Authors:
Bessonneau-Gaborit, V; Cruard, J, Guerin-Charbonnel, C, Derrien, J, Alberge, J.-B, Douillard, E, Devic, M, Deshayes, S, Campion, L, Westermann, F, Moreau, P, Herrmann, C, Bourdon, J, Magrangeas, F, Minvielle, S. Pages: e202302195 - e202302195 Abstract: Among glucocorticoids (GCs), dexamethasone (Dex) is widely used in treatment of multiple myelomas. However, despite a definite benefit, all patients relapse. Moreover, the molecular basis of glucocorticoid efficacy remains elusive. To determine genomic response to Dex in myeloma cells, we generated bulk and single-cell multi-omics data and high-resolution contact maps of active enhancers and target genes. We show that a minority of glucocorticoid receptor-binding sites are associated with enhancer activity gains, increased interaction loops, and transcriptional activity. We identified and characterized a predominant enhancer enriched in cohesin (RAD21) and more accessible upon Dex exposure. Analysis of four gene-specific networks revealed the importance of the CTCF–cohesin couple and the synchronization of regulatory sequence openings for efficient transcription in response to Dex. Notably, these epigenomic changes are associated with cell-to-cell transcriptional heterogeneity, in particular, lineage-specific genes. As consequences, BCL2L11-encoding BIM critical for Dex-induced apoptosis and CXCR4 protective from chemotherapy-induced apoptosis are rather up-regulated in different cells. In summary, our work provides new insights into the molecular mechanisms involved in Dex escape. Keywords: Cancer PubDate: 2023-07-31T10:23:53-07:00 DOI: 10.26508/lsa.202302195 Issue No:Vol. 6, No. 9 (2023)
Authors:
Karl, L. A; Galanti, L, Bantele, S. C, Metzner, F, Safaric, B, Rajappa, L, Foster, B, Pires, V. B, Bansal, P, Chacin, E, Basquin, J, Duderstadt, K. E, Kurat, C. F, Bartke, T, Hopfner, K.-P, Pfander, B. Pages: e202201790 - e202201790 Abstract: Fun30 is the prototype of the Fun30-SMARCAD1-ETL subfamily of nucleosome remodelers involved in DNA repair and gene silencing. These proteins appear to act as single-subunit nucleosome remodelers, but their molecular mechanisms are, at this point, poorly understood. Using multiple sequence alignment and structure prediction, we identify an evolutionarily conserved domain that is modeled to contain a SAM-like fold with one long, protruding helix, which we term SAM-key. Deletion of the SAM-key within budding yeast Fun30 leads to a defect in DNA repair and gene silencing similar to that of the fun30 mutant. In vitro, Fun30 protein lacking the SAM-key is able to bind nucleosomes but is deficient in DNA-stimulated ATPase activity and nucleosome sliding and eviction. A structural model based on AlphaFold2 prediction and verified by crosslinking-MS indicates an interaction of the long SAM-key helix with protrusion I, a subdomain located between the two ATPase lobes that is critical for control of enzymatic activity. Mutation of the interaction interface phenocopies the domain deletion with a lack of DNA-stimulated ATPase activation and a nucleosome-remodeling defect, thereby confirming a role of the SAM-key helix in regulating ATPase activity. Our data thereby demonstrate a central role of the SAM-key domain in mediating the activation of Fun30 catalytic activity, thus highlighting the importance of allosteric activation for this class of enzymes. Keywords: Molecular Biology, Structural Biology, Chromatin & Epigenetics PubDate: 2023-07-19T07:52:05-07:00 DOI: 10.26508/lsa.202201790 Issue No:Vol. 6, No. 9 (2023)
Authors:
Kolitsida, P; Nolic, V, Zhou, J, Stumpe, M, Niemi, N. M, Dengjel, J, Abeliovich, H. Pages: e202302149 - e202302149 Abstract: The mitophagic degradation of mitochondrial matrix proteins in Saccharomyces cerevisiae was previously shown to be selective, reflecting a pre-engulfment sorting step within the mitochondrial network. This selectivity is regulated through phosphorylation of mitochondrial matrix proteins by the matrix kinases Pkp1 and Pkp2, which in turn appear to be regulated by the phosphatase Aup1/Ptc6. However, these same proteins also regulate the phosphorylation status and catalytic activity of the yeast pyruvate dehydrogenase complex, which is critical for mitochondrial metabolism. To understand the relationship between these two functions, we evaluated the role of the pyruvate dehydrogenase complex in mitophagic selectivity. Surprisingly, we identified a novel function of the complex in regulating mitophagic selectivity, which is independent of its enzymatic activity. Our data support a model in which the pyruvate dehydrogenase complex directly regulates the activity of its associated kinases and phosphatases. This regulatory interaction then determines the phosphorylation state of mitochondrial matrix proteins and their mitophagic fates. Keywords: Cell Biology PubDate: 2023-07-13T09:49:59-07:00 DOI: 10.26508/lsa.202302149 Issue No:Vol. 6, No. 9 (2023)
Authors:
