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5,819 results found. For more information, click each entry to expand.

5641: Characterization of human embryonic stem cells in animal component-free medium
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Posted 15 Dec 2020

Characterization of human embryonic stem cells in animal component-free medium
89 downloads bioRxiv cell biology

Masakazu Machida, Rie Abutani, Hiroshi Miyajima, Tetsuji Sasaki, Yoshiko Abe, Hidenori Akutsu, Akihiro Umezawa

Clinical use of human embryonic stem cells (ESCs) as a raw material requires good manufacturing practice-compliant axillary materials such as culture medium. To this end, animal components should not be used and contamination of virus/bacteria/fungus and allergens are a concern. In addition, animal components such as albumin and fetal bovine serum pose difficulties such as a lot-to-lot variation. However, only a limited number of animal component-free media have been developed to date. In this study, we investigated whether SEES2 ESCs can be stably propagated for 16 passages (54 population doublings) over a period of 60 days in a newly established Stem-Partner ACF medium. SEES2 ESC maintained their intact karyotype, i.e. 46,XX, and their undifferentiated phenotypes after long-term culture. An in vitro differentiation assay revealed that SEES2 ESCs exhibited multipotency, i.e. endodermal, mesodermal and ectodermal differentiation. Subcutaneous implantation of SEES2 ESCs generated mature teratomas without malignant transformation. These results show that SEES2 ESCs in the Stem-Partner ACF medium can be used to establish master cell banks for future regenerative medicine as well as other ESCs in the previously reported culture medium.

5642: Molecular and structural mechanisms of ZZ domain-mediated cargo recognition by autophagy receptor Nbr1
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Posted 07 Feb 2021

Molecular and structural mechanisms of ZZ domain-mediated cargo recognition by autophagy receptor Nbr1
89 downloads bioRxiv cell biology

Ying-Ying Wang, Jianxiu Zhang, Xiao-Man Liu, Meng-Qiu Dong, Keqiong Ye, Li-Lin Du

In selective autophagy, cargo selectivity is determined by autophagy receptors. However, it remains scarcely understood how autophagy receptors recognize specific protein cargos. In the fission yeast Schizosaccharomyces pombe, a selective autophagy pathway termed Nbr1-mediated vacuolar targeting (NVT) employs Nbr1, an autophagy receptor conserved across eukaryotes including humans, to target cytosolic hydrolases into the vacuole. Here, we identify two new NVT cargos, the mannosidase Ams1 and the aminopeptidase Ape4, that bind competitively to the first ZZ domain of Nbr1 (Nbr1-ZZ1). High-resolution cryo-EM analyses reveal how a single ZZ domain recognizes two distinct protein cargos. Nbr1-ZZ1 not only recognizes the N-termini of cargos via a conserved acidic pocket, similar to other characterized ZZ domains, but also engages additional parts of cargos in a cargo-specific manner. Our findings unveil a single-domain bispecific mechanism of autophagy cargo recognition, elucidate its underlying structural basis, and expand the understanding of ZZ domain-mediated protein-protein interactions.

5643: Escape from mitotic catastrophe by actin-dependent nuclear displacement in fission yeast
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Posted 04 Dec 2020

Escape from mitotic catastrophe by actin-dependent nuclear displacement in fission yeast
88 downloads bioRxiv cell biology

Masashi Yukawa, Yasuhiro Teratani, Takashi Toda

Proper nuclear positioning is essential for the execution of a wide variety of cellular processes in eukaryotic cells (Gundersen and Worman, 2013; Kopf et al., 2020; Lele et al., 2018). In proliferating mitotic cells, nuclear positioning is crucial for successful cell division. The bipolar spindle, which pulls sister chromatids towards two opposite poles, needs to assemble in the geometrical center of the cell. This ensures symmetrical positioning of the two nuclei that are reformed upon mitotic exit, by which two daughter cells inherit the identical set of the chromosomes upon cytokinesis. In fission yeast, the nucleus is positioned in the cell center during interphase; cytoplasmic microtubules interact with both the nucleus and the cell tips, thereby retaining the nucleus in the medial position of the cell (Daga et al., 2006; Tran et al., 2001). By contrast, how the nucleus is positioned during mitosis remains elusive. Here we show that several cell-cycle mutants that arrest in mitosis all displace the nucleus towards one end of the cell axis. Intriguingly, the actin cytoskeleton, not the microtubule counterpart, is responsible for the asymmetric movement of the nucleus. Time-lapse live imaging indicates that mitosis-specific F-actin cables interact with the nuclear membrane, thereby possibly generating an asymmetrical pushing force. In addition, constriction of the actomyosin ring further promotes nuclear displacement. This nuclear movement is beneficial, because if the nuclei were retained in the cell center, subsequent cell division would impose the lethal cut phenotype (Hirano et al., 1986; Yanagida, 1998), in which chromosomes are intersected by the contractile actin ring and the septum. Thus, fission yeast escapes from mitotic catastrophe by means of actin-dependent nuclear movement.

