bioRxiv - Cell Biology最新文献

{"title":"Influence of formaldehyde on signaling pathways when used in mammalian cell culture","authors":"Katharina Ostmann, Annette Kraegeloh, Wilfried Weber","doi":"10.1101/2024.09.17.613450","DOIUrl":"https://doi.org/10.1101/2024.09.17.613450","url":null,"abstract":"Formaldehyde is the smallest existing aldehyde, a highly reactive color less gas at room temperature and ubiquitously present in our atmosphere. Because of its reactivity leading to the crosslinking of macromolecules like proteins, it is widely used in industrial applications, but also in cell biology in order to preserve cells and tissues for further analysis. In this work, we show that formaldehyde releasing solutions commonly used for fixation of cells, can diffuse via the gas phase to the neighboring well and influence signaling processes in the therein cultured cells. To analyze this effect, we utilized a stable reporter cell line for YAP signaling or a gene expression-based reporter for activation of the NF-kappaB pathway. Especially the stable reporter cell line can also be used as sensor for bioavailable formaldehyde. The observed impact of formaldehyde on cellular signaling underscores the need for careful planning of experimental protocols and emphasizes the importance of implementing proper controls when utilizing this reagent in cellular signaling analyses.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265325","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"MEK1/2 kinases cooperate with c-Myc:MAX to prevent polycomb repression of TERT in human pluripotent stem cells","authors":"Spandana T Kotian, Lindsay F Rizzardi, Josh Lewis Stern","doi":"10.1101/2024.09.16.613267","DOIUrl":"https://doi.org/10.1101/2024.09.16.613267","url":null,"abstract":"Telomerase counteracts telomere shortening, enabling human embryonic stem cells (hESC) to undergo long-term proliferation. MAPK signaling plays a major role in regulating the self-renewal of hESC, and previous studies in induced pluripotent stem cells (iPSC) suggested that expression of TERT, the gene encoding the catalytic subunit of telomerase, relies on MAPK signaling. We examined whether MEK-ERK signaling regulated TERT transcription in a model of normal hESC. Kinase inhibitors of MEK1 and MEK2 (MEKi) or ERK1 and ERK2 (ERKi) significantly repressed TERT mRNA levels. Using chromatin immunoprecipitation (ChIP) we observed that MEKi induced the accumulation of the repressive histone mark histone 3 lysine 27 trimethylation (H3K27me3) at the TERT proximal promoter. This increase corresponded with a loss of histone 3 lysine 27 acetylation (H3K27ac) which is associated with transcriptionally active loci. Inhibition of the polycomb repressive complex 2 (PRC2), which deposits H3K27me3, partially rescued the loss of TERT expression, indicating that MEK1/2 activity can limit PRC2 activity at TERT. Inhibition of MEK/ERK kinases also repressed expression of c-Myc, a transcription factor reported to regulate TERT in other immortalized cells. Consistent with a key role for c-Myc in regulating TERT, low doses of a c-Myc:MAX dimerization inhibitor induced a striking and rapid gain of H3K27me3 at TERT and repressed TERT transcription in hESC. Inhibiting c-Myc:MAX dimerization also resulted in lower MAX recruitment to TERT, suggesting that this complex acts in cis at TERT. Our study using a model of normal human pluripotent stem cells identifies new regulators and mechanisms controlling transcription of an important, developmentally regulated gene involved in telomere protection.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265323","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mid-infrared chemical imaging of living cells enabled by plasmonic metasurfaces","authors":"Steven H. Huang, Po-Ting Shen, Aditya Mahalanabish, Giovanni Sartorello, Gennady Shvets","doi":"10.1101/2024.09.17.613596","DOIUrl":"https://doi.org/10.1101/2024.09.17.613596","url":null,"abstract":"Mid-Infrared (MIR) chemical imaging provides rich chemical information of biological samples in a label-free and non-destructive manner. Yet, its adoption to live-cell analysis is limited by the strong attenuation of MIR light in water, often necessitating cell culture geometries that are incompatible with the prolonged viability of cells and with standard high-throughput workflow. Here, we introduce a new approach to MIR microscopy, where cells are imaged through their localized near-field interaction with a plasmonic metasurface. Chemical contrast of distinct molecular groups provided sub-cellular resolution images of the proteins, lipids, and nucleic acids in the cells that were collected using an inverted MIR microscope. Time-lapse imaging of living cells demonstrated that their behaviors, including motility, viability, and substrate adhesion, can be monitored over extended periods of time using low-power MIR light. The presented approach provides a method for the non-perturbative MIR imaging of living cells, which is well-suited for integration with modern high-throughput screening technologies for the label-free, high-content chemical imaging of living cells.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265197","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Reducing oxidative stress improves ex vivo polymer-based human haematopoietic stem and progenitor cell culture and gene editing","authors":"Yavor Bozhilov, Elizabeth Brown, Ian Hsu, Indranil Singh, Alejo Rodriguez-Fraticelli, Anindita Roy, Satoshi Yamazaki, Adam C Wilkinson","doi":"10.1101/2024.09.17.613552","DOIUrl":"https://doi.org/10.1101/2024.09.17.613552","url":null,"abstract":"Self-renewing multipotent haematopoietic stem cells (HSCs) have the unique capacity to stably regenerate the entire blood and immune systems following transplantation. HSCs are used clinically to reconstitute a healthy blood system in patients suffering from a range of haematological diseases. However, HSCs are very rare and have been challenging to grow ex vivo, which has hampered efforts to collect large numbers of HSCs for both basic research and clinical therapies. Polymer-based culture conditions have recently been developed to support expansion of mouse and human haematopoietic stem and progenitor cells (HSPCs). While mouse HSPCs expanded rapidly in polymer-based cultures, growth speeds for human HSPCs in polymer-based cultures was limited to ~70-fold over 4-weeks. Here we have found that reducing oxidative stress improves human HSPC growth in these conditions. We describe an optimised culture condition that improves growth to 250-1400-fold over 4-weeks through reducing oxygen tension and the addition of antioxidants. These conditions also enable efficient gene editing in these polymer-based cultures. We envision these improved culture conditions will support a range of research into human HSPC biology and provide a platform for clinical-scale HSPC expansion and gene editing.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265199","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The lysosomal membrane protein LAMP2B mediates microlipophagy to target obesity-related disorders","authors":"Ryohei Sakai, Shu Aizawa, Hyeon-Cheol Lee-Okada, Katsunori Hase, Hiromi Fujita, Hisae Kikuchi, Yukiko U. Inoue, Takayoshi Inoue, Chihana Kabuta, Takehiko Yokomizo, Tadafumi Hashimoto, Keiji Wada, Tatsuo Mano, Ikuko Koyama-Honda, Tomohiro Kabuta","doi":"10.1101/2024.09.18.613587","DOIUrl":"https://doi.org/10.1101/2024.09.18.613587","url":null,"abstract":"Lifestyle diseases, such as obesity, diabetes, and metabolic syndrome, are leading health problems, most of which are related to abnormal lipid metabolism. Lysosomes can degrade lipid droplets (LDs) via microautophagy. Here, we report the molecular mechanism and pathophysiological roles of microlipophagy, regulated by the lysosomal membrane protein LAMP2B. Our study revealed that LAMP2B interacts with phosphatidic acid, facilitating lysosomal-LD interactions and enhancing lipid hydrolysis via microlipophagy depending on endosomal sorting complexes required for transport. Correlative light and electron microscopy demonstrated direct LDs uptake into lysosomes at contact sites. Moreover, LAMP2B overexpression in mice prevents high-fat diet-induced obesity, insulin resistance, and adipose tissue inflammation; liver lipidomics analysis suggested enhanced triacylglycerol hydrolysis. Overall, the findings of this study elucidated the mechanism of microlipophagy, which could be promising for the treatment of obesity and related disorders.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265322","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Seipin Regulates Caveolin-1 Trafficking and Organelle Crosstalk","authors":"Maxime Carpentier, Mohyeddine Omrane, Jennica Trager, Mehdi Zouiouich, Rola Shaaban, Xavier Prieur, Marie Palard, Naima El Khallouki, Francesca Giordano, Takeshi Harayama, Corinne Vigouroux, Soazig Le Lay, Abdou Rachid THIAM","doi":"10.1101/2024.09.17.613438","DOIUrl":"https://doi.org/10.1101/2024.09.17.613438","url":null,"abstract":"Caveolin-1 (CAV1), the main structural component of caveolae, is essential in various biological processes, including mechanotransduction, lipid metabolism, and endocytosis. Deregulation of CAV1 dynamics is linked to various pathologies, including cellular senescence, cancer, insulin resistance, and lipodystrophy. However, mechanisms regulating CAV1 trafficking and function remain poorly understood. Here, we show that seipin, a crucial lipid droplet (LD) biogenesis factor, modulates CAV1 trafficking. Deletion of seipin resulted in the accumulation of saturated lipids, leading to ceramide and sphingomyelin overproduction, which disrupted the membrane order of the trans-Golgi network (TGN). In seipin deficiency, CAV1 location to the plasma membrane (PM) was impaired, reducing caveolae. Instead, CAV1 