authors: Tung, V. S. K., Mathews, F., Boruk, M., Suppa, G., Foronjy, R., Pato, M., Pato, C., Knowles, J. A., Evgrafov, O. V. Abstract: Study of the neurodevelopmental molecular mechanisms of schizophrenia requires the development of adequate biological models such as patient-derived cells and their derivatives. We previously used cell lines with neural progenitor properties (CNON) derived from superior or middle turbinates of patients with schizophrenia and control groups to study gene expression specific to schizophrenia. In this study, we compared single cell-RNA seq data from two CNON cell lines, one derived from an individual with schizophrenia (SCZ) and the other from a control group, with two biopsy samples from the middle turbinate (MT), also from an individual with SCZ and a control. In addition, we compared our data with previously published data from olfactory neuroepithelium (1). Our data demonstrated that CNON originated from a single cell type which is present both in middle turbinate and olfactory neuroepithelium. CNON express multiple markers of mesenchymal cells. In order to define relatedness of CNON to the developing human brain, we also compared CNON datasets with scRNA-seq data of embryonic brain (2) and found that the expression profile of CNON very closely matched one of the cell types in the embryonic brain. Finally, we evaluated differences between SCZ and control samples to assess usability and potential benefits of using single cell RNA-seq of CNON to study etiology of schizophrenia. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534729v1?rss=1 Authors: Brooks, J. W., Tillu, V., Verma, S., Collins, B. M., Parton, R. G., Yap, A. S. Abstract: As physical barriers, epithelia must preserve their integrity when challenged by mechanical stresses. Cell-cell junctions linked to the cortical cytoskeleton play key roles in this process, often with mechanotransduction mechanisms that reinforce tissues. Caveolae are mechanosensitive organelles that buffer tension via disassembly. Loss of caveolae, through caveolin-1 or cavin1 depletion, causes activation of PtdIns(4, 5)P2 signalling, recruitment of FMNL2 formin, and enhanced cortical actin assembly. How this equates to physiological responses in epithelial cells containing endogenous caveolae is unknown. Here we examined the effect of mechanically-inducing acute disassembly of caveolae in epithelia. We show that perturbation of caveolae, through direct mechanical stress, reinforces the actin cortex at adherens junctions. Increasing interactions with membrane lipids by introducing multiple phosphatidylserine-binding undecad cavin1 (UC1) repeat domains into cavin1 rendered caveolae more stable to mechanical stimuli. This molecular stabilization blocked cortical reinforcement in response to mechanical stress. Cortical reinforcement elicited by the mechanically-induced disassembly of caveolae increased epithelial resilience against tensile stresses. These findings identify the actin cortex as a target of caveola mechanotransduction that contributes to epithelial integrity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.29.514378v1?rss=1 Authors: Wang, T., Ling, A. H., Billings, S. E., Hosseini, D. K., Vaisbuch, Y., Kim, G. S., Atkinson, P. J., Sayyid, Z. N., Aaron, K. A., Wagh, D., Pham, N., Scheibinger, M., Ishiyama, A., Santa Maria, P., Blevins, N. H., Jackler, R. K., Heller, S., Lopez, I. A., Grillet, N., Jan, T. A., Cheng, A. G. Abstract: Mammalian inner ear hair cell loss leads to permanent hearing and balance dysfunction. In contrast to the cochlea, vestibular hair cells of the murine utricle have some regenerative capacity. Whether human utricular hair cells regenerate remains unknown. Here we procured live, mature utricles from organ donors and vestibular schwannoma patients, and present a validated single-cell transcriptomic atlas at unprecedented resolution. We describe previously unknown markers of 25 sensory and non-sensory cell types, with genes of hair cell and supporting cell subtypes displaying striking divergence between mice and humans. We further uncovered transcriptomes unique to hair cell precursors, which we validated to be 14-fold more robust in vestibular schwannoma utricles, representing ongoing regeneration in humans. Lastly, trajectory analysis of the supporting cell-hair cell axis revealed 5 distinct patterns of dynamic gene expression and associated pathways, including mTOR signaling and synaptogenesis. Our dataset constitutes a foundational resource, accessible via a web-based interface, serving to advance knowledge of the normal and diseased human inner ears and tools to stimulate human inner ear regeneration. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534670v1?rss=1 Authors: Wang, Y., Ruan, L., Zhu, J., Zhang, X., Chang, A., Tomaszewski, A., Li, R. Abstract: Mitochondria are the cellular energy hub and central target of metabolic regulation. Mitochondria also facilitate proteostasis through pathways such as the mitochondria as guardian in cytosol (MAGIC) whereby cytosolic misfolded proteins are imported into and degraded inside mitochondria. In this study, a genome-wide screen in yeast uncovered that Snf1, the yeast AMP-activated protein kinase (AMPK), inhibits the import of misfolded proteins into mitochondria while promoting mitochondrial biogenesis under glucose starvation. We show that this inhibition requires a downstream transcription factor regulating mitochondrial gene expression and is likely to be conferred through substrate competition and mitochondrial import channel selectivity. We further show that Snf1/AMPK activation protects mitochondrial fitness in yeast and human cells under stress induced by misfolded proteins such as those associated with neurodegenerative diseases. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.27.534417v1?rss=1 Authors: Itkin, T., Houghton, S., Schreiner, R., Lin, Y., Badwe, C., Voisin, V., Murison, A., Seyedhassantehrani, N., Kaufmann, K. B., Garcia-Prat, L., Booth, G. T., Geng, F., Liu, Y., Gomez-Salinero, J. M., Shieh, J.-H., Redmond, D., Xiang, J. Z., Josefowicz, S. Z., Trapnell, C., Spencer, J. A., Zangi, L., Hadland, B., Dick, J. E., Xie, S. Z., Rafii, S. Abstract: Transition between activation and quiescence programs in hematopoietic stem and progenitor cells (HSC/HSPCs) is perceived to be governed intrinsically and by microenvironmental co-adaptation. However, HSC programs dictating both transition and adaptability, remain poorly defined. Single cell multiome analysis divulging differential transcriptional activity between distinct HSPC states, indicated for the exclusive absence of Fli-1 motif from quiescent HSCs. We reveal that Fli-1 activity is essential for HSCs during regenerative hematopoiesis. Fli-1 directs activation programs while manipulating cellular sensory and output machineries, enabling HSPCs co-adoptability with a stimulated vascular niche. During regenerative conditions, Fli-1 presets and enables propagation of niche-derived Notch1 signaling. Constitutively induced Notch1 signaling is sufficient to recuperate functional HSC impairments in the absence of Fli-1. Applying FLI-1 modified-mRNA transduction into lethargic adult human mobilized HSPCs, enables their vigorous niche-mediated expansion along with superior engraftment capacities. Thus, decryption of stem cell activation programs offers valuable insights for immune regenerative medicine. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.27.534302v1?rss=1 Authors: Yordanov, T. E., Martinez, M. A. E., Esposito, T., Tefft, J. B., Labzin, L. I., Stehbens, S. J., Rowan, A., Hogan, B. M., Chen, C. S., Lauko, J., Lagendijk, A. K. Abstract: Cerebral Cavernous Malformations (CCMs) are vascular lesions that predominantly form in blood vessels of the central nervous system (CNS) upon loss of the CCM multimeric protein complex. The endothelial cells (ECs) within CCM lesions are characterised by overactive MEKK3 kinase and KLF2/4 transcription factor signalling, leading to pathological changes such as increased EC spreading and reduced junctional integrity. Concomitant to aberrant EC signalling, non-autonomous signals from the extracellular matrix (ECM) have also been implicated in CCM lesion growth and these factors might explain why CCM lesions mainly develop in the CNS. Here, we adapted a three dimensional (3D) microfluidic system to examine CCM1 deficient human micro-vessels in distinctive ECMs. We validate that EC pathological hallmarks are maintained in this 3D model. We further show that supplementing the ECM with distinct forms of Hyaluronic Acid (HA), a major ECM component of the CNS, alters CCM1 biology, independent of KLF2/4. This study provides a proof-of-principle that ECM embedded 3D microfluidic models are ideally suited to identify how changes in ECM structure and signalling impact vascular malformations. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534690v1?rss=1 Authors: Islam, A., Chen, X.-C., Weng, C.-W., Chen, C.-Y., Wang, C.-W., Chen, M.-K., Tikhomirov, A. S., Shchekotikhin, A. E., Chueh, P. J. Abstract: The antibiotic heliomycin (resistomycin), which is generated from Streptomyces resistomycificus, has multiple activities, including anticancer effects. Heliomycin was first described in the 1960s, but its clinical applications have been hindered by extremely low solubility. A series of 4-aminomethyl derivatives of heliomycin were synthesized to increase water solubility; studies showed that they had anti-proliferative effects, but the drug targets remained unknown. In this study, we conducted cellular thermal shift assays and molecular docking simulations to identify and validate the intracellular targets of heliomycin and its water-soluble derivative, 4-(dimethylaminomethyl)heliomycin (designated compound 4-dmH), in p53-functional SAS and p53-mutated HSC-3 oral cancer cells. Consistent with our in silico studies, our cellular thermal shift assays (CETSA) revealed that, in addition to SIRT1, the water-soluble 4-dmH preferentially targeted a tumor-associated NADH oxidase called tNOX or ENOX2. The direct binding of 4-dmH to tNOX inhibited the activity of tNOX and enhanced its ubiquitin-proteasomal protein degradation in both SAS and HSC-3 cells. Moreover, the inhibition of tNOX by 4-dmH decreased the oxidation of NADH to NAD+ which diminished NAD+-dependent SIRT1 deacetylase activity, ultimately inducing apoptosis and significant cytotoxicity in both cell types. We also observed that tNOX and SIRT1 were both upregulated in tumor tissues of oral cancer patients compared to adjacent normal tissues, suggesting their clinical relevance. Finally, the better therapeutic efficacy of 4-dmH was confirmed in tumor-bearing mice, which showed greater tNOX and SIRT1 downregulation and tumor volume reduction when treated with 4-dmH compared to heliomycin. Taken together, our in vitro and in vivo findings suggest that the multifaceted properties of water-soluble 4-dmH enable it to offer superior antitumor value compared to parental heliomycin, and indicated that it functions through targeting the tNOX-NAD+-SIRT1 axis to induce apoptosis in oral cancer cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.27.534352v1?rss=1 Authors: Cai, P., Casas, C. J., Hua, L. L., Mikawa, T. Abstract: Individual homologous chromosomes are spatially segregated into haploid chromosome sets along the centrosome axis in an antipairing configuration. Disruption of the antipairing pattern occurs in cancer cells. However, little is known about how this spatial organization of chromosomes is established or maintained. Here, we report that there is a zone of diminished interchromosomal linkage and centromere components between haploid sets in primary and established human epithelial cell lines. Using 4-Dimensional live cell imaging analysis of centromere and centrosome tracking, we show ipsilateral restriction of chromosome oscillations along the diminished zone, coincident with the centrosome and apical-basal axis from mitosis onset to G1 interphase. We propose a biophysical model of axis-dependent ipsilateral restriction of chromosome oscillations for haploid set organization. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534715v1?rss=1 Authors: van Deventer, S. J., Hoogvliet, I. A., van de Voort, M., Arnold, F., van Spriel, A. Abstract: Tetraspanin proteins play an important role in many cellular processes as they are key organizers of different plasma membrane receptors. Most tetraspanins are highly glycosylated, but the function of this post-translational modification remains largely unstudied. In this study we investigated the glycosylation of CD37 and CD53, two tetraspanins important for cellular and humoral immunity. Broad and cell-specific repertoires of N-glycosylated CD37 and CD53 were observed in human B cells. We generated different glycosylation mutants and analyzed their localization, nanoscale organization and protein interactions. Abrogation of glycosylation in CD37 revealed the importance of this modification for CD37 surface expression, whereas neither surface expression nor nanoscale organization of CD53 was affected by its glycosylation. CD37 interaction with its known partner proteins, CD20 and IL-6R, was not affected by glycosylation, other than via its changed subcellular localization. Surprisingly, glycosylation was found to inhibit the interaction between CD53 and its partner proteins CD45 and CD20. Together, our data show that tetraspanin glycosylation affects their function in immune cells, which adds another layer of regulation to tetraspanin-mediated membrane organization. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534651v1?rss=1 Authors: Ng, J. Q., Jafarov, T. H., Little, C. B., Wang, T., Ali, A., Ma, Y., Radford, G., Vrbanac, L., Ichinose, M., Whittle, S., Hunter, D., Lannagan, T., Suzuki, N., Goyne, J. M., Kobayashi, H., WANG, T. C., Haynes, D., Menicanin, D., Gronthos, S., Worthley, D. L., Woods, S. L., Mukherjee, S. Abstract: Osteoarthritis (OA), which carries an enormous disease burden across the world, is characterised by irreversible degeneration of articular cartilage (AC), and subsequently bone. The cellular cause of OA is unknown. Here, using lineage tracing in mice, we show that the BMP-antagonist Gremlin 1 (Grem1) marks a novel chondrogenic progenitor (CP) cell population in the articular surface that generates joint cartilage and subchondral bone during development and adulthood. Notably, this CP population is depleted in injury-induced OA, and with age. OA is also induced by toxin-mediated ablation of Grem1 CP cells in young mice. Transcriptomic analysis and functional modelling in mice revealed articular surface Grem1-lineage cells are dependent on Foxo1; ablation of Foxo1 in Grem1-lineage cells led to early OA. This analysis identified FGFR3 signalling as a therapeutic