5644: A non-olfactory shark adenosine receptor activates CFTR with unique pharmacology and structural features
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Posted 02 Nov 2020

A non-olfactory shark adenosine receptor activates CFTR with unique pharmacology and structural features
88 downloads bioRxiv cell biology

Sumeet Bhanot, Gabriele Hemminger, Cole L. Martin, Stephen G. Aller, John N. Forrest

Adenosine receptors (ADORs) are G-protein coupled purinoceptors that have several functions including regulation of chloride secretion via CFTR in human airway and kidney. We cloned an ADOR from Squalus acanthias (shark) that likely regulates CFTR in the rectal gland. Phylogenic- and expression- analyses indicate that elasmobranch ADORs are non-olfactory, and appear to represent extant predecessors of mammalian ADORs. We therefore designate the shark ADOR as the A0 receptor. We co-expressed A0 with CFTR in Xenopus laevis oocytes and characterized the coupling of A0 to the chloride channel. Two electrode voltage clamping was performed and current-voltage (I-V) responses were recorded to monitor CFTR status. Only in A0- and CFTR- co-injected oocytes did adenosine analogs produce a significant concentration-dependent activation of CFTR consistent with its electrophysiological signature. A pharmacological profile for A0 was obtained for ADOR agonists and antagonists that differed markedly from all mammalian ADOR subtypes (agonists: R-PIA > S-PIA > CGS21680 > CPA > 2ClADO > CV1808 = DPMA > NECA) and (antagonists: DPCPX > PD115199 > 8PT > CGC > CGS15943). Structures of human ADORs permitted a high-confidence homology model of the shark A0 core which revealed unique structural features of ancestral receptors. We conclude: (1) A0 is a novel and unique adenosine receptor ancestor by functional and structural criteria; (2) A0 likely activates CFTR in vivo and this receptor activates CFTR in oocytes indicating an evolutionary coupling between ADORs and chloride secretion; and (3) A0 appears to be a non-olfactory evolutionary ancestor of all four mammalian ADOR subtypes.

5645: Functionally non-redundant paralogs spe-47 and spe-50 encode FB-MO associated proteins and interact with him-8
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Posted 17 Mar 2020

Functionally non-redundant paralogs spe-47 and spe-50 encode FB-MO associated proteins and interact with him-8
88 downloads bioRxiv cell biology

Jessica N. Clark, Gaurav Prajapati, Fermina Aldaco, Thomas J. Sokolich, Steven Keung, Sarojani Austin, Ángel A. Valdés, Craig W. LaMunyon

The activation of C. elegans spermatids to crawling spermatozoa is affected by a number of genes including spe-47 . Here, we investigate a paralog to spe-47 : spe-50 , which has a highly conserved sequence and expression, but which is not functionally redundant to spe-47 . Phylogenetic analysis indicates that the duplication event that produced the paralogs occurred prior to the radiation of the Caenorhabditis species included in the analysis, allowing a long period for the paralogs to diverge in function. Furthermore, we observed that knockout mutations in both genes, either alone or together, have little effect on sperm function. However, hermaphrodites harboring both knockout mutations combined with a third mutation in the him-8 gene are nearly self-sterile due to a sperm defect, even though they have numerous apparently normal sperm within their spermathecae. We suggest that the sperm in these triple mutants are defective in fusing with oocytes, and that the effect of the him-8 mutation is due to its role in chromatin remodeling.

5646: Ca2+ signaling driving pacemaker activity in submucosal interstitial cells of Cajal in the colon
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Posted 26 Oct 2020

Ca2+ signaling driving pacemaker activity in submucosal interstitial cells of Cajal in the colon
88 downloads bioRxiv cell biology

Sal Baker, Wesley A Leigh, Inigo F De Yturriaga, Sean M Ward, Caroline A Cobine, Bernard T Drumm, Kent Sanders

Interstitial cells of Cajal (ICC) generate pacemaker activity responsible for phasic contractions in colonic segmentation and peristalsis. ICC along the submucosal border (ICC-SM) contributing to mixing and more complex patterns of colonic motility. We show the complex patterns of Ca2+ signaling in ICC-SM and the relationship between ICC-SM Ca2+ transients and activation of SMCs using optogenetic tools. ICC-SM displayed rhythmic firing of Ca2+ transients ~15 cpm and paced adjacent SMCs. The majority of spontaneous activity occurred in regular Ca2+ transients clusters (CTCs) that propagated through the network. CTCs were organized and dependent upon Ca2+ entry through voltage-dependent Ca2+ conductances, L- and T-type Ca2+ channels. Removal of Ca2+ from the external solution abolished CTCs. Ca2+ release mechanisms reduced the duration and amplitude of Ca2+ transients but did not block CTCs. These data reveal how colonic pacemaker ICC-SM exhibit complex Ca2+ firing patterns and drive smooth muscle activity and overall colonic contractions.

5647: Calaxin is essential for the transmission of Ca2+-dependent asymmetric waves in sperm flagella
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Posted 22 Sep 2020

Calaxin is essential for the transmission of Ca2+-dependent asymmetric waves in sperm flagella
87 downloads bioRxiv cell biology

Kogiku Shiba, Shoji A Baba, Eiji Fujiwara, Kazuo Inaba

Regulation of waveform asymmetry in sperm flagella is critical for changes in sperm swimming trajectory as seen during sperm chemotaxis towards eggs. Ca2+ is known as an important regulator of asymmetry in flagellar waveforms. A calcium sensor protein, calaxin, which is associated with the outer arm dynein, plays a key role in the sperm waveform regulation in a Ca2+-dependent manner. However, the molecular mechanism underlying the regulation of asymmetric waves by Ca2+ and calaxin remains unclear. We performed experiments using caged ATP to elucidate the formation and propagation of asymmetric flagellar waves in the sperm of the ascidian Ciona intestinalis . Demembranated sperm cells were suspended in a solution containing caged ATP and reactivated using UV flash photolysis. Initial bends were formed at the base and propagated towards the tip of flagella; however, the bend direction was different between asymmetric and symmetric waves. A calaxin inhibitor, repaglinide, had no effect on initial bend formation, but significantly inhibited the generation of the second flagellar bend in the reverse direction, resulting in the failure of asymmetric wave formation and propagation. These results suggest that calaxin plays a critical role in Ca2+-dependent transmission of flagellar asymmetric waveforms.

5648: Decreased calmodulin recruitment triggers PMCA4 dysfunction and pancreatic injury in cystic fibrosis
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Posted 11 Sep 2020

Decreased calmodulin recruitment triggers PMCA4 dysfunction and pancreatic injury in cystic fibrosis
87 downloads bioRxiv cell biology

Tamara Madácsy, Árpad Varga, Noémi Papp, Barnabás Deák, Bálint Tél, Petra Pallagi, Viktória Szabó, Júlia Fanczal, Zoltan Rakonczay, Zsolt Rázga, Meike Hohwieler, Alexander Kleger, Mike Gray, Péter Hegyi, József Maléth

Exocrine pancreatic damage is a common complication of cystic fibrosis (CF), which can significantly debilitate the quality of life and life expectancy of CF patients. The cystic fibrosis transmembrane conductance regulator (CFTR) has a major role in pancreatic ductal ion secretion, however, it presumably has an influence on intracellular signaling as well. Here we describe in multiple model systems, including iPSC-derived human pancreatic organoids from CF patients, that the activity of PMCA4 is impaired by the decreased expression of CFTR in ductal cells. The regulation of PMCA4, which colocalizes and physically interacts with CFTR on the apical membrane of the ductal cells, is dependent on the calmodulin binding ability of CFTR. Moreover, CFTR seems to be involved in the process of the apical recruitment of calmodulin, which enhances its role in calcium signaling and homeostasis. Sustained intracellular Ca2+ elevation in CFTR KO cells undermined the mitochondrial function and increased apoptosis. Based on these, the prevention of sustained intracellular Ca2+ overload may improve the exocrine pancreatic function and may have a potential therapeutic aspect in CF.

5649: Establishment and characterization of fibroblast cultures derived from a female common hippopotamus (Hippopotamus amphibius) skin biopsy
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Posted 14 Oct 2020

Establishment and characterization of fibroblast cultures derived from a female common hippopotamus (Hippopotamus amphibius) skin biopsy
87 downloads bioRxiv cell biology

Tao Wang, Zelong Li, Jinpu Wei, Dongmin Zheng, Chen Wang, Chang Xu, Wu Chen, Bo Wang

The population decline in the common hippopotamus (Hippopotamus amphibius) has necessitated the preservation of their genetic resources for species conservation and research. Of all actions, cryopreservation of fibroblast cell cultures derived from animal biopsy is considered a simple but efficient means. Nevertheless, preserving viable cell cultures of the common hippopotamus has not been achieved to our knowledge. To this end, we detailed a method to establish fibroblast cell cultures from a female common hippopotamus fetus in this study. By combining the classic tissue explant direct culture and enzymatic digestion methods, we isolated a great number of cells with typical fibroblastic morphology and high viability. Characterization of the fibroblast cultures was carried out using different techniques. In short, neither bacteria/fungi nor mycoplasma was detectable in the cell cultures throughout the study. The population doubling time was 23.9 h according to the growth curve. Karyotyping based on Giemsa staining showed that cultured cells were diploid with 36 chromosomes in all, one pair of which was sex chromosomes. Mitochondrial cytochrome C oxidase subunit I gene sequence of the cultured cells was 99.26% identical with the Hippopotamus amphibius complete mitochondrial DNA sequence registered in GenBank, confirming the cells were derived from a common hippopotamus. Flow cytometry and immunofluorescence staining results revealed that the detected cells were positive for fibroblast markers, S100A4 and Vimentin. In conclusion, we isolated and characterized a new fibroblast cell culture from a common hippopotamus skin sample and the cryopreserved cells could be useful genetic materials for the future research. ### Competing Interest Statement The authors have declared no competing interest.

5650: PP2A-Cdc55 is responsible for mitotic arrest in DNA re-replicating cells in S. cerevisiae
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Posted 27 Aug 2020

PP2A-Cdc55 is responsible for mitotic arrest in DNA re-replicating cells in S. cerevisiae
87 downloads bioRxiv cell biology

Amy E. Ikui, Shoily Khondker

The cell cycle is an ordered process in which cells replicate their DNA in S-phase and divide them into two identical daughter cells in mitosis. DNA replication takes place only once per cell cycle to preserve genome integrity, which is tightly regulated by Cyclin Dependent Kinase (CDK). Formation of the pre-replicative complex, a platform for origin licensing, is inhibited through CDK-dependent phosphorylation. Failure of this control leads to re-licensing, re-replication and DNA damage. Eukaryotic cells have evolved surveillance mechanisms to maintain genome integrity, termed cell cycle checkpoints. It has been shown that the DNA damage checkpoint is activated upon the induction of DNA re-replication and arrests cell cycle in mitosis in S. cerevisiae . In this study, we show that PP2A-Cdc55 is responsible for the metaphase arrest induced by DNA re-replication, leading to dephosphorylation of APC component, Exclusion of Cdc55 from the nucleus bypassed the mitotic arrest and resulted in enhanced cell lethality in re-replicating cells. The metaphase arrest in re-replication cells was retained in the absence of Mad2, a key component of the spindle assembly checkpoint. Moreover, re-replicating cells showed the same rate of DNA damage induction in the presence or absence of Cdc55. These results indicate that PP2A-Cdc55 maintains metaphase arrest upon DNA re-replication and DNA damage through APC inhibition.

5651: Interdomain interactions regulate the localization of a lipid transfer protein at ER-PM contact sites
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Posted 19 Oct 2020

Interdomain interactions regulate the localization of a lipid transfer protein at ER-PM contact sites
87 downloads bioRxiv cell biology

Padinjat Raghu, Harini Krishnan

During phospholipase C-β (PLC-β) signalling in Drosophila photoreceptors, the phosphatidylinositol transfer protein (PITP) RDGB, is required for lipid transfer at endoplasmic reticulum (ER)-plasma membrane (PM) contact sites (MCS). Depletion of RDGB or its mis-localization away from the ER-PM MCS results in multiple defects in photoreceptor function. Previously, the interaction between the FFAT motif of RDGB and the integral ER protein dVAP-A was shown to be essential for accurate localization to ER-PM MCS. Here, we report that the FFAT/dVAP-A interaction alone is insufficient to localize RDGB accurately; this also requires the function of the C-terminal domains, DDHD and LNS2. Mutations in each of these domains results in mis-localization of RDGB leading to loss of function. While the LNS2 domain is necessary, it is not sufficient for the correct localization of RDGB, which also requires the C-terminal DDHD domain. The function of the DDHD domain is mediated through an intramolecular interaction with the LNS2 domain. Thus, interactions between the additional domains in a multi-domain PITP together lead to accurate localization at the MCS and signalling function. ### Competing Interest Statement The authors have declared no competing interest.

5652: Endothelial SOCS3 maintains homeostasis and promotes survival in endotoxemic mice
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Posted 28 Dec 2020

Endothelial SOCS3 maintains homeostasis and promotes survival in endotoxemic mice
87 downloads bioRxiv cell biology

Nina Martino, Ramon Bossardi Ramos, Shuhan Lu, Kara Leyden, Lindsay Tomaszek, Ariel Jaitovich, Peter A. Vincent, Alejandro P. Adam

SOCS3 is the main inhibitor of the JAK/STAT3 pathway. This pathway is activated by interleukin 6 (IL-6), a major mediator of the cytokine storm during shock. To determine its role in the vascular response to shock, we challenged mice lacking SOCS3 in the adult endothelium (SOCS3iEKO) with a non-lethal dose of lipopolysaccharide (LPS). SOCS3iEKO mice died 16-24 hours post-injection after severe kidney failure. Loss of SOCS3 led to an LPS-induced type I interferon-like program, and high expression of pro-thrombotic and pro-adhesive genes. Consistently, we observed intraluminal leukocyte adhesion and NETosis, as well as retinal venular leukoembolization. Notably, heterozygous mice displayed an intermediate phenotype, suggesting a gene dose effect. In vitro studies were performed to study the role of SOCS3 protein levels in the regulation of the inflammatory response. In HUVEC, pulse-chase experiments showed that SOCS3 protein has a half-life below 20 minutes. Inhibition of SOCS3 ubiquitination and proteasomal degradation leads to protein accumulation and a stronger inhibition of IL-6 signaling and barrier function loss. Together, our data demonstrates that the regulation of SOCS3 protein levels is critical to inhibit IL-6-mediated endotheliopathy during shock and provides a promising new therapeutic avenue to prevent MODS though stabilization of endothelial SOCS3. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/424586v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@17b0733org.highwire.dtl.DTLVardef@15d223org.highwire.dtl.DTLVardef@971fe1org.highwire.dtl.DTLVardef@1ae2ba7_HPS_FORMAT_FIGEXP M_FIG C_FIG

5653: Artificial Intelligence Approaches to Assessing Primary Cilia
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Posted 04 Feb 2021

Artificial Intelligence Approaches to Assessing Primary Cilia
87 downloads bioRxiv cell biology

Ruchi Bansal, Staci E. Engle, Tisianna K. Kamba, Kathryn Brewer, Wesley R. Lewis, Nicolas F. Berbari

Cilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. Cilia length is highly conserved, tightly regulated, and varies between different cell types and tissues and has been implicated in directly impacting their signaling capacity. For example, cilia have been shown to alter their lengths in response to activation of ciliary G protein-coupled receptors. However, accurately and reproducibly measuring the lengths of numerous cilia is a time-consuming and labor-intensive procedure. Current approaches are also error and bias prone. Artificial intelligence (Ai) programs can be utilized to overcome many of these challenges due to capabilities that permit assimilation, manipulation, and optimization of extensive data sets. Here, we demonstrate that an Ai module can be trained to recognize neuronal cilia in images from both in vivo and in vitro samples. After using our trained Ai to identify cilia, we are able to design and rapidly utilize applications that analyze hundreds of cilia in a single sample for length, fluorescence intensity and co-localization. This unbiased approach increased our confidence and rigor when comparing samples from different primary neuronal preps in vitro as well as across different brain regions within an animal and between animals. Moreover, this technique can be used to reliably analyze cilia dynamics from any cell type and tissue in a high-throughput manner across multiple samples and treatment groups. Ultimately, Ai-based approaches will likely become standard as most fields move toward less biased and more reproducible approaches for image acquisition and analysis.

5654: SiCEP3, a C-terminally encoded peptides from Setaria italica, promotes ABA import and signaling pathway
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Posted 31 Jan 2021

SiCEP3, a C-terminally encoded peptides from Setaria italica, promotes ABA import and signaling pathway
87 downloads bioRxiv cell biology

Chang Ai Wu, Yue Ren, Yiman Wan, Qian Xu, Shizhong Zhang, Guodong Yang, Jingguang Huang, Kang Yan, Cheng-Chao Zheng, Lei Zhang

C-terminally encoded peptides (CEPs) are small peptides, typically post-translationally modified, and highly conserved in many species. CEPs are known to play roles in inhibition of plant growth and regulation of development, but the mechanisms are not well understood. In this study, we searched for CEP peptides in foxtail millet (Setaria italica). The 14 peptides we identified are divided into two subfamilies. The transcripts of most SiCEPs were more abundant in roots than in other tissues. SiCEP3, SiCEP4, and SiCEP5 were also expressed at high levels in panicles. Moreover, expression of all SiCEPs was induced by biotic stress and phytohormones. SiCEP3 overexpression and application of biosynthetic SiCEP3 both inhibited the growth of seedlings. In the presence of ABA, growth inhibition and ABA content of seedlings increased with the concentration of SiCEP3. Transcripts encoding two ABA transporters and one ABA receptor were induced by SiCEP3, ABA, and the two in combination. Further analysis revealed that SiCEP3 promoted ABA transport via NRT1.2 and ABCG40. In addition, SiCEP3, ABA, or the combination inhibited the kinase activities of CEP receptors CEPR1/2. Taken together, our results indicated that the CEP-CEPR module mediates ABA signaling by regulating ABCG40, NRT1.2, and PYL4 in planta.

5655: Papillary And Reticular Fibroblasts Generate Distinct Microenvironments That Differentially Impact Angiogenesis
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Posted 29 Nov 2020

Papillary And Reticular Fibroblasts Generate Distinct Microenvironments That Differentially Impact Angiogenesis
87 downloads bioRxiv cell biology

Adele Mauroux, Pauline Joncour, Benjamin Gillet, Sandrine Hugues, Corinne Ardidie-Robouant, Laetitia Marchand, Athanasia Liabotis, Philippe Mailly, Catherine Monnot, Stephane Germain, Sylvie Bordes, Brigitte Closs, Florence Ruggiero, Laurent Muller

Papillary and reticular dermis show distinct extracellular matrix (ECM) and vascularization, and fibroblasts isolated from these compartments have different gene expression patterns and behaviour in vitro. However, due to lack of relevant models, the contribution of skin fibroblast sub-populations to vascularization remains unknown. We thus cultured human papillary and reticular fibroblasts as cell sheets. Differential transcriptomic analysis was performed by RNA sequencing to characterize their microenvironment. Bioinformatic analysis revealed that each fibroblast population expressed specific angiogenesis and matrisome gene expression signatures resulting in specific ECM that differed both in composition and structure. The impact of secreted and ECM-bound factors was then assessed using 3D angiogenesis assays. When co-cultivated with endothelial cells, the papillary and reticular microenvironments induced the formation of distinct capillary networks mimicking the characteristics of vasculature of native dermis subcompartments (vessel diameter and density, number of branch points). Whereas conditioned media of papillary fibroblasts displayed intrinsic high angiogenic potential, reticular ones only contributed to capillary formation induced by exogenous VEGF. These results show that skin fibroblast populations regulate angiogenesis via both secreted and ECM-bound factors. Our work emphasizes the importance of papillary and reticular fibroblasts, not only for modelling dermis microenvironment but also for its vascularization.

5656: ACVR1R206H increases osteogenic/ECM gene expression and impairs myofiber formation in human skeletal muscle stem cells
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Posted 19 Jan 2021

ACVR1R206H increases osteogenic/ECM gene expression and impairs myofiber formation in human skeletal muscle stem cells
86 downloads bioRxiv cell biology

Emilie Barruet, Steven M Garcia, Jake Wu, Blanca M Morales, Stanley Tamaki, Tania Moody, Jason H Pomerantz, Edward C Hsiao

Abnormalities in skeletal muscle repair lead to poor function and complications such as scarring or heterotopic ossification (HO). Here, we use fibrodysplasia ossificans progressiva (FOP), a disease of progressive HO caused by ACVR1R206H (Activin receptor type-1 receptor) mutation, to elucidate how ACVR1 affects skeletal muscle repair. Rare and unique primary FOP human muscle stem cells (Hu-MuSCs) isolated from cadaveric skeletal muscle demonstrated increased ECM marker expression, and showed skeletal muscle-specific impaired engraftment and regeneration ability. Human induced pluripotent stem cell (iPSC)-derived muscle stem/progenitor cells (iMPCs) Single cell transcriptome analyses from FOP also revealed unusually increased ECM and osteogenic marker expression compared to control iMPCs. These results show that iMPCs can recapitulate many aspects of Hu-MuSCs for detailed in vitro study, that ACVR1 is a key regulator of Hu-MuSC function and skeletal muscle repair; and that ACVR1 activation in iMPCs or Hu-MuSCs contributes to HO by changing the local tissue environment.

5657: STAT3 inhibits Myocardin induced cardiac hypertrophy
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Posted 22 Nov 2020

STAT3 inhibits Myocardin induced cardiac hypertrophy
86 downloads bioRxiv cell biology

Xing-Hua Liao, Yuan Xiang, Jia-Peng Li, Hui Li, You Huang, Chao Shen, Zhang zhang, Tong-Cun Zhang

Background: In order to explore the molecular mechanism of cardiomyocyte-dependent myocardial gene expression and cardiomyocyte differentiation in cardiac hypertrophy, and to provide new insights for cardiac hypertrophy. Methods: Cardiac myocytes were isolated from day 1-3 Sprague-Dawley rat pups. Real time quantitative PCR, western blot and immunocytochemistry Assay were used to detect the expression and localization of related genes. CO-IP was used to detect direct protein interactions between Myocardin and STAT3. Luciferase reporter assay and chromatin immunoprecipitation were used to detect the binding of Myocardin to the promoter of a downstream target gene. Microinjection of zebrafish embryos was used to examine the effects of STAT3 and Myocardin interactions on cardiac development in vivo. Results: The N-terminus of STAT3 directly binds to the basic domain of myocardin and inhibits the transcriptional activity of Myocardin-mediated cardiac-specific genes ANF and -actinin, thereby inhibiting their expression, and further inhibit myocardin-mediated cardiac hypertrophy in vivo. Conclusions: In summary, our report states that signal transduction and transcriptional activation factor 3 (STAT3) are inhibitors of the major cardiac hypertrophic transcription factor Myocardiin, which is required for cardiomyocyte differentiation. The STAT3-cardiacin interaction identified nuclear hormone receptor-mediated and cardiac-specific gene-regulated convergence sites and suggested a possible mechanism for cardioprotective effects.

5658: EFFECT OF ACUTE LOW OXYGEN EXPOSURE ON THE PROLIFERATION RATE, VIABILITY, AND GENE EXPRESSION OF C2C12 MYOBLASTS IN VITRO
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Posted 09 Jul 2020

EFFECT OF ACUTE LOW OXYGEN EXPOSURE ON THE PROLIFERATION RATE, VIABILITY, AND GENE EXPRESSION OF C2C12 MYOBLASTS IN VITRO
86 downloads bioRxiv cell biology

Jack V Sharkey

INTRODUCTION: Changes in the oxygen concentration of cellular microenvironments play a significant role in regulating cell function during muscle regeneration. Generally, most in-vitro cell culture experiments have been carried out in atmospheric conditions with 21% O2, which compared to the actual micro-environment of mature skeletal muscle of between 1% and 10% pO2 is extremely hyperoxic (Li et al, 2007). Culturing skeletal muscle cells in vitro within their typical physiologically hypoxic environment in situ (2-10% pO2) has been shown to increase proliferation rate, reduce apoptosis and increase multiple MRF gene expressions, compared to culturing in a normoxic environment (21% O2). However, chronic exposure (>24 hr) to a semi-severe hypoxic environment (<5% O2) can lead to a decrease in cell proliferation and differentiation (Chakravarthy et al, 2001). The effects of acute hypoxic exposure (24 h) has limited research and could be important in understanding the effects of hypoxia on skeletal muscle during brief exposures such as those observed within intermittent hypoxic training programmes. The purpose of this work was to examine the role of acute hypoxia (24 h) on C2C12 proliferation and relevant gene expression in 2D culture. METHODS: C2C12 mouse myoblast cells were seeded into six well plates. The cells were maintained in DMEM with 20% FCS. C2C12 myoblasts were either exposed to 21% or 5% O2. The effects of acute hypoxic exposure (24hours) at different time points during the proliferative phase of myogenesis, rather than chronic exposure, on cell proliferation, cellular viability and myogenic regulatory factor gene expression was examined. At 24, 48, 72 and 96 hours RNA was extracted using the Trizol method and mRNA expression of myogenic regulatory factors, myoD, myf5 and myogenin were detected using the 2-ΔΔCT method. Cell counts and cell viability were also quantified RESULTS: No significant difference was found between cells cultured in normoxic conditions (21%) and those that were exposed to low oxygen conditions for 24hours at various time points over a 96 hour culture period, with regards to proliferation rate, cell viability, and myogenic regulatory gene expression (Myf5, MyoD and Myogenin). DISCUSSION: The effect of acute low oxygen exposure lasting 24hours appears to not be insufficient in having an effect on the proliferation rate, viability or transcription factor expression of C2C12 cells during the proliferative phase of myogenesis. ### Competing Interest Statement The authors have declared no competing interest.

5659: SMAD2 promotes myogenin expression and terminal myogenic differentiation.
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Posted 29 Jul 2020

SMAD2 promotes myogenin expression and terminal myogenic differentiation.
86 downloads bioRxiv cell biology

Émilie Lamarche, Hamood AlSudais, Rashida Rajgara, Dechen Fu, Saadeddine Omaiche, Nadine Wiper-Bergeron

SMAD2 is a transcription factor whose activity is regulated by members of the Transforming Growth Factor beta (TGFβ) superfamily. While activation of SMAD2 and SMAD3 downstream of TGFβ or myostatin signaling is known to inhibit myogenesis, we find that SMAD2 in the absence of TGFβ signaling promotes terminal myogenic differentiation. We find that during myogenic differentiation, SMAD2 expression is induced. Knockout of SMAD2 expression in primary myoblasts did not affect the efficiency of myogenic differentiation but produced smaller myotubes with reduced expression of the terminal differentiation marker myogenin. Conversely, overexpression of SMAD2 stimulated myogenin expression, and enhanced both differentiation and fusion, and these effects were independent of classical activation by the TGFβ receptor complex. Loss of Smad2 in muscle satellite cells in vivo resulted in decreased muscle fiber caliber and impaired regeneration after acute injury. Taken together, we demonstrate that SMAD2 is an important positive regulator of myogenic differentiation, in part through the regulation of Myog.

5660: l-DOPA stimulates the dopaminergic phenotype in human retina
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Posted 14 Oct 2020

l-DOPA stimulates the dopaminergic phenotype in human retina
86 downloads bioRxiv cell biology

Bojana Radojevic, Margarita Mauro-Herrera, Lea D Bennett

Retinal organoids derived from inducible pluripotent stem cells were used to gain insight into the role of l-DOPA during human retinal development. Dopaminergic gene expression was indicated by assessing two dopamine receptors ( DRD1 and DRD2 ), DOPA decarboxylase ( DDC ), and tyrosine hydroxylase ( TH ) via quantitative reverse transcription-polymerase chain reaction at various developmental stages. TH transcript levels started to express around day (D) 42, reached maximal expression ~D63 and then decreased thereafter. At D29, proliferating retinal progenitors expressed DRD1, DRD2, and DDC at various levels of mRNA throughout the day. In the presence of l-DOPA, D29 retinal organoids expressed DRD1 but DRD2 mRNA expression was suppressed. Additionally, l-DOPA upregulated TH mRNA prior to dopaminergic amacrine cell (DAC) development. After the appearance of DACs, l-DOPA phase shifted expression of DRD2 and synchronized mRNA expression of DDC, DRD2, and TH . The present results suggest unique mechanisms for DA signaling at different stages of development in the human retina. ### Competing Interest Statement The authors have declared no competing interest.

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