accumulated in TGN and late endosome compartments, which fused with LDs and delivered the protein. In wild-type (WT) cells, this process was minimal but significantly enhanced by treatment with palmitate, ceramide, or Stearoyl-CoA desaturase-1 (SCD1) inhibition. Conversely, in seipin-deficient cells, inhibiting Fatty Acid Synthase (FASN) or overexpressing SCD1 restored CAV1 localization to the PM and reduced its accumulation in LDs. Our findings reveal that seipin controls the funneling of palmitate toward glycerolipids synthesis and storage in LDs versus conversion to ceramides in the ER. This balance is crucial to cellular protein trafficking by controlling the TGN membrane order. Therefore, our study identifies seipin as a critical regulator of cellular lipid metabolism, protein trafficking, and organelle homeostasis. These findings shed light on the processes regulating CAV1 trafficking and show that convergent pathophysiological mechanisms associated with defects in CAV1 and seipin contribute to metabolic disorders, including insulin resistance and lipodystrophies.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265321","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CRISPR screen for rAAV production implicates genes associated with infection","authors":"Emily O'Driscoll, Sakshi Arora, Jonathan Lang, Beverly Davidson, Ophir Shalem","doi":"10.1101/2024.09.17.613356","DOIUrl":"https://doi.org/10.1101/2024.09.17.613356","url":null,"abstract":"Recombinant adeno-associated virus (rAAV) vectors are an effective and well-established tool in the growing gene therapy field, with five approved AAV-mediated gene therapies already on the market and numerous more in clinical trials. However, manufacturing rAAV vectors is an expensive, timely, and labor-intensive process that limits the commercial use of AAV-mediated gene therapies. To address this limitation, we screened producer cells for genes that could be targeted to increase rAAV yield. Specifically, we performed a CRISPR-based genome-wide knockout screen in HEK 293 cells using an antibody specific to intact AAV2 capsids coupled with flow cytometry to identify genes that modulate rAAV production. We discovered that the knockout of a group of heparan-sulfate biosynthesis genes previously implicated in rAAV infectivity decreased rAAV production. Additionally, we identified several vesicular trafficking proteins for which knockout in HEK 293 cells increased rAAV yields. Our findings provide evidence that host proteins associated with viral infection may have also been co-opted for viral assembly and that the genetic makeup of viral producer cells can be manipulated to increase particle yield.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265198","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Select autosomal dominant DFNA11 deafness mutations activate Myo7A in epithelial cells","authors":"Prashun Acharya, Gaima Thapa, Xiayi Liao, Samaneh Matoo, Maura J Graves, Sarah Y Atallah, Ashna K Tipirneni, Tram Nguyen, Niki M Chhabra, Jaden Maschack, Mackenzie R Herod, Favour Ohaezu, Alder Robison, Ashwini Mudaliyar, Jasvinderkaur Bharaj, Nicole Roeser, Katherine Holmes, Vishwaas Nayak, Rayah Alsayed, Benjamin J Perrin, Scott W Crawley","doi":"10.1101/2024.09.17.613491","DOIUrl":"https://doi.org/10.1101/2024.09.17.613491","url":null,"abstract":"Myosin-7A (Myo7A) is a motor protein crucial for the organization and function of stereocilia, specialized actin-rich protrusions on the surface of inner ear hair cells that mediate hearing. Mutations in Myo7A cause several forms of genetic hearing loss, including autosomal dominant DFNA11 deafness. Despite its importance, the structural elements of Myo7A that control its motor activity within cells are not well understood. In this study, we used cultured kidney epithelial cells to screen for mutations that activate the motor-dependent targeting of Myo7A to the tips of apical microvilli on these cells. Our findings reveal that Myo7A is regulated by specific IQ motifs within its lever arm, and that this regulation can function at least partially independent of its tail sequence. Importantly, we demonstrate that many of the DFNA11 deafness mutations reported in patients activate Myo7A targeting, providing a potential explanation for the autosomal dominant genetics of this form of deafness.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265319","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Detailed Colocalization Analysis of A- and B-type Nuclear Lamins: a Workflow Using Super-Resolution STED Microscopy and Deconvolution","authors":"Merel Stiekema, Owen N. Gibson, Rogier J.A. Veltrop, Frans C.S. Ramaekers, Jos L.V. Broers, Marc A.M.J. van Zandvoort","doi":"10.1101/2024.09.17.613415","DOIUrl":"https://doi.org/10.1101/2024.09.17.613415","url":null,"abstract":"The inner nuclear membrane is covered by a filamentous network, the nuclear lamina, consisting of A- and B-type lamins as its major components. The A- and B-type lamins form independent but interacting and partially overlapping networks, as demonstrated by previous super-resolution studies. The nuclear lamina in fibroblast cultures derived from laminopathy patients shows an increased segregation of the A- and B-type lamin networks, which can be quantitatively expressed by the Pearson's Correlation Coefficient (PCC). Blurring and noise (convolution), however, significantly affect the quality of microscopy images, which led us to optimize the deconvolution approach for Confocal Scanning Laser Microscopy (CSLM) and Stimulated Emission Depletion (STED) microscopy images. For that purpose, the differences in using a theoretical, experimental, or semi-experimental Point Spread Function (PSF), an important parameter for deconvolution, was evaluated for its use in deconvolution of CSLM and STED microscopy images of double immunolabeled healthy and laminopathy patient fibroblasts. The semi-experimental is a new PSF introduced in this study, which combines the theoretical and experimental PSF to solve issues that arise from noisy PSF recordings due to very small and thereby low intensity fluorescent beads. From these deconvoluted images, the colocalization of the lamin networks could not only be quantified at the level of the nucleus as a whole, but also at a subnuclear level. The latter was achieved by dividing the nucleus into multiple equal rectangles using a custom-made ImageJ macro in Fiji. In this detailed analysis, we found heterogeneity in the colocalization of lamins A/C and B1 within and between nuclei in both healthy and laminopathy dermal fibroblasts, which cannot be detected in one single analysis for the entire nucleus.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265324","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Afterload-induced Decreases in Fatty Acid Oxidation Develop Independently of Increased Glucose Utilization","authors":"Hande Piristine, Herman I May, Nan Jiang, Daniel Daou, Francisco Olivares-Silva, Abdallah Elnwasany, Pamela Szweda, Caroline Kinter, Michael Kinter, Gaurav Sharma, Xiaodong Wen, Craig R Malloy, Michael E. Jessen, Thomas G. Gillette, Joseph A Hill","doi":"10.1101/2024.09.17.613531","DOIUrl":"https://doi.org/10.1101/2024.09.17.613531","url":null,"abstract":"Background: Metabolic substrate utilization in HFpEF (heart failure with preserved ejection fraction), the leading cause of heart failure worldwide, is pivotal to syndrome pathogenesis and yet remains ill defined. Under resting conditions, oxidation of free fatty acids (FFA) is the predominant energy source of the heart, supporting its unremitting contractile activity. In the context of disease-related stress, however, a shift toward greater reliance on glucose occurs. In the setting of obesity or diabetes, major contributors to HFpEF pathophysiology, the shift in metabolic substrate use toward glucose is impaired, sometimes attributed to the lower oxygen requirement of glucose oxidation versus fat metabolism. This notion, however, has never been tested conclusively. Furthermore, whereas oxygen demand increases in the setting of increased afterload, myocardial oxygen availability remains adequate for fatty acid oxidation (FAO). Therefore, a preference for glucose has been proposed.\u0000Methods and Results: Pyruvate dehydrogenase complex (PDC) is the rate-limiting enzyme linking glycolysis to the TCA cycle. As PDK4 (PDC kinase 4) is up-regulated in HFpEF, we over-expressed PDK4 in cardiomyocytes, ensuring that PDC is phosphorylated and thereby inhibited. This leads to diminished use of pyruvate as energy substrate, mimicking the decline in glucose oxidation in HFpEF. Importantly, distinct from HFpEF-associated obesity, this model positioned us to abrogate the load-induced shift to glucose utilization in the absence of systemic high fat conditions. As expected, PDK4 transgenic mice manifested normal cardiac performance at baseline. However, they manifested a rapid and severe decline in contractile performance when challenged with modest increases in afterload triggered either by L-NAME or surgical transverse aortic constriction (TAC). This decline in function was not accompanied by an exacerbation of the myocardial hypertrophic growth response. Surprisingly, metabolic flux analysis revealed that, after TAC, fractional FAO decreased, even when glucose/pyruvate utilization was clamped at very low levels. Additionally, proteins involved in the transport and oxidation of FFA were paradoxically downregulated after TAC regardless of genotype. Conclusions: These data demonstrate that cardiomyocytes in a setting in which glucose utilization is robustly diminished and prevented from increasing do not compensate for the deficit in glucose utilization by up-regulating FFA use.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265318","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}