target, and injection of its activator, FGF18, caused proliferation of Grem1-lineage CP cells, increased cartilage thickness, and reduced OA pathology. We propose that OA arises from the loss of CP cells at the articular surface secondary to an imbalance in progenitor cell homeostasis and present a new progenitor population as a locus for OA therapy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.534618v1?rss=1 Authors: Govindaraj, K., Kannan, S., Karperien, M., Post, J. N. Abstract: The multi-lineage differentiation capacity of human mesenchymal stem cells (hMSCs) enables its potential for tissue engineering and regenerative medicine. Master transcription factors play a key role during development, differentiation, homeostasis and disease pathology. RUNX2 is the master transcription factor for bone development, and it regulates several important signaling pathways during chondrogenic and osteogenic differentiation of hMSCs. However, modulation of RUNX2 activity during hMSC differentiation into various lineages is not yet fully described. We differentiated hMSCs into chondro-, osteo-, and adipogenic lineages and studied RUNX2 protein dynamics using Transcription Factor - Fluorescence Recovery After Photobleaching (TF-FRAP) at different time points. The TF-FRAP method can capture the dynamic changes of RUNX2 protein mobility at the single cell level resolution, and cluster analysis shows how RUNX2 dynamics change at subpopulation level in proliferating and differentiating hMSCs. Our data show that although whole hMSC population is exposed to differentiation stimuli, some subpopulations in hMSCs do not respond to environmental cues. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534749v1?rss=1 Authors: van Soest, D. M. K., Polderman, P. E., den Toom, W. T., Zwakenberg, S., De Henau, S., Burgering, B. M. T., Dansen, T. B. Abstract: Reactive Oxygen Species (ROS) derived from mitochondrial respiration are frequently cited as a major source of genomic DNA damage and subsequent mutations that contribute to cancer development and aging. However, experimental evidence showing that ROS released by mitochondrial can directly damage nuclear DNA under (patho)physiological conditions has been largely lacking. In this study we modeled the effects of mitochondrial H2O2 release and compared this to H2O2 production at the nucleosomes in an untransformed human cell line. We used a chemogenetic approach to produce localized H2O2 and combined it with a new method we developed to directly quantify the amount of H2O2 produced. This enabled us to precisely investigate to what extent DNA damage occurs downstream of near- and supraphysiological amounts of localized H2O2 generation. Nuclear H2O2 production gives rise to DNA strand breaks, subsequent activation of the DNA damage response, cell cycle arrest and eventually senescence. Release of H2O2 from mitochondria on the other hand shows none of these effects, even at levels that are orders of magnitude higher than what mitochondria normally produce. Artificially high levels of mitochondrial H2O2 release do result in DNA strand breaks, but in parallel invariably cause ferroptosis-mediated cell death, preventing propagation of DNA damage-induced mutations. This study shows that H2O2 released from mitochondria is unlikely to directly damage genomic DNA, limiting its contribution to oncogenic transformation and aging. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.534893v1?rss=1 Authors: Carvalho, A. M., Silva, B., Pereira, F. B., Kim, H.-N., Almeida, M., Sardao, V. A. Abstract: Estrogen deficiency increases bone resorption and is a major contributor to osteoporosis. However, the molecular mechanisms mediating the effects of estrogen on osteoclasts remain unclear. This study aimed at elucidating the early metabolic effects of RANKL, the essential cytokine for osteoclastogenesis, and 17-beta-estradiol (E2) on osteoclast progenitor cells, using RAW 264.7 macrophage cell line and bone marrow-derived macrophages as biological models. RANKL stimulated complex I activity, oxidative phosphorylation (OXPHOS), and mitochondria-derived ATP production, as early as 3 to 6 h. This up-regulation of mitochondrial bioenergetics was associated with an increased capacity to oxidize TCA cycle substrates, fatty acids, and amino-acids. E2 inhibited all effects of RANKL on mitochondria metabolism. In the presence of RANKL, E2 also decreased cell number and stimulated the mitochondrial-mediated apoptotic pathway, detected as early as 3h. Surprisingly, the pro-apoptotic effects of E2 were associated with an accumulation of p392S-p53 in mitochondria. These findings elucidate early effects of RANKL on osteoclast progenitor metabolism and suggest novel p53-mediated mechanisms that contribute to postmenopausal osteoporosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.31.514611v1?rss=1 Authors: Plazen, L., Al Rahbani, J., Brown, C. M., Khadra, A. Abstract: In mesenchymal cell motility, several migration patterns have been observed, including directional, exploratory and stationary. Two key members of the Rho-family of GTPases, Rac and Rho, along with an adaptor protein called paxillin, have been particularly implicated in the formation of such migration patterns and in regulating adhesion dynamics. Together, they form a key regulatory network that involves the mutual inhibition exerted by Rac and Rho on each other and the promotion of Rac activation by phosphorylated paxillin. Although this interaction is sufficient to generating wave-pinning that underscores cellular polarization comprised of cellular front (high active Rac) and back (high active Rho), it remains unclear how they interact collectively to induce other modes of migration detected in Chinese hamster Ovary (CHO-K1) cells. We previously developed a 6D reaction-diffusion model describing the interactions of these three proteins (in their active/phosphorylated and inactive/unphosphorylated forms) along with other auxiliary proteins, to decipher their role in generating wave-pinning. In this study, we explored, through computational modeling and image analysis, how differences in timescales within this molecular network can potentially produce the migration patterns in CHO-K1 cells and how switching between them could occur. To do so, the 6D model was reduced to an excitable 4D spatiotemporal model possessing three different timescales. The model produced not only wave-pinning in the presence of diffusion, but also mixed-mode oscillations (MMOs) and relaxation oscillations (ROs). Implementing the model using the Cellular Potts Model (CPM) produced outcomes in which protrusions in cell membrane changed Rac-Rho localization, resulting in membrane oscillations and fast directionality variations similar to those seen in CHO-K1 cells. The latter was assessed by comparing the migration patterns of CHO-K1 cells with CPM cells using four metrics: instantaneous cell speed, exponent of mean square-displacement (called -value), directionality ratio and protrusion rate. Variations in migration patterns induced by mutating paxillin's serine 273 residue was also captured by the model and detected by a machine classifier, revealing that this mutation alters the dynamics of the system from MMOs to ROs or nonoscillatory behaviour through variation in the concentration of an active form of an adhesion protein called p21-Activated Kinase 1 (PAK). These results thus suggest that MMOs and adhesion dynamics are the key ingredients underlying CHO-K1 cell motility. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.531699v1?rss=1 Authors: Hosseini, A., Lindholm, H. T., Chen, R., Mehdipour, P., Marhon, S. A., Ishak, C. A., De Carvalho, D. D. Abstract: Viral mimicry describes the immune response induced by endogenous stimuli such as dsRNA formed by endogenous retroelements. Activation of viral mimicry has the potential to kill cancer cells or augment anti-tumor immune response. Paradoxically, cancer cells frequently present a dysregulated epigenome, leading to increased expression of retroelements. We previously found that ADAR1 p150 upregulation is an adaptation mechanism to tolerate high retroelement-derived dsRNA levels, leading to a druggable dependency. Here, we systematically identified novel mechanisms of viral mimicry adaptation associated with cancer cell dependencies. We correlated the gene knockout sensitivity from the DepMap dataset and interferon stimulated gene (ISG) expression in the Cancer Cell Line Encyclopedia (CCLE) dataset of 1005 human cell lines and identified pathways such as RNA modification and nucleic acid metabolism. Among the top hits was the RNA decay protein XRN1 as an essential gene for the survival of a subset of cancer cell lines. XRN1-sensitive cancer cell lines have a high level of cytosolic dsRNA and high ISG expression. Furthermore, sensitivity to XRN1 knockout was mediated by MAVS and PKR activation, indicating that the cells die due to XRN1-dependent induction of viral mimicry. XRN1-resistant cell lines had low basal dsRNA levels, but became synthetically dependent on XRN1 upon treatment with viral mimicry inducing drugs such as 5-AZA-CdR or palbociclib. Finally, XRN1-dependency is partly independent of ADAR1 activity. These results confirm the potential for our ISG correlation analysis to discover novel regulators of viral mimicry and show that XRN1 activation is an adaptive mechanism to control high dsRNA stress induced by dysregulated retroelements in cancer cells and creates a dependency that can be explored for novel cancer therapies. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.534941v1?rss=1 Authors: Ingram, S., DeCorte, A., Gentry, A. E., Philpott, M. K., Moldenhauer, T., Stadler, S., Steinberg, C., Millman, J., Ehrhardt, C. J. Abstract: Analysis of DNA mixtures from sexual assault evidence is an ongoing challenge for DNA casework laboratories. There is a significant need for new techniques that can provide information as to the source of DNA, particularly for sexual assault samples that do not involve semen. The goal of this study was to develop a new biological signature system that provides additional probative value to samples comprised of mixtures of epidermal and vaginal cells, as may be observed in cases involving digital penetration. Signatures were based on morphological and autofluorescence properties of individual cells collected through Imaging Flow Cytometry (IFC). Comparisons to reference cell populations from vaginal tissue and epidermal cells collected from hands showed strong multivariate differences across greater than 80 cellular measurements. These differences were used to build a predictive framework for classifying unknown cell populations as originating from epithelial cells associated with digital penetration or epidermal tissue. As part of the classification scheme, posterior probabilities of specific tissue group membership were calculated for each cell, along with multivariate similarity to that tissue type. We tested this approach on cell populations from reference tissue as well as mock casework samples involving digital penetration. Many more cells classifying as non-epidermal tissue were detected in digital penetration samples than control hand swabbings. Minimum interpretation thresholds were developed to minimize false positives; these thresholds were also effective when screening licked hands, indicating the potential utility of this method for a variety of biological mixture types and depositional events relevant to forensic casework. Results showed that samples collected subsequent to digital penetration possessed markedly higher numbers of cells classifying as vaginal tissue as well as higher posterior probabilities for vaginal tissue ( greater than or equal to 0.90) compared to cell populations collected from hands without prior contact with vaginal tissue. Additionally, digital penetration cell populations may be resolved from saliva cell populations and other non-target tissue types. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535084v1?rss=1 Authors: Otto, T. A., Bergsma, T., Dekker, M., Mouton, S., Gallardo, P., Wolters, J. C., Steen, A., Onck, P. R., Veenhoff, L. L. Abstract: Transport through the NPC relies on intrinsically disordered FG-Nups forming a selective barrier. Away from the NPC, FG-Nups readily form condensates and aggregates, and we address how this behavior is surveilled in cells. FG-Nups, including Nsp1, together with nuclear transport receptor Kap95, form a native cytosolic condensate in yeast. In aged cells this condensate disappears as cytosolic Nsp1 levels decline. Biochemical assays and modeling show that Nsp1 is a modulator of FG-Nup liquid-liquid phase separation, promoting a liquid-like state. Nsp1s presence in the cytosol and condensates is critical, as a reduction of cytosolic levels in young cells induces NPC assembly and transport defects and a general decline in protein quality control, all quantitatively mimicking aging phenotypes. Excitingly, these phenotypes can be rescued by cytosolic Nsp1. We conclude that Nsp1 is a phase state regulator that surveils FG-Nups and impacts general protein homeostasis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.534946v1?rss=1 Authors: Hutchins, J. R. A., Peiffer, I., Urbach, S., Mergny, J.-L., Marin, P., Maiorano, D., MECHALI, M. Abstract: In metazoan cells, replication of genomic DNA initiates from thousands of discrete chromosomal loci known as origins. Proteins such as the Origin Recognition Complex (ORCs) associate with origins, but this does not show clear sequence specificity for DNA binding. Genome-wide origin mapping studies have shown that the region surrounding the replication initiation site contains motifs such as the Origin G-rich Repeated Element (OGRE), proximal to the majority of origins. Here, using an approach coupling DNA affinity purification to quantitative proteomics, we identified proteins that interact specifically with an OGRE. Three of the top-scoring interactors, Dhx36, Pura and Tial1, were selected for further study. We show that Dhx36 and Tial1 localise to the nucleus and their knockdown decreased cells in S-phase resulting in their accumulation in the G1 phase of the cell cycle. Altogether these results indicate that these OGRE-binding factors may play roles in DNA synthesis in mammalian cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.531359v1?rss=1 Authors: Ramzan, F., Abrar, F., Kumar, A., Liao, L. M. Q., Campbell, Z. E., Gray, R. V., Akanni, O., Guyn, C. M., Martin, D. D. O. Abstract: Multi-system proteinopathy (MSP) is a rare dominantly-inherited disorder that includes a cluster of diseases, including frontotemporal dementia, inclusion body myopathy, and Paget's disease of bone. MSP is caused by mutations in the gene encoding Valosin-containing protein (VCP). Patients with the same mutation, even within the same family, can present with a different combination of any or all of these diseases, along with amyotrophic lateral sclerosis (ALS). The pleiotropic effects may be linked to the greater than 50 VCP co-factors that direct VCP's many roles in the cell. Small VCP-Interacting Protein (SVIP) is a small protein that directs VCP to autophagosomes and lysosomes. We found that SVIP directs VCP localization to autophagosomes in an acylation-dependent manner. We demonstrate that SVIP is myristoylated at glycine 2 and palmitoylated at cysteines 4 and 7. Acylation of SVIP was required to mediate cell death in the presence of the MSP- associated VCP variant (R155H-VCP), whereby blocking SVIP myristoylation rescues cytotoxicity. Therefore, SVIP acylation may present a novel target in MSP. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535068v1?rss=1 Authors: Shi, Q., Zheng, L. Abstract: Long-term space flight will lead to bone loss and osteoblasts dysfunction. The underlying mechanism is still far to reveal. Frizzled-9 (Fzd9) is a Wnt receptor which is essential to osteoblasts differentiation and bone formation. Here we investigate whether Fzd9 plays a role in simulated microgravity (SMG) induced osteoblasts dysfunction. After 1-3 days of SMG, the osteogenic markers were decreased which accompanied the decline of Fzd9 expression. Fzd9 also decreased in the femur of the rats after 3 weeks of hindlimb unloading. Overexpression of Fzd9 will counteract SMG-induced osteoblasts dysfunction. However, Fzd9 overexpression did not affect SMG induced pGSK3 and -catenin expression or sublocalization. Overexpression of Fzd9 regulates the phosphorylation of Akt and ERK, as well as induces F-actin polymerization to form the actin cap, presses the nuclei, and increases the nuclear pore size, which promotes nuclear translocation of YAP. Our study provides mechanistic insights into the role of Fzd9 regulates YAP in SMG-mediated osteoblasts dysfunction and indicates Fzd9 as a potential target to restore osteoblast function in bone diseases and space flight. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535108v1?rss=1 Authors: Skoufou-Papoutsaki, N., Adler, S., D'Santos, P., Mannion, L., Mehmed, S., Kemp, R., Smith, A., Perrone, F., Nayak, K., Russell, A., Zilbauer, M., Winton, D. J. Abstract: Organoids are currently one of the most widely used ex vivo models in epithelial biology. Combined with genetic editing strategies, organoids offer a promise of rapid and efficient investigation of gene function in many models of human disease. However, to date, the editing efficiency of organoids with the use of non-viral electroporation methods has been only up to 30%, with implications for the subsequent need for selection including including turnaround time and exhaustion or adaptation of the organoid population. Here, we describe an efficient method of intestinal organoid editing using a Ribonucleoprotein CRISPR-based approach. Editing efficiencies of up to 98% in target genes were robustly achieved across different anatomical gut locations and developmental timepoints from multiple patient samples with no off-target editing. The method allowed us to study the effect of the loss of the tumour suppressor gene, PTEN, in normal human intestinal cells. Analysis of PTEN deficient organoids defined phenotypes that likely relate to its tumour suppressive function in vivo, such as a proliferative advantage and increased organoid budding. Transcriptional profiling revealed differential expression of genes in pathways commonly known to be associated with PTEN loss including mTORC1 activation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.533363v1?rss=1 Authors: Iuliani, I., Mbemba, G., Lagomarsino, M. C., Sclavi, B. Abstract: A long-standing hypothesis sees DNA replication control in E. coli as a central cell cycle oscillator at whose core is the DnaA protein. The consensus is that the activity of the DnaA protein, which is dependent on its nucleotide bound state, is an effector of initiation of DNA replication and a sensor of cell size. However, while several processes are known to regulate the change in DnaA activity, the oscillations in DnaA production and DnaA activity have never been observed at the single cell level, and their correlation with cell volume has yet to be established. Here, we measured the volume-specific production rate of a reporter protein under control of the dnaAP2 promoter in single cells. By a careful dissection of the effects of DnaA-ATP- and SeqA-dependent regulation of dnaAP2 promoter activity two distinct cell-cycle oscillators emerge. The first one, driven by both DnaA activity and SeqA repression, is strongly coupled to cell cycle and cell size, and its minima show the same "adder" behaviour as initiation events. The second, a reporter of DnaA activity in the absence of SeqA binding, is still coupled with cell size but not to the time of cell division, and its minima (corresponding to DnaA activity peaks) show a "sizer-like" behavior, hence deviating from actual initiations. These findings indicate that production of DnaA is tightly coupled to cell volume through the timing of gene duplication, positive and negative regulation by DnaA-ATP itself and SeqA repression, and that DnaA activity peaks are a necessary but not sufficient condition to trigger replication initiation, posing firmer quantitative bases for a mechanistic understanding of cell cycle progression in bacteria. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534814v1?rss=1 Authors: Chang, W.-J., Harpel, Z., Circelli, J., Chen, R., Chang, I., Rivera, J., Wu, S., Wei, Z. Abstract: Tetrahymena are ciliated protists that have been used to study the effects of toxic chemicals, including anticancer drugs. In this study, we tested the inhibitory effects of six pyrimidine analogs (5-fluorouracil, floxuridine, 5-deoxy-5-fluorouridine, 5-fluorouridine, gemcitabine, cytarabine) on wild-type CU428 and conditional mutant NP1 Tetrahymena thermophila at room temperature and the restrictive temperature (37{degrees}C) where NP1 does not form the oral apparatus. We found that cytarabine was the only tested analog that did not inhibit growth, and phagocytosis was not required for pyrimidine analog entry. IC50 values did not significantly differ between strains for the same analog at either temperature. To investigate the mechanism of inhibition, we used two pyrimidine bases (uracil and thymine) and three nucleosides (uridine, thymidine, 5-methyluridine) to help determine whether the inhibitory effects from analogs were reversible. We found that the inhibitory effects from 5-fluorouracil could be reversed by uracil and thymine, from floxuridine could be reversed by thymidine, and from 5-deoxy-5-fluorouridine could be reversed by uracil. None of the tested nucleobases or nucleosides could reverse the inhibitory effects of gemcitabine or 5-fluorouridine. Our results suggest that the five pyrimidine analogs act on different sites to inhibit T. thermophila growth and that nucleobases and nucleosides are metabolized differently. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.534530v1?rss=1 Authors: Sun, X.