Balendra, R; Ruiz de los Mozos, I, Odeh, H. M, Glaria, I, Milioto, C, Wilson, K. M, Ule, A. M, Hallegger, M, Masino, L, Martin, S, Patani, R, Shorter, J, Ule, J, Isaacs, A. M. Pages: e202201824 - e202201824 Abstract: An intronic GGGGCC repeat expansion in C9orf72 is a common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeats are transcribed in both sense and antisense directions to generate distinct dipeptide repeat proteins, of which poly(GA), poly(GR), and poly(PR) have been implicated in contributing to neurodegeneration. Poly(PR) binding to RNA may contribute to toxicity, but analysis of poly(PR)-RNA binding on a transcriptome-wide scale has not yet been carried out. We therefore performed crosslinking and immunoprecipitation (CLIP) analysis in human cells to identify the RNA binding sites of poly(PR). We found that poly(PR) binds to nearly 600 RNAs, with the sequence GAAGA enriched at the binding sites. In vitro experiments showed that poly(GAAGA) RNA binds poly(PR) with higher affinity than control RNA and induces the phase separation of poly(PR) into condensates. These data indicate that poly(PR) preferentially binds to poly(GAAGA)-containing RNAs, which may have physiological consequences. Keywords: Neuroscience PubDate: 2023-07-12T08:09:47-07:00 DOI: 10.26508/lsa.202201824 Issue No:Vol. 6, No. 9 (2023)
Authors:
Cheng, L.-C; Zhang, X, Baboo, S, Nguyen, J. A, Martinez-Bartolome, S, Loose, E, Diedrich, J, Yates, J. R, Gerace, L. Pages: e202301998 - e202301998 Abstract: The nuclear envelope (NE) is a subdomain of the ER with prominent roles in nuclear organization, which are largely mediated by its distinctive protein composition. We developed methods to reveal low-abundance transmembrane (TM) proteins concentrated at the NE relative to the peripheral ER. Using label-free proteomics that compared isolated NEs with cytoplasmic membranes, we first identified proteins with apparent NE enrichment. In subsequent authentication, ectopically expressed candidates were analyzed by immunofluorescence microscopy to quantify their targeting to the NE in cultured cells. Ten proteins from a validation set were found to associate preferentially with the NE, including oxidoreductases, enzymes for lipid biosynthesis, and regulators of cell growth and survival. We determined that one of the validated candidates, the palmitoyltransferase Zdhhc6, modifies the NE oxidoreductase Tmx4 and thereby modulates its NE levels. This provides a functional rationale for the NE concentration of Zdhhc6. Overall, our methodology has revealed a group of previously unrecognized proteins concentrated at the NE and additional candidates. Future analysis of these can potentially unveil new mechanistic pathways associated with the NE. Keywords: Methods & Resources, Molecular Biology, Cell Biology PubDate: 2023-07-11T08:07:16-07:00 DOI: 10.26508/lsa.202301998 Issue No:Vol. 6, No. 9 (2023)
Authors:
Verhoeven, D; Grinwis, L, Marsman, C, Jansen, M. H, T2B Consortium, Van Leeuwen, E. M, Kuijpers, T. W. Pages: e202302214 - e202302214 Abstract: B cell–targeted therapies, such as CD20-targeting mAbs, deplete B cells but do not target the autoantibody-producing plasma cells (PCs). PC-targeting therapies such as daratumumab (anti-CD38) form an attractive approach to treat PC-mediated diseases. CD38 possesses enzymatic and receptor capabilities, which may impact a range of cellular processes including proliferation and differentiation. However, very little is known whether and how CD38 targeting affects B-cell differentiation, in particular for humans beyond cancer settings. Using in-depth in vitro B-cell differentiation assays and signaling pathway analysis, we show that CD38 targeting with daratumumab demonstrated a significant decrease in proliferation, differentiation, and IgG production upon T cell–dependent B-cell stimulation. We found no effect on T-cell activation or proliferation. Furthermore, we demonstrate that daratumumab attenuated the activation of NF-B in B cells and the transcription of NF-B–targeted genes. When culturing sorted B-cell subsets with daratumumab, the switched memory B-cell subset was primarily affected. Overall, these in vitro data elucidate novel non-depleting mechanisms by which daratumumab can disturb humoral immune responses. Affecting memory B cells, daratumumab may be used as a therapeutic approach in B cell–mediated diseases other than the currently targeted malignancies. Keywords: Immunology, Medical Research PubDate: 2023-07-07T08:50:21-07:00 DOI: 10.26508/lsa.202302214 Issue No:Vol. 6, No. 9 (2023)
Authors:
Preiss, N. K; Kamal, Y, Wilkins, O. M, Li, C, Kolling, F. W, Trask, H. W, Usherwood, Y.-K, Cheng, C, Frost, H. R, Usherwood, E. J. Pages: e202201683 - e202201683 Abstract: Members of the BTB-ZF transcription factor family regulate the immune system. Our laboratory identified that family member Zbtb20 contributes to the differentiation, recall responses, and metabolism of CD8 T cells. Here, we report a characterization of the transcriptional and epigenetic signatures controlled by Zbtb20 at single-cell resolution during the effector and memory phases of the CD8 T cell response. Without Zbtb20, transcriptional programs associated with memory CD8 T cell formation were up-regulated throughout the CD8 T response. A signature of open chromatin was associated with genes controlling T cell activation, consistent with the known impact on differentiation. In addition, memory CD8 T cells lacking Zbtb20 were characterized by open chromatin regions with overrepresentation of AP-1 transcription factor motifs and elevated RNA- and protein-level expressions of the corresponding AP-1 components. Finally, we describe motifs and genomic annotations from the DNA targets of Zbtb20 in CD8 T cells identified by cleavage under targets and release under nuclease (CUT&RUN). Together, these data establish the transcriptional and epigenetic networks contributing to the control of CD8 T cell responses by Zbtb20. Keywords: Genomics & Functional Genomics, Immunology, Chromatin & Epigenetics PubDate: 2023-07-06T08:56:21-07:00 DOI: 10.26508/lsa.202201683 Issue No:Vol. 6, No. 9 (2023)
Authors:
Ujiie, N; Norden, P. R, Fang, R, Beckmann, L, Cai, Z, Kweon, J, Liu, T, Tan, C, Kuhn, M. S, Stamer, W. D, Aoto, K, Quaggin, S. E, Zhang, H. F, Kume, T. Pages: e202201721 - e202201721 Abstract: Impaired development and maintenance of Schlemm’s canal (SC) are associated with perturbed aqueous humor outflow and intraocular pressure. The angiopoietin (ANGPT)/TIE2 signaling pathway regulates SC development and maintenance, whereas the molecular mechanisms of crosstalk between SC and the neural crest (NC)-derived neighboring tissue, the trabecular meshwork (TM), are poorly understood. Here, we show NC-specific forkhead box (Fox)c2 deletion in mice results in impaired SC morphogenesis, loss of SC identity, and elevated intraocular pressure. Visible-light optical coherence tomography analysis further demonstrated functional impairment of the SC in response to changes in intraocular pressure in NC-Foxc2-/- mice, suggesting altered TM biomechanics. Single-cell RNA-sequencing analysis identified that this phenotype is predominately characterized by transcriptional changes associated with extracellular matrix organization and stiffness in TM cell clusters, including increased matrix metalloproteinase expression, which can cleave the TIE2 ectodomain to produce soluble TIE2. Moreover, endothelial-specific Foxc2 deletion impaired SC morphogenesis because of reduced TIE2 expression, which was rescued by deleting the TIE2 phosphatase VE-PTP. Thus, Foxc2 is critical in maintaining SC identity and morphogenesis via TM–SC crosstalk. Keywords: Genetics, Gene Therapy & Genetic Disease, Medical Research, Development PubDate: 2023-07-06T08:56:21-07:00 DOI: 10.26508/lsa.202201721 Issue No:Vol. 6, No. 9 (2023)
Authors:
Zhang, X; Li, Z, Zhang, Y, Liu, Y, Wang, J, Liu, B, Chen, Q, Wang, Q, Fu, L, Wang, P, Zhong, X, Jin, L, Yan, Q, Chen, L, He, J, Zhao, J, Xiong, X. Pages: e202201796 - e202201796 Abstract: SARS-CoV-2 spike protein (S) is structurally dynamic and has been observed by cryo-EM to adopt a variety of prefusion conformations that can be categorized as locked, closed, and open. S-trimers adopting locked conformations are tightly packed featuring structural elements incompatible with RBD in the "up" position. For SARS-CoV-2 S, it has been shown that the locked conformations are transient under neutral pH. Probably because of their transience, locked conformations remain largely uncharacterized for SARS-CoV-1 S. In this study, we introduced x1, x2, and x3 disulfides into SARS-CoV-1 S. Some of these disulfides have been shown to preserve rare locked conformations when introduced to SARS-CoV-2 S. Introduction of these disulfides allowed us to image a variety of locked and other rare conformations for SARS-CoV-1 S by cryo-EM. We identified bound cofactors and structural features that are associated with SARS-CoV-1 S locked conformations. We compare newly determined structures with other available spike structures of SARS-related CoVs to identify conserved features and discuss their possible functions. Keywords: Immunology, Microbiology, Virology & Host Pathogen Interaction, Structural Biology PubDate: 2023-07-04T15:24:30-07:00 DOI: 10.26508/lsa.202201796 Issue No:Vol. 6, No. 9 (2023)
Authors:
Engler, M; Albers, D, Von Maltitz, P, Gross, R, Münch, J, Cirstea, I. C. Pages: e202201880 - e202201880 Abstract: SARS-CoV-2 triggered the most severe pandemic of recent times. To enter into a host cell, SARS-CoV-2 binds to the angiotensin-converting enzyme 2 (ACE2). However, subsequent studies indicated that other cell membrane receptors may act as virus-binding partners. Among these receptors, the epidermal growth factor receptor (EGFR) was hypothesized not only as a spike protein binder, but also to be activated in response to SARS-CoV-2. In our study, we aim at dissecting EGFR activation and its major downstream signaling pathway, the mitogen-activated signaling pathway (MAPK), in SARS-CoV-2 infection. Here, we demonstrate the activation of EGFR–MAPK signaling axis by the SARS-CoV-2 spike protein and we identify a yet unknown cross talk between ACE2 and EGFR that regulated ACE2 abundance and EGFR activation and subcellular localization, respectively. By inhibiting the EGFR-MAPK activation, we observe a reduced infection with either spike-pseudotyped particles or authentic SARS-CoV-2, thus indicating that EGFR serves as a cofactor and the activation of EGFR-MAPK contributes to SARS-CoV-2 infection. Keywords: Cell Biology PubDate: 2023-07-04T15:24:30-07:00 DOI: 10.26508/lsa.202201880 Issue No:Vol. 6, No. 9 (2023)
Authors:
Li, Y; Tian, L, Zhang, Y, Shi, Y. Pages: e202302056 - e202302056 Abstract: In Caenorhabditis elegans (C. elegans), onset of programmed cell death is marked with the activation of CED-3, a process that requires assembly of the CED-4 apoptosome. Activated CED-3 forms a holoenzyme with the CED-4 apoptosome to cleave a wide range of substrates, leading to irreversible cell death. Despite decades of investigations, the underlying mechanism of CED-4–facilitated CED-3 activation remains elusive. Here, we report cryo-EM structures of the CED-4 apoptosome and three distinct CED-4/CED-3 complexes that mimic different activation stages for CED-3. In addition to the previously reported octamer in crystal structures, CED-4, alone or in complex with CED-3, exists in multiple oligomeric states. Supported by biochemical analyses, we show that the conserved CARD–CARD interaction promotes CED-3 activation, and initiation of programmed cell death is regulated by the dynamic organization of the CED-4 apoptosome. Keywords: Structural Biology PubDate: 2023-07-04T15:24:30-07:00 DOI: 10.26508/lsa.202302056 Issue No:Vol. 6, No. 9 (2023)
Authors:
Iyer, S. S; Sun, Y, Seyfferth, J, Manjunath, V, Samata, M, Alexiadis, A, Kulkarni, T, Gutierrez, N, Georgiev, P, Shvedunova, M, Akhtar, A. Pages: e202302194 - e202302194 Abstract: The NSL complex is a transcriptional activator. Germline-specific knockdown of NSL complex subunits NSL1, NSL2, and NSL3 results in reduced piRNA production from a subset of bidirectional piRNA clusters, accompanied by widespread transposon derepression. The piRNAs most transcriptionally affected by NSL2 and NSL1 RNAi map to telomeric piRNA clusters. At the chromatin level, these piRNA clusters also show decreased levels of H3K9me3, HP1a, and Rhino after NSL2 depletion. Using NSL2 ChIP-seq in ovaries, we found that this protein specifically binds promoters of telomeric transposons HeT-A, TAHRE, and TART. Germline-specific depletion of NSL2 also led to a reduction in nuclear Piwi in nurse cells. Our findings thereby support a role for the NSL complex in promoting the transcription of piRNA precursors from telomeric piRNA clusters and in regulating Piwi levels in the Drosophila female germline. Keywords: Chromatin & Epigenetics PubDate: 2023-06-30T08:06:13-07:00 DOI: 10.26508/lsa.202302194 Issue No:Vol. 6, No. 9 (2023)
Authors:
Salem, O; Jia, S, Qian, B.-Z, Hansen, C. G. Pages: e202201620 - e202201620 Abstract: The Hippo signalling pathway is a master regulator of cell growth, proliferation, and cancer. The transcriptional coregulators of the Hippo pathway, YAP and TAZ, are central in various cancers. However, how YAP and TAZ get activated in most types of cancers is not well understood. Here, we show that androgens activate YAP/TAZ via the androgen receptor (AR) in prostate cancer (PCa), and that this activation is differential. AR regulates YAP translation while inducing transcription of the TAZ encoding gene, WWTR1. Furthermore, we show that AR-mediated YAP/TAZ activation is regulated by the RhoA GTPases transcriptional mediator, serum response factor (SRF). Importantly, in prostate cancer patients, SRF expression positively correlates with TAZ and the YAP/TAZ target genes CYR61 and CTGF. We demonstrate that YAP/TAZ are not essential for sustaining AR activity, however, targeting YAP/TAZ or SRF sensitize PCa cells to AR inhibition in anchorage-independent growth conditions. Our findings dissect the cellular roles of YAP, TAZ, and SRF in prostate cancer cells. Our data emphasize the interplay between these transcriptional regulators and their roles in prostate tumorigenesis and highlight how these insights might be exploited therapeutically. Keywords: Cancer, Cell Biology PubDate: 2023-06-29T09:54:22-07:00 DOI: 10.26508/lsa.202201620 Issue No:Vol. 6, No. 9 (2023)
Authors:
Zuo, Z; Roux, M. E, Chevalier, J. R, Dagdas, Y. F, Yamashino, T, Hojgaard, S. D, Knight, E, Ostergaard, L, Rodriguez, E, Petersen, M. Pages: e202302090 - e202302090 Abstract: Multicellular organisms perceive and transduce multiple cues to optimize development. Key transcription factors drive developmental changes, but RNA processing also contributes to tissue development. Here, we report that multiple decapping deficient mutants share developmental defects in apical hook, primary and lateral root growth. More specifically, LATERAL ORGAN BOUNDARIES DOMAIN 3 (LBD3)/ASYMMETRIC LEAVES 2-LIKE 9 (ASL9) transcripts accumulate in decapping deficient plants and can be found in complexes with decapping components. Accumulation of ASL9 inhibits apical hook and lateral root formation. Interestingly, exogenous auxin application restores lateral roots formation in both ASL9 over-expressors and mRNA decay–deficient mutants. Likewise, mutations in the cytokinin transcription factors type-B ARABIDOPSIS RESPONSE REGULATORS (B-ARRs) ARR10 and ARR12 restore the developmental defects caused by over-accumulation of capped ASL9 transcript upon ASL9 overexpression. Most importantly, loss-of-function of asl9 partially restores apical hook and lateral root formation in both dcp5-1 and pat triple decapping deficient mutants. Thus, the mRNA decay machinery directly targets ASL9 transcripts for decay, possibly to interfere with cytokinin/auxin responses, during development. Keywords: Plant Science, Cell Biology, Development PubDate: 2023-06-29T09:54:22-07:00 DOI: 10.26508/lsa.202302090 Issue No:Vol. 6, No. 9 (2023)
Authors:
Liu, H; Lu, P, He, S, Luo, Y, Fang, Y, Benkaci, S, Wu, B, Wang, Y, Zhou, B. Pages: e202302163 - e202302163 Abstract: Endocardial cushion formation is essential for heart valve development and heart chamber separation. Abnormal endocardial cushion formation often causes congenital heart defects. β-Catenin is known to be essential for endocardial cushion formation; however, the underlying cellular and molecular mechanisms remain incompletely understood. Here, we show that endothelial-specific deletion of β-catenin in mice resulted in formation of hypoplastic endocardial cushions due to reduced cell proliferation and impaired cell migration. By using a β-cateninDM allele in which the transcriptional function of β-catenin is selectively disrupted, we further reveal that β-catenin regulated cell proliferation and migration through its transcriptional and non-transcriptional function, respectively. At the molecular level, loss of β-catenin resulted in increased expression of cell cycle inhibitor p21 in cushion endocardial and mesenchymal cells in vivo. In vitro rescue experiments with HUVECs and pig aortic valve interstitial cells confirmed that β-catenin promoted cell proliferation by suppressing p21. In addition, one savvy negative observation is that β-catenin was dispensable for endocardial-to-mesenchymal fate change. Taken together, our findings demonstrate that β-catenin is essential for cell proliferation and migration but dispensable for endocardial cells to gain mesenchymal fate during endocardial cushion formation. Mechanistically, β-catenin promotes cell proliferation by suppressing p21. These findings inform the potential role of β-catenin in the etiology of congenital heart defects. Keywords: Development PubDate: 2023-06-29T09:54:22-07:00 DOI: 10.26508/lsa.202302163 Issue No:Vol. 6, No. 9 (2023)
Authors:
Yasuda, T; Uchiyama, T, Watanabe, N, Ito, N, Nakabayashi, K, Mochizuki, H, Onodera, M. Pages: e202201881 - e202201881 Abstract: Niemann–Pick disease type C1 (NPC1) is a fatal lysosomal storage disorder characterized by progressive neuronal degeneration. Its key pathogenic events remain largely unknown. We have, herein, found that neonatal BM–derived cell transplantation can ameliorate Purkinje cell degeneration in NPC1 mice. We subsequently addressed the impact of the peripheral immune system on the neuropathogenesis observed in NPC1 mice. The depletion of mature lymphocytes promoted NPC1 phenotypes, thereby suggesting a neuroprotective effect of lymphocytes. Moreover, the peripheral infusion of CD4-positive cells (specifically, of regulatory T cells) from normal healthy donor ameliorated the cerebellar ataxic phenotype and enhanced the survival of Purkinje cells. Conversely, the depletion of regulatory T cells enhanced the onset of the neurological phenotype. On the other hand, circulating inflammatory monocytes were found to be involved in the progression of Purkinje cell degeneration, whereas the depletion of resident microglia had little effect. Our findings reveal a novel role of the adaptive and the innate immune systems in NPC1 neuropathology. Keywords: Neuroscience PubDate: 2023-06-27T10:04:46-07:00 DOI: 10.26508/lsa.202201881 Issue No:Vol. 6, No. 9 (2023)
Authors:
Alberici Delsin, L. E; Plutoni, C, Clouvel, A, Keil, S, Marpeaux, L, Elouassouli, L, Khavari, A, Ehrlicher, A. J, Emery, G. Pages: e202302196 - e202302196 Abstract: Collective cell migration is not only important for development and tissue homeostasis but can also promote cancer metastasis. To migrate collectively, cells need to coordinate cellular extensions and retractions, adhesion sites dynamics, and forces generation and transmission. Nevertheless, the regulatory mechanisms coordinating these processes remain elusive. Using A431 carcinoma cells, we identify the kinase MAP4K4 as a central regulator of collective migration. We show that MAP4K4 inactivation blocks the migration of clusters, whereas its overexpression decreases cluster cohesion. MAP4K4 regulates protrusion and retraction dynamics, remodels the actomyosin cytoskeleton, and controls the stability of both cell–cell and cell–substrate adhesion. MAP4K4 promotes focal adhesion disassembly through the phosphorylation of the actin and plasma membrane crosslinker moesin but disassembles adherens junctions through a moesin-independent mechanism. By analyzing traction and intercellular forces, we found that MAP4K4 loss of function leads to a tensional disequilibrium throughout the cell cluster, increasing the traction forces and the tension loading at the cell–cell adhesions. Together, our results indicate that MAP4K4 activity is a key regulator of biomechanical forces at adhesion sites, promoting collective migration. Keywords: Cell Biology PubDate: 2023-06-27T10:04:46-07:00 DOI: 10.26508/lsa.202302196 Issue No:Vol. 6, No. 9 (2023)
Authors:
Schmitter, C; Di-Luoffo, M, Guillermet-Guibert, J. Pages: e202201862 - e202201862 Abstract: In living organisms, cells sense mechanical forces (shearing, tensile, and compressive) and respond to those physical cues through a process called mechanotransduction. This process includes the simultaneous activation of biochemical signaling pathways. Recent studies mostly on human cells revealed that compressive forces selectively modulate a wide range of cell behavior, both in compressed and in neighboring less compressed cells. Besides participating in tissue homeostasis such as bone healing, compression is also involved in pathologies, including intervertebral disc degeneration or solid cancers. In this review, we will summarize the current scattered knowledge of compression-induced cell signaling pathways and their subsequent cellular outputs, both in physiological and pathological conditions, such as solid cancers. Keywords: Cell Biology PubDate: 2023-06-26T06:19:58-07:00 DOI: 10.26508/lsa.202201862 Issue No:Vol. 6, No. 9 (2023)
Authors:
Ozgencil, M; Dullovi, A, Christiane Higos, R. C, Horejsi, Z, Bellelli, R. Pages: e202301933 - e202301933 Abstract: The maintenance of genome integrity relies on replication fork stabilization upon encountering endogenous and exogenous sources of DNA damage. How this process is coordinated with the local chromatin environment remains poorly defined. Here, we show that the replication-dependent histone H1 variants interact with the tumour suppressor BRCA1 in a replication stress–dependent manner. Transient loss of the replication-dependent histones H1 does not affect fork progression in unchallenged conditions but leads to the accumulation of stalled replication intermediates. Upon challenge with hydroxyurea, cells deficient for histone H1 variants fail to recruit BRCA1 to stalled replication forks and undergo MRE11-dependent fork resection and collapse, which ultimately leads to genomic instability and cell death. In summary, our work defines an essential role of the replication-dependent histone H1 variants in mediating BRCA1-dependent fork protection and genome stability. Keywords: Molecular Biology PubDate: 2023-06-26T06:19:58-07:00 DOI: 10.26508/lsa.202301933 Issue No:Vol. 6, No. 9 (2023)
Authors:
Minet, N; Boschat, A.-C, Lane, R, Laughton, D, Beer, P, Asnagli, H, Soudais, C, Bourne, T, Fischer, A, Martin, E, Latour, S. Pages: e202302066 - e202302066 Abstract: The CTP nucleotide is a key precursor of nucleic acids metabolism essential for DNA replication. De novo CTP production relies on CTP synthetases 1 and 2 (CTPS1 and CTPS2) that catalyze the conversion of UTP into CTP. CTP synthetase activity is high in proliferating cells including cancer cells; however, the respective roles of CTPS1 and CTPS2 in cell proliferation are not known. By inactivation of CTPS1 and/or CTPS2 and complementation experiments, we showed that both CTPS1 and CTPS2 are differentially required for cell proliferation. CTPS1 was more efficient in promoting proliferation than CTPS2, in association with a higher intrinsic enzymatic activity that was more resistant to inhibition by 3-deaza-uridine, an UTP analog. The contribution of CTPS2 to cell proliferation was modest when CTPS1 was expressed but essential in absence of CTPS1. Public databases analysis of more than 1,000 inactivated cancer cell lines for CTPS1 or CTPS2 confirmed that cell growth is highly dependent of CTPS1 but less or not of CTPS2. Therefore, our results demonstrate that CTPS1 is the main contributor to cell proliferation. Keywords: Metabolism PubDate: 2023-06-22T08:38:32-07:00 DOI: 10.26508/lsa.202302066 Issue No:Vol. 6, No. 9 (2023)
Authors:
Xu, Y; Song, D, Wang, W, Li, S, Yue, T, Xia, T, Shi, Y. Pages: e202301938 - e202301938 Abstract: Monosodium uric acid (MSU) crystal, the etiological agent of gout, has been shown to trigger innate immune responses via multiple pathways. It is known that MSU-induced lipid sorting on plasma membrane promotes the phosphorylation of Syk and eventually leads to the activation of phagocytes. However, whether this membrane lipid-centric mechanism is regulated by other processes is unclear. Previous studies showed that Clec12a, a member of the C-type lectin receptor family, is reported to recognize MSU and suppresses this crystalline structure-induced immune activation. How this scenario is integrated into the lipid sorting-mediated inflammatory responses by MSU, and particularly, how Clec12a intercepts lipid raft-originated signaling cascade remains to be elucidated. Here, we found that the ITIM motif of Clec12a is dispensable for its inhibition of MSU-mediated signaling; instead, the transmembrane domain of Clec12a disrupts MSU-induced lipid raft recruitment and thus attenuates downstream signals. Single amino acid mutagenesis study showed the critical role of phenylalanine in the transmembrane region for the interactions between C-type lectin receptors and lipid rafts, which is critical for the regulation of MSU-mediated lipid sorting and phagocyte activation. Overall, our study provides new insights for the molecular mechanisms of solid particle-induced immune activation and may lead to new strategies in inflammation control. Keywords: Immunology, Biophysics, Cell Biology PubDate: 2023-06-20T08:17:22-07:00 DOI: 10.26508/lsa.202301938 Issue No:Vol. 6, No. 9 (2023)
Authors:
Pasquier, C; Guerlais, V, Pallez, D, Rapetti-Mauss, R, Soriani, O. Pages: e202201550 - e202201550 Abstract: The identification of condition-specific gene sets from transcriptomic experiments is important to reveal regulatory and signaling mechanisms associated with a given cellular response. Statistical methods of differential expression analysis, designed to assess individual gene variations, have trouble highlighting modules of small varying genes whose interaction is essential to characterize phenotypic changes. To identify these highly informative gene modules, several methods have been proposed in recent years, but they have many limitations that make them of little use to biologists. Here, we propose an efficient method for identifying these active modules that operates on a data embedding combining gene expressions and interaction data. Applications carried out on real datasets show that our method can identify new groups of genes of high interest corresponding to functions not revealed by traditional approaches. Software is available at https://github.com/claudepasquier/amine. Keywords: Molecular Biology, Systems & Computational Biology, Cancer PubDate: 2023-06-20T06:13:22-07:00 DOI: 10.26508/lsa.202201550 Issue No:Vol. 6, No. 9 (2023)
Authors:
de Vries, I; Ammerlaan, D, Heidebrecht, T, Celie, P. H, Geerke, D. P, Joosten, R. P, Perrakis, A. Pages: e202302150 - e202302150 Abstract: Base-J (β-D-glucopyranosyloxymethyluracil) is a modified DNA nucleotide that replaces 1% of thymine in kinetoplastid flagellates. The biosynthesis and maintenance of base-J depends on the base-J-binding protein 1 (JBP1) that has a thymidine hydroxylase domain and a J-DNA-binding domain (JDBD). How the thymidine hydroxylase domain synergizes with the JDBD to hydroxylate thymine in specific genomic sites, maintaining base-J during semi-conservative DNA replication, remains unclear. Here, we present a crystal structure of the JDBD including a previously disordered DNA-contacting loop and use it as starting point for molecular dynamics simulations and computational docking studies to propose recognition models for JDBD binding to J-DNA. These models guided mutagenesis experiments, providing additional data for docking, which reveals a binding mode for JDBD onto J-DNA. This model, together with the crystallographic structure of the TET2 JBP1-homologue in complex with DNA and the AlphaFold model of full-length JBP1, allowed us to hypothesize that the flexible JBP1 N-terminus contributes to DNA-binding, which we confirmed experimentally. A high-resolution JBP1:J-DNA complex, which must involve conformational changes, would however need to be determined experimentally to further understand this unique underlying molecular mechanism that ensures replication of epigenetic information. Keywords: Structural Biology PubDate: 2023-06-16T08:54:53-07:00 DOI: 10.26508/lsa.202302150 Issue No:Vol. 6, No. 9 (2023)
Authors:
Kim, J. Y; Atanassov, I, Dethloff, F, Kroczek, L, Langer, T. Pages: e202302127 - e202302127 Abstract: Mitochondrial dysfunction and cellular senescence are hallmarks of aging. However, the relationship between these two phenomena remains incompletely understood. In this study, we investigated the rewiring of mitochondria upon development of the senescent state in human IMR90 fibroblasts. Determining the bioenergetic activities and abundance of mitochondria, we demonstrate that senescent cells accumulate mitochondria with reduced OXPHOS activity, resulting in an overall increase of mitochondrial activities in senescent cells. Time-resolved proteomic analyses revealed extensive reprogramming of the mitochondrial proteome upon senescence development and allowed the identification of metabolic pathways that are rewired with different kinetics upon establishment of the senescent state. Among the early responding pathways, the degradation of branched-chain amino acid was increased, whereas the one carbon folate metabolism was decreased. Late-responding pathways include lipid metabolism and mitochondrial translation. These signatures were confirmed by metabolic flux analyses, highlighting metabolic rewiring as a central feature of mitochondria in cellular senescence. Together, our data provide a comprehensive view on the changes in mitochondrial proteome in senescent cells and reveal how the mitochondrial metabolism is rewired in senescent cells. Keywords: Metabolism, Methods & Resources, Cell Biology PubDate: 2023-06-15T11:26:42-07:00 DOI: 10.26508/lsa.202302127 Issue No:Vol. 6, No. 9 (2023)
Authors:
Ghosh, A; Venugopal, A, Shinde, D, Sharma, S, Krishnan, M, Mathre, S, Krishnan, H, Saha, S, Raghu, P. Pages: e202301920 - e202301920 Abstract: Phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol 5-phosphate (PI5P) are low-abundance phosphoinositides crucial for key cellular events such as endosomal trafficking and autophagy. Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) is an enzyme that regulates PI5P in vivo but can act on both PI5P and PI3P in vitro. In this study, we report a role for PIP4K in regulating PI3P levels in Drosophila. Loss-of-function mutants of the only Drosophila PIP4K gene show reduced cell size in salivary glands. PI3P levels are elevated in dPIP4K29 and reverting PI3P levels back towards WT, without changes in PI5P levels, can rescue the reduced cell size. dPIP4K29 mutants also show up-regulation in autophagy and the reduced cell size can be reverted by depleting Atg8a that is required for autophagy. Lastly, increasing PI3P levels in WT can phenocopy the reduction in cell size and associated autophagy up-regulation seen in dPIP4K29. Thus, our study reports a role for a PIP4K-regulated PI3P pool in the control of autophagy and cell size. Keywords: Metabolism PubDate: 2023-06-14T09:27:24-07:00 DOI: 10.26508/lsa.202301920 Issue No:Vol. 6, No. 9 (2023)
Authors:
Naas, S; Krüger, R, Knaup, K. X, Naas, J, Grampp, S, Schiffer, M, Wiesener, M, Schödel, J. Pages: e202302078 - e202302078 Abstract: The interplay between genetic and environmental factors influences the course of chronic kidney disease (CKD). In this context, genetic alterations in the kidney disease gene MUC1 (Mucin1) predispose to the development of CKD. These variations comprise the polymorphism rs4072037, which alters splicing of MUC1 mRNA, the length of a region with variable number of tandem repeats (VNTR), and rare autosomal-dominant inherited dominant-negative mutations in or 5' to the VNTR that causes autosomal dominant tubulointerstitial kidney disease (ADTKD-MUC1). As hypoxia plays a pivotal role in states of acute and chronic kidney injury, we explored the effects of hypoxia-inducible transcription factors (HIF) on the expression of MUC1 and its pathogenic variants in isolated primary human renal tubular cells. We defined a HIF-binding DNA regulatory element in the promoter-proximal region of MUC1 from which hypoxia or treatment with HIF stabilizers, which were recently approved for an anti-anemic therapy in CKD patients, increased levels of wild-type MUC1 and the disease-associated variants. Thus, application of these compounds might exert unfavorable effects in patients carrying MUC1 risk variants. Keywords: Genetics, Gene Therapy & Genetic Disease, Physiology, Chromatin & Epigenetics PubDate: 2023-06-14T09:27:24-07:00 DOI: 10.26508/lsa.202302078 Issue No:Vol. 6, No. 9 (2023)
Authors:
Tan, C; Shi, W, Zhang, Y, Liu, C, Hu, T, Chen, D, Huang, J. Pages: e202201732 - e202201732 Abstract: The present study focused on the effect of miR-93-5p on apoptosis of retinal neurons in acute ocular hypertension (AOH) model by regulating PDCD4 and explored its related mechanism. We detected that miR-93-5p expression was decreased and PDCD4 expression was increased in the AOH retina by qRT-PCR. Therefore, we explored the role of miR-93-5p and PDCD4. MiR-93-5p overexpression inhibited the apoptosis of retinal neurons and the expression of PDCD4 in vivo and in vitro. Inhibiting the expression of PDCD4 via transfected interfering RNA decreased the apoptosis of retinal cells and increased the expression of PI3K/Akt pathway–related proteins in vitro. However, the addition of PI3K protein inhibitor LY294002 reversed this effect, leading to a decrease of PI3K/Akt pathway protein expression and an increase of apoptosis-related protein Bax/Bcl-2 expression ratio. Finally, up-regulating miR-93-5p or down-regulating PDCD4 increased the expression of PI3K/Akt pathway protein in vivo. In conclusion, under the condition of AOH injury, miR-93-5p-inhibiting PDCD4 expression reduced the apoptosis of retinal neurons by activating PI3K/Akt pathway. Keywords: Neuroscience PubDate: 2023-06-12T07:55:25-07:00 DOI: 10.26508/lsa.202201732 Issue No:Vol. 6, No. 9 (2023)
Authors:
Moron-Oset, J; Fischer, L. K, Carcole, M, Giblin, A, Zhang, P, Isaacs, A. M, Grönke, S, Partridge, L. Pages: e202201739 - e202201739 Abstract: Hexanucleotide repeat expansions in the C9orf72 gene are the most prevalent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Transcripts of the expansions are translated into toxic dipeptide repeat (DPR) proteins. Most preclinical studies in cell and animal models have used protein-tagged polyDPR constructs to investigate DPR toxicity but the effects of tags on DPR toxicity have not been systematically explored. Here, we used Drosophila to assess the influence of protein tags on DPR toxicity. Tagging of 36 but not 100 arginine-rich DPRs with mCherry increased toxicity, whereas adding mCherry or GFP to GA100 completely abolished toxicity. FLAG tagging also reduced GA100 toxicity but less than the longer fluorescent tags. Expression of untagged but not GFP- or mCherry-tagged GA100 caused DNA damage and increased p62 levels. Fluorescent tags also affected GA100 stability and degradation. In summary, protein tags affect DPR toxicity in a tag- and DPR-dependent manner, and GA toxicity might be underestimated in studies using tagged GA proteins. Thus, including untagged DPRs as controls is important when assessing DPR toxicity in preclinical models. Keywords: Neuroscience PubDate: 2023-06-12T07:55:25-07:00 DOI: 10.26508/lsa.202201739 Issue No:Vol. 6, No. 9 (2023)
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