-M., Miles, G. J., Craxton, A., Powley, I. R., Galavotti, S., Chernova, T., Dawson, A., Nakas, A., Willis, A. E., Cain, K., MacFarlane, M. Abstract: Malignant pleural mesothelioma (MPM) is an aggressive malignancy linked to asbestos exposure and highly resistant to chemotherapy, potentially due to upregulated expression of the pro-survival proteins, BCL2/BCL-XL/MCL-1. Using clinically-relevant models of MPM we show that patient-derived primary MPM cell lines and ex-vivo 3D tumour explants are highly resistant to apoptosis induced by the BCL2/BCL-XL inhibitor, ABT-737. Importantly, we discover that 2-deoxyglucose (2DG), a glycolytic inhibitor, can sensitize MPM cells to ABT-737 and show this correlates with loss of the pro-survival protein, MCL-1. siRNA knockdown of MCL-1 (MCL-1 KD) combined with ABT-737 induced BAX/BAK-dependent, but BIM/PUMA-independent apoptosis, mimicking 2DG/ABT-737 treatment. MCL-1 KD/ABT-737 induced mitochondrial cytochrome c release and caspase-independent inhibition of mitochondrial respiration. Moreover, we observed a hitherto unreported caspase-dependent cleavage of glycolytic enzymes and subsequent inhibition of glycolysis. 2DG inhibited ERK/STAT3 activity, decreased MCL-1 mRNA and protein levels, with concurrent activation of AKT, which limited loss of MCL-1 protein. However, co-treatment with a specific AKT inhibitor, AZD5363, and 2DG/ABT-737 potently induced cell death and inhibited clonogenic cell survival, while in MPM 3D tumour explants MCL-1 protein expression decreased significantly following 2DG or 2DG/AZD5363 treatment. Notably, a similar synergy was observed in MPM cell lines and MPM 3D tumour explants using ABT-737 in combination with the recently developed MCL-1 inhibitor, S63845. Importantly, our study provides a mechanistic explanation for the chemoresistance of MPM and highlights how this can be overcome by a combination of metabolic reprogramming and/or simultaneous targeting of MCL-1 and BCL-2/BCL-XL using BH3-mimetics. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.31.514578v1?rss=1 Authors: Boyer, C. K., Zhang, J., Wang, Y., Stephens, S. B. Abstract: The pancreatic islet beta-cell's preference for release of newly synthesized insulin requires careful coordination of insulin exocytosis with sufficient insulin granule production to ensure that insulin stores exceed peripheral demands for glucose homeostasis. Thus, the cellular mechanisms regulating insulin granule production are critical to maintaining beta-cell function. In this report, we utilized the synchronous protein trafficking system, RUSH, in primary beta-cells to evaluate proinsulin transit through the secretory pathway leading to insulin granule formation. We demonstrate that the trafficking, processing, and secretion of the proinsulin RUSH reporter, proCpepRUSH, are consistent with current models of insulin maturation and release. Using a rodent dietary model of hyperglycemia and beta-cell dysfunction, we show that proinsulin trafficking is impeded at the Golgi and coincides with the decreased appearance of nascent insulin granules at the plasma membrane. Ultrastructural analysis of beta-cells from diabetic leptin receptor deficient mice revealed gross morphological changes in Golgi structure, including shortened and swollen cisternae, and partial Golgi vesiculation, which are consistent with defects in secretory protein export. Collectively, this work highlights the utility of the proCpepRUSH reporter in studying proinsulin trafficking dynamics and suggests that altered Golgi export function contributes to beta-cell secretory defects in the pathogenesis of Type 2 diabetes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535067v1?rss=1 Authors: Jokl, E., Mullan, A., Simpson, K., Birchall, L., Pearmain, L., Martin, K., Pritchett, J., Shah, R., Hodson, N., Williams, C., Camacho, E., Zeef, L., Donaldson, I., Athwal, V., Hanley, N., Piper Hanley, K. Abstract: Myofibroblasts are responsible for scarring and organ stiffness during fibrosis. The scar propagates mechanical signals inducing a radical transformation in myofibroblast cell state linked to an increasingly pro-fibrotic phenotype. Here, we have discovered mechanical stress from progressive scarring induces nuclear softening and de-repression of heterochromatin. The parallel loss of H3K9Me3 enables a permissive state for distinct chromatin accessibility and profibrotic gene regulation. By integrating chromatin accessibility profiles (ATAC sequencing) we provide insight into the transcription network and open chromatin landscape underlying the switch in profibrotic myofibroblast states, emphasizing mechanoadaptive pathways linked to PAK1 as key drivers. Through genetic manipulation in liver and lung fibrosis, uncoupling PAK1-dependant signaling impaired the mechanoadaptive response in vitro and dramatically improved fibrosis in vivo. Moreover, we provide human validation for mechanisms underpinning PAK1 mediated mechanotransduction in liver and lung fibrosis. Collectively, these observations provide new insight into the nuclear mechanics driving the profibrotic chromatin landscape in fibrosis, highlighting actomyosin-dependent mechanisms linked to chromatin organisation as urgently needed therapeutic targets in fibrosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535027v1?rss=1 Authors: Armendariz, I., Lopez de Heredia, U., Soler, M., Puigdemont, A., Ruiz, M. M., Jove, P., Soto, A., Serra, O., Figueras, M. Abstract: The periderm is basic for land plants due to its protective role during radial growth, which is achieved by the polymers deposited in the cell walls. In most trees, like holm oak, the periderm is frequently replaced by subsequent internal periderms yielding a heterogeneous outer bark made of a mixture of periderms and phloem tissues, known as rhytidome. Exceptionally, cork oak forms a persistent or long-lived periderm which results in a homogeneous outer bark of thick phellem cell layers known as cork. Here we use the outer bark of cork oak, holm oak, and their natural hybrids to analyse the chemical composition, the anatomy and the transcriptome, and further understand the mechanisms underlying periderm development. The inclusion of hybrid samples showing rhytidome-type and cork-type barks is valuable to approach to cork and rhytidome development, allowing an accurate identification of candidate genes and processes. The present study underscores that biotic stress and cell death signalling are enhanced in rhytidome-type barks whereas lipid metabolism and cell cycle are enriched in cork-type barks. Development-related DEGs, showing the highest expression, highlight cell division, cell 47 expansion, and cell differentiation as key processes leading to cork or rhytidome-type barks. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535077v1?rss=1 Authors: Santos, C., Rogers, S. L., Carter, A. P. Abstract: Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identity of these objects and what causes their accumulation has not been conclusively established. Here, we used cryogenic electron tomography (cryoET) of Drosophila S2 cell protrusions and found filaments inside the microtubule lumen, which resemble those reported recently in human HAP1 cells. The frequency of these filaments increased upon inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) with the small-molecule drug thapsigargin. Subtomogram averaging showed that the luminal filaments adopt a helical structure reminiscent of cofilin-bound actin (cofilactin). Consistent with this, cofilin was activated in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNAi knock-down of cofilin reduced the frequency of luminal filaments with cofilactin morphology. These results suggest that cofilin activation stimulates its accumulation on actin filaments inside the microtubule lumen. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534783v1?rss=1 Authors: Bychkov, I., Topchu, I., Makhov, P., Kudinov, A., Patel, J. D., Boumber, Y. Abstract: Lung cancer is the most frequently diagnosed cancer type and the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) represents most of the lung cancer. Vascular endothelial growth factor receptor-2 (VEGFR2) is a member of the VEGF family of receptor tyrosine kinase proteins, expressed on both endothelial and tumor cells which is one of the key proteins contributing to cancer development and involved in drug resistance. We previously showed that Musashi-2 (MSI2) RNA-binding protein is associated with NSCLC progression by regulating several signaling pathways relevant to NSCLC. In this study, we performed Reverse Protein Phase Array (RPPA) analysis of murine lung cancer which nominated VEGFR2 protein as strongly positively regulated by MSI2. Next, we validated VEGFR2 protein regulation by MSI2 in several human NSCLC cell line models. Additionally, we found that MSI2 affected AKT signaling via negative PTEN mRNA translation regulation. In silico prediction analysis suggested that both VEGFR2 and PTEN mRNAs have predicted binding sites for MSI2. We next performed RNA immunoprecipitation coupled with quantitative PCR which confirmed that MSI2 directly binds to VEGFR2 and PTEN mRNAs, suggesting direct regulation mechanism. Finally, MSI2 expression positively correlated with VEGFR2 and VEGF-A protein levels in human NSCLC samples. We conclude that MSI2/VEGFR2 axis contributes to NSCLC progression and is worth further investigations and therapeutic targeting. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535147v1?rss=1 Authors: Hladyshau, S., Stoop, J. P., Kamada, K., Nie, S., Tsygankov, D. V. Abstract: Rho-GTPases are central regulators within a complex signaling network that controls the cytoskeletal organization and cell movement. This network includes multiple GTPases, such as the most studied Rac1, Cdc42, and RhoA, and their numerous effectors that provide mutual regulation and feedback loops. Here we investigate the temporal and spatial relationship between Rac1 and Cdc42 during membrane ruffling using a simulation model which couples GTPase signaling with cell morphodynamics to capture the GTPase behavior observed with FRET-based biosensors. We show that membrane velocity is regulated by the kinetic rate of GTPase activation rather than the concentration of active GTPase. Our model captures both uniform and polarized ruffling. We also show that cell-type specific time delays between Rac1 and Cdc42 activation can be reproduced with a single signaling motif, in which the delay is controlled by feedback from Cdc42 to Rac1. The resolution of our simulation output matches those of the time-lapsed recordings of cell dynamics and GTPase activity. This approach allows us to validate simulation results with quantitative precision using the same pipeline for the analysis of simulated and experimental data. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.534966v1?rss=1 Authors: Wang, C., Ma, H., Baserga, S., Pederson, T., Huang, S. Abstract: The nucleolus is a multi-functional nuclear body. To tease out the roles of nucleolar structure without resorting to multi-action drugs, we knocked down RNA polymerase I subunit RPA194 in HeLa cells by siRNA. Loss of RPA194 resulted in nucleolar structural segregation and effects on both nucleolus-proximal and distal nuclear components. The perinucleolar compartment was disrupted, centromere-nucleolus interactions were significantly reduced, and the intranuclear locations of specific genomic loci were altered. Moreover, Cajal bodies, distal from nucleoli, underwent morphological and compositional changes. To distinguish whether these global reorganizations are the results of nucleolar structural disruption or inhibition of ribosome synthesis, the pre-ribosomal RNA processing factor, UTP4, was also knocked down, which did not lead to nucleolar segregation, nor the intranuclear effects seen with RPA195A knockdown, demonstrating that they do not arise from a cessation of ribosome synthesis. These findings point to a commutative system that links nucleolar structure to the maintenance and spatial organization of certain nuclear bodies and genomic loci. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535026v1?rss=1 Authors: Deolal, P., Ramalingam, K., Das, B., Mishra, K. Abstract: When yeast cells are exposed to nutrient-limiting conditions, they undergo transcriptional and translational reprogramming that results in the remodeling of metabolite utilization and organelle architecture. Organelle membranes and contacts also undergo structural and functional alterations. In the budding yeast Saccharomyces cerevisiae, regulated expression of Uip4 is shown to be a critical effector of nuclear shape and function, particularly during the stationary phase. In this work, we demonstrate that the absence of UIP4 affects the morphology of multiple other organelles including mitochondria, endoplasmic reticulum, vacuole and the distribution of lipid droplets. The results show that modulating carbon source, nitrogen availability and cellular energy state impact Uip4 expression. This expression of Uip4 is controlled by the transcription factor Msn2, downstream of Sch9 signaling pathway. Cells lacking Uip4 have poor survival in the stationary phase of the growth cycle. These cellular changes are concomitant with dysregulation of the global lipidome profile and aberrant organelle interaction. We propose that the dynamic and regulated expression of Uip4 is required to maintain lipid homeostasis and organelle architecture which is ultimately required to survive in nutrient-limiting conditions such as stationary phase. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.534956v1?rss=1 Authors: Oprescu, S. N., Baumann, N., Chen, X., Sun, Q., Zhao, Y., Yue, F., Wang, H., Kuang, S. Abstract: Transcription factors (TFs) play key roles in regulating the differentiation and function of stem cells, including muscle satellite cells (MuSCs), a resident stem cell population responsible for postnatal regeneration of the skeletal muscle. Sox11 belongs to the Sry-related HMG-box (SOX) family of TFs that play diverse roles in stem cell behavior and tissue specification. Analysis of single-cell RNA-sequencing (scRNA-seq) datasets identify a specific enrichment of Sox11 mRNA in differentiating but not quiescent MuSCs. Consistent with the scRNA-seq data, Sox11 levels increase during differentiation of murine primary myoblasts in vitro. scRNA-seq data comparing muscle regeneration in young and old mice further demonstrate that Sox11 expression is reduced in aged MuSCs. Age-related decline of Sox11 expression is associated with reduced chromatin contacts within the topologically associated domains. Unexpectedly, Myod1Cre-driven deletion of Sox11 in embryonic myoblasts has no effects on muscle development and growth, resulting in apparently healthy muscles that regenerate normally. Pax7CreER or Rosa26CreER driven (MuSC-specific or global) deletion of Sox11 in adult mice similarly has no effects on MuSC differentiation or muscle regeneration. These results identify Sox11 as a novel myogenic differentiation marker with reduced expression in quiescent and aged MuSCs, but the specific function of Sox11 in myogenesis remain to be elucidated. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.534746v1?rss=1 Authors: Kettel, P., Marosits, L., Spinetti, E., Rechberger, M., Radler, P., Niedermoser, I., Fischer, I., Versteeg, G. A., Loose, M., Covino, R., Karagoz, G. E. Abstract: Upon accumulation of unfolded proteins at the endoplasmic reticulum (ER), IRE1 activates the unfolded protein response (UPR) to restore protein-folding homeostasis. During ER stress, the ER lumenal domain (LD) of IRE1 drives its clustering on the ER membrane to initiate signaling. How IRE1 LD assembles into high-order oligomers remains largely unknown. By in vitro reconstitution experiments we show that human IRE1 LD forms dynamic biomolecular condensates. IRE1 LD condensates were stabilized when IRE1 LD was tethered to model membranes and upon binding of unfolded polypeptide ligands. Molecular dynamics simulations suggested that weak multivalent interactions are involved in IRE1 LD assemblies. Mutagenesis showed that disordered regions in IRE1 LD control its clustering in vitro and in cells. Importantly, dysregulated clustering led to defects in IRE1 signaling. Our results reveal that membranes and unfolded polypeptides act as scaffolds to assemble dynamic IRE1 condensates into stable, signaling competent clusters. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535113v1?rss=1 Authors: Litschel, T., Kelley, C. F., Cheng, X., Babl, L., Mizuno, N., Case, L. B., Schwille, P. Abstract: Focal adhesions form liquid-like assemblies around activated integrin receptors at the plasma membrane. Made up of hundreds of proteins, focal adhesions are dynamic structures which can assemble and disassemble quickly, withstand strong actomyosin-applied forces, and form highly stable complexes. How they achieve these flexible characteristics is not well understood. Here, we use recombinant focal adhesion proteins to reconstitute the core structural machinery in vitro, with the goal of understanding the underlying protein dynamics and interactions. We observe liquid-liquid phase separation of the core focal adhesion proteins talin and vinculin for a spectrum of conditions and in combination with several interaction partners. Intriguingly, we show that membrane binding triggers phase separation of these proteins on the membrane, which in turn induces the enrichment of integrin in the clusters. We also introduce a novel experimental setup to probe talin-membrane interactions down to the single protein level. Our results suggest that membrane composition triggers condensate assembly at the membrane, a regulatory mechanism which could widely apply to membrane-localized biomolecular condensates and provide a pathway of how spatial organization of lipids within the membrane can couple into the cytosol. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.31.514487v1?rss=1 Authors: Nedachi, T., Bonod, C., Rorteau, J., Chinoune, W., Ischiuchi, Y., Hugues, S., Gillet, B., Sigaudo-Roussel, D., Lamartine, J. Abstract: The disturbance of intercellular communication is one of the hallmarks of aging. The goal of this study is to clarify the impact of chronological aging on extracellular vesicles (EVs), a key mode of communication in mammalian tissues. We focused on epidermal keratinocytes, the main cells of the outer protective layer of the skin which is strongly impaired in the skin of elderly. EVs were purified from conditioned medium of primary keratinocytes isolated from infant or aged adult skin. A significant increase of the relative number of EVs released from aged keratinocytes was observed whereas their size distribution was not modified. By small RNA sequencing, we described a specific microRNA (miRNA) signature of aged EVs with an increase abundance of miR-30a, a key regulator of barrier function in human epidermis. EVs from aged keratinocytes were found to be able to reduce the proliferation of young keratinocytes, to impact their organogenesis properties in a reconstructed epidermis model and to slow down the early steps of skin wound healing in mice, three features observed in aged epidermis. This work reveals that intercellular communication mediated by EVs is modulated during aging process in keratinocytes and might be involved in the functional defects observed in aged skin. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535033v1?rss=1 Authors: Aslan, M. K., Fourneaux, C., Yilmaz, A., Stavros, S., Parmentier, R., Paldi, A., Gonin-Giraud, S., deMello, A. J., Gandrillon, O. Abstract: Cell lineage tracking is a long-standing and unresolved problem in biology. Microfluidic technologies have the potential to address this problem, by virtue of their ability to manipulate and process single-cells in a rapid, controllable and efficient manner. Indeed, when coupled with traditional imaging approaches, microfluidic systems allow the experimentalist to follow single-cell divisions over time. Herein, we present a valve-based microfluidic system able to probe the decision-making processes of single-cells, by tracking their lineage over multiple generations. The system operates by trapping single-cells within growth chambers, allowing the trapped cells to grow and divide, isolating sister cells after a user-defined number of divisions and finally extracting them for downstream transcriptome analysis. The platform incorporates multiple cell manipulation operations, image processing-based automation for cell loading and growth monitoring, reagent addition and device washing. To demonstrate the efficacy of the microfluidic workflow, 6C2 (chicken erythroleukemia) and T2EC (primary chicken erythrocytic progenitors) cells are tracked inside the microfluidic device over two generations, with a cell viability rate in excess of 90%. Sister cells are successfully isolated after division and extracted within a 500 nL volume, which is compatible with downstream single-cell RNA sequencing analysis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535139v1?rss=1 Authors: Connor, O. M., Matta, S. K., Friedman, J. R. Abstract: Mitochondria are highly dynamic double membrane-bound organelles that maintain their shape in part through fission and fusion. Mitochondrial fission is performed by the dynamin-related protein Dnm1 (Drp1 in humans), a large GTPase that constricts and divides the mitochondria in a GTP hydrolysis-dependent manner. However, it is unclear whether factors inside mitochondria help coordinate the process and if Dnm1/Drp1 activity alone is sufficient to complete fission of both mitochondrial membranes. Here, we identify an intermembrane space protein required for mitochondrial fission in yeast, which we propose to name Mdi1. Loss of Mdi1 leads to hyper-fused mitochondria networks due to defects in mitochondrial fission, but not lack of Dnm1 recruitment to mitochondria. Mdi1 plays a conserved role in fungal species and its homologs contain a putative amphipathic alpha-helix, mutations in which disrupt mitochondrial morphology. One model to explain these findings is that Mdi1 associates with and distorts the mitochondrial inner membrane to enable Dnm1 to robustly complete fission. Our work reveals that Dnm1 cannot efficiently divide mitochondria without the coordinated function of a protein that resides inside mitochondria. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC