Biology subjects

Lehmann, M.

Publications and source records attributed to Lehmann, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Repeated cigarette smoke exposure attenuates the proliferation rate of primary human lung fibroblasts in vitro

BackgroundLung fibroblasts are crucial structural cells involved in airway remodelling and tissue maintenance. The study aimed to analyze the effect of cigarette smoke extract (CSE) on primary human lung fibroblasts at various stages of proliferation to assess whether an initial exposure could exert a protective effect against subsequent exposures. MethodsWe examined the effect of CSE on fibroblasts isolated from 15 donors who had undergone lung surgery. Fibroblast cultures were exposed to 2% CSE early (day 5-7), late (day 9-11), or at both time points (double). Cell counts and viability tests were performed every 2 days. Changes in proliferation rates were determined immediately at the end of each 2-day exposure as well as 2 days afterwards, until the end of the experiment with a plateau of proliferation. Differences between exposures were analyzed by comparing proliferation rates by ANOVA with adjusted post-hoc analyses. ResultsEarly-, late- and double-CSE exposed cultures showed lower proliferation rates (p<0.05 each) compared to unexposed cultures. The response to late-CSE exposure varied depending on whether an initial CSE exposure had occurred, suggesting a protective effect from the early exposure. Changes observed over the 2-day exposure period and the subsequent 2 days (a total of 4 days) yielded similar results to those recorded during just the 2-day exposure period. Proliferation rates varied among cells from different donors, but responses correlated across different exposure regimens, indicating inherent differences in susceptibility to CSE. ConclusionsRepeated CSE exposures attenuated the proliferation of primary human lung fibroblasts more strongly than a single exposure, despite the fact that an early CSE exposure appeared to protect against the CSE in repetitively exposed fibroblast cultures. Our results highlight that while significant differences may occur among primary cells from different donors in vitro, responses can remain consistent across various exposure conditions.

cell biology↗

bioRxiv2024

New therapeutic combination to enhance endocytosis of antibodies and nucleic-acid aptamers targeting EGFR in glioblastoma cells

Active targeting is based on the binding of ligands to receptors present on the surface of targeted cells, in order to promote the internalization of the drugs conjugated to the ligands. Several conjugates are already in use or under development for active targeting of tumors, the most widely known being antibody-drug conjugates (ADC). They combine the specificity of monoclonal antibodies with the cytotoxicity of chemotherapeutic molecules. Other than antibodies, nucleic-acid aptamers, are promising ligands to deliver conjugated drugs by active targeting in tumor cells. The therapeutic efficacy of conjugates largely depends on their endocytosis and vesicular trafficking. However, so far, no therapeutic approach to enhance endocytosis of conjugates is available. In recent studies, we showed that gefitinib, a tyrosine kinase inhibitor directed against the epidermal growth factor receptor EGFR, induces a massive, non-physiological endocytosis of EGFR, known as gefitinib-mediated endocytosis (GME), in different glioblastoma cell lines. We thus hypothesized that besides promoting endocytosis of EGFR, gefitinib could also promote endocytosis of its ligands. In this study, we proved by quantitative fluorescence bioimaging, that gefitinib is indeed able to strengthen the endocytosis of fluorophore-conjugated EGFR-specific antibodies and aptamers. We also showed that the GME potentiates the toxicity of an antibody-drug conjugate, even at low concentrations. Our results suggest the development of a new therapeutic combination, of ADC and gefitinib, to potentiate the delivery of ADC and likely other conjugates targeting EGFR in glioblastoma, while limiting side effects on non-targeted cells.

cell biology↗

bioRxiv2024

Endogenous tagging of Munc13-1 with a SNAP tag as a tool for monitoring synapse nanoarchitecture

ABSTRACTSynaptic function is governed by highly regulated protein machineries, whose abundance and spatial localization change continually. Studies to determine the dynamic changes in synaptic proteins nanoarchitecture typically rely on immunolabeling, or on the expression of fluorescent proteins. The former employs chemical fluorophores and signal amplification, but requires fixation. The latter enables monitoring of proteins in live microscopy, but uses suboptimal fluorophores. Self-labeling tags have been introduced to combine the advantages of these two approaches, and here we introduce a knock-in mouse line where the essential presynaptic protein Munc13-1 is endogenously fused to the self-labeling SNAP tag. We demonstrate efficient Munc13-1-SNAP labeling in fixed neurons and brain sections by various SNAP dyes, as well as by a novel far-red and cell impermeable compound, SBG-SiR-d12. We introduce and characterize SBG-SiR-d12 here as a highly-efficient dye for SNAP-tag labeling of extracellular epitopes, and of intracellular proteins in fixed and permeabilized tissue. Finally, we show that Munc13-1-SNAP can be labeled in living neurons and monitored through live-cell imaging using confocal- and super resolution microscopy. We conclude that the Unc13aSNAP mouse line is a useful tool for the analysis of presynaptic nanoarchitectural dynamics, with a potential for wide adoption.

neuroscience↗

bioRxiv2024

Megakaryocytes build a cage of extracellular matrix that controls their maturation and anchoring to the vascular niche

Megakaryocytes, the progenitor cells of blood platelets, play a crucial role in hemostasis by residing in the bone marrow and ensuring continuous platelet production. Unlike other hematopoietic cells, megakaryocytes do not enter the blood circulation intact. They remain anchored within the bone marrow while extending cytoplasmic protrusions called proplatelets through the sinusoidal endothelial barrier. These proplatelets subsequently fragment into functional platelets. This unique process of intravasation facilitates efficient platelet production while maintaining the megakaryocyte cell body within the bone marrow niche, thus preventing potential thrombotic complications. How the extracellular matrix (ECM) influences the delicate balance between megakaryocyte retention and proplatelet extension remains largely unknown. Here, we investigate the spatial organization and functional role of ECM components in the megakaryocyte vascular niche. Our findings reveal that laminin and collagen IV form three-dimensional (3D) ECM cages encompassing megakaryocytes and anchor them to the sinusoidal basement membrane. Gene deletion shows the existence of laminin 4 in the ECM cage that is necessary to maintain megakaryocyte-sinusoid interactions. Notably, megakaryocytes actively contribute to the ECM cage assembly; {beta}1/{beta}3 integrin knockout weakens these structures, increasing intravasation and entire megakaryocyte entry into circulation. The retention of megakaryocytes by these 3D ECM cages depends on dynamic remodeling processes. Inhibition of ECM proteolysis results in denser cage formation, increasing the frequence of immature megakaryocytes with impaired demarcation membrane system (DMS) development. Thus, the ECM cage represents a novel concept of an active and dynamic 3D microenvironment that is continuously remodeled and essential for maintaining megakaryocyte perivascular positioning. This specific microarchitecture guides megakaryocyte maturation and intravasation, underscoring the critical role of ECM microarchitecture and dynamics in megakaryocyte function. Key PointsO_LIMegakaryocytes form a three-dimensional (3D) cage composed of laminin and collagen IV connected to the basement membrane surrounding them. This microarchitecture stabilizes megakaryocytes within their vascular niche. C_LIO_LI{beta}1/{beta}3 integrins and MMP are key ECM cage regulators that assist megakaryocyte maturation and intravasation at the bone marrow-blood interface. C_LI

physiology↗

bioRxiv2024

A one-step protocol to generate impermeable fluorescent HaloTag substrates for in situ live cell application and super-resolution imaging

Communication between cells is largely orchestrated by proteins on the cell surface, which allow information transfer across the cell membrane. Super-resolution and single-molecule visualization of these proteins can be achieved by genetically grafting HTP (HaloTag Protein) into the protein of interest followed by brief incubation of cells with a dye-HTL (dye-linked HaloTag Ligand). This approach allows for use of cutting-edge fluorophores optimized for specific optical techniques or a cell-impermeable dye-HTL to selectively label surface proteins without labeling intracellular copies. However, these two goals often conflict, as many high-performing dyes exhibit membrane permeability. Traditional methods to eliminate cell permeability face synthetic bottlenecks and risk altering photophysical properties. Here we report that dye-HTL reagents can be made cell-impermeable by inserting a charged sulfonate directly into the HTL, leaving the dye moiety unperturbed. This simple, one-step method requires no purification and is compatible with both the original HTL and second-generation HTL.2, the latter offering accelerated labeling. We validate such compounds, termed dye-SHTL ( dye shuttle) conjugates, in live cells via widefield microscopy, demonstrating exclusive membrane staining of extracellular HTP fusion proteins. In transduced primary hippocampal neurons, we label mGluR2, a neuromodulatory G protein-coupled receptor (GPCR), with dyes optimized for stimulated emission by depletion (STED) super-resolution microscopy, allowing unprecedented accuracy in distinguishing surface and receptors from those in internal compartments of the presynaptic terminal, important in neural communication. This approach offers broad utility for surface-specific protein labelling.

pharmacology and toxicology↗

bioRxiv2024

Integrative Molecular Dynamics Simulations Untangle Cross-Linking Data to Unveil Mitochondrial Protein Distributions

Cross-linking mass spectrometry (XL-MS) enables the mapping of protein-protein interactions on the cellular level. When applied to all compartments of mitochondria, the sheer number of cross-links and connections can be over-whelming, rendering simple cluster analyses convoluted and uninformative. To address this limitation, we integrate the XL-MS data, 3D electron microscopy data, and localization annotations with a supra coarse-grained molecular dynamics simulation to sort all data, making clusters more accessible and interpretable. In the context of mitochondria, this method, through a total of 6.9 milliseconds of simulations, successfully identifies known, suggests unknown protein clusters, and reveals the distribution of inner mitochondrial membrane proteins allowing a more precise localization within compartments. Our integrative approach suggests, that two so-far ambigiously placed proteins FAM162A and TMEM126A are localized in the cristae, which is validated through super resolution microscopy. Together, this demonstrates the strong potential of the presented approach. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/612425v2_ufig1.gif" ALT="Figure 1"> View larger version (74K): org.highwire.dtl.DTLVardef@1a3236dorg.highwire.dtl.DTLVardef@16ad120org.highwire.dtl.DTLVardef@1c79bfdorg.highwire.dtl.DTLVardef@1d20e51_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOEntry for the Table of ContentsC_FLOATNO The approach combines cross-linking mass spectrometry, electron microscopy, and coarse-grained molecular dynamics simulations to map protein-protein interactions in mitochondria. This integration makes complex data clusters accessible, revealing protein distributions and localizations. Notably, it allows the localization of FAM162A and TMEM126A through super-resolution microscopy, showcasing the synergy of experimental and computational methods. C_FIG

biophysics↗

bioRxiv2024

Morphological Profiling Dataset of EU-OPENSCREEN Bioactive Compounds Over Multiple Imaging Sites and Cell Lines

Morphological profiling with the Cell Painting assay has emerged as a promising method in drug discovery research. The assay captures morphological changes across various cellular compartments enabling the rapid identification of the effect of compounds. We present a comprehensive morphological profiling dataset using the carefully curated and well-annotated EU-OPENSCREEN Bioactive Compound Set. Our profiling dataset was generated across multiple imaging sites with high-throughput confocal microscopes using the Hep G2 as well as the U2 OS cell line. We employed an extensive assay optimization process to achieve high data quality across the different imaging sites. An analysis of the four replicates validates the robustness of the generated data. We compare morphological features of the different cell lines and map the profiles to activity, toxicity, and basic compound targets to further describe the dataset as well as to demonstrate the potential of this dataset to be used for mechanism of action exploration.

cell biology↗

bioRxiv2024

Flavonoid metabolism and its different physiological roles in plant cold acclimation

Flavonoids represent a diverse group of plant secondary metabolites which are also discussed in context of dietary health and inflammatory response. Numerous studies have revealed that flavonoids play a central role in plant acclimation to abiotic factors like low temperature or high light, but their structural and functional diversity frequently prevents a detailed mechanistic understanding. Further complexity in analysing flavonoid metabolism arises from the different subcellular compartments which are involved in biosynthesis and storage. In the present study, non-aqueous fractionation of Arabidopsis leaf tissue was combined with metabolomics and proteomics analysis to reveal subcellular regulation during cold acclimation in two flavonoid-deficient mutants. During the first three days of a two-week cold acclimation period, flavonoid deficiency was observed to affect pyruvate, citrate and glutamate metabolism which indicated a role in stabilising C/N metabolism and photosynthesis. Also, tetrahydrofolate metabolism was found to be affected which had significant effects on regulation of the proteome of the photorespiratory pathway. In the late stage of cold acclimation, flavonoid deficiency was found to affect protein stability, folding and proteasomal degradation which resulted in a significant decrease of total protein amounts in both mutants. In summary, these findings suggest that flavonoid metabolism plays different roles in early and late stages of plant cold acclimation and significantly contributes to establishing a new protein homeostasis in a changing environment.

plant biology↗

bioRxiv2024

Septin-associated PIPKIγ splice variants drive centralspindlin association with the midbody via PI(4,5)P2

Mammalian cytokinesis critically depends on the phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] which serves as docking site for crucial components of the cytokinetic machinery at the plasma membrane. PI(4,5)P2 supports several stages of cytokinesis, including actomyosin ring assembly and constriction, membrane tethering of spindle microtubules, and midbody organization. How these various activities of PI(4,5)P2 and the underlying mechanisms of local PI(4,5)P2 synthesis are orchestrated in space and time has remained elusive. Here, we identify a pivotal role of septin-binding splice variants of PIPKI{gamma} that couple nanoscale PI(4,5)P2 synthesis at the ingressing cleavage furrow to late midbody formation. Depletion of PIPKI{gamma} isoforms causes multinucleation and perturbs anillin and septin deposition at the intercellular bridge and at the midbody. These defects are rescued by wild-type kinase, but not by septin binding-deficient or catalytically inactive PIPKI{gamma} variants. We further show that both, septins and PIPKI{gamma} form a complex with centralspindlin, and thereby facilitate the recruitment of centralspindlin to the midbody. Taken together, our findings establish septin-associated PIPKI{gamma} isoforms as key regulators of midbody organization that act through generating a local pool of PI4,5P2 required for centralspindlin recruitment and maintenance at the midbody, and for septin association with microtubules.

cell biology↗

bioRxiv2023

Claudin-4 polymerizes after the incorporation of just two extracellular claudin-3 residues

Tight junctions play a pivotal role in the functional integrity of the human body by forming barriers crucial for tissue compartmentalization and protecting the body from external threats. Essential components of tight junctions are the transmembrane claudin proteins, which can polymerize into tight junction strands and meshworks. This study delves into the structural determinants of claudin polymerization, utilizing the close homology yet strong difference in polymerization capacity between claudin-3 and claudin-4. Through a combination of sequence alignment and structural modeling, critical residues in the second extracellular segment are pinpointed. Molecular dynamics simulations provide insights into the interactions of and the conformational changes induced by the identified extracellular segment 2 residues, shedding light on the intricacies of claudin polymerization. Live-STED imaging demonstrates that introduction of these residues from claudin-3 into claudin-4 significantly enhances polymerization in non-epithelial cells. In tight junction-deficient epithelial cells, mutated claudin-4 not only influences tight junction morphology but also partially restores barrier function. Understanding the structural basis of claudin polymerization is of paramount importance, as it offers insights into the dynamic nature of tight junctions. This knowledge could be applied to targeted therapeutic interventions, offering insight to repair or prevent barrier defects associated with pathological conditions, or introduce temporary barrier openings during drug delivery.

cell biology↗

bioRxiv2023

Ergosterol mediates aggregation of natamycin in the yeast plasma membrane

Polyene macrolides are antifungal substances, which interact with cells in a sterol-dependent manner. While being widely used, their mode of action is poorly understood. Here, we employ ultraviolet-sensitive (UV) microscopy to show that the antifungal polyene natamycin binds to the yeast plasma membrane (PM) and causes permeation of propidium iodide into cells. Right before membrane permeability becomes compromised, we observed clustering of natamycin in the PM that was independent of PM protein domains. Aggregation of natamycin was paralleled by cell deformation and membrane blebbing as revealed by soft X-ray microscopy. Substituting ergosterol for cholesterol decreased natamycin binding and resulted in reduced clustering of natamycin in the PM. Blocking of ergosterol synthesis necessitates sterol import via the ABC transporters Aus1/Pdr11 to ensure natamycin binding. Quantitative imaging of dehydroergosterol (DHE) and cholestatrienol (CTL), two analogs of ergosterol and cholesterol, respectively, revealed a largely homogeneous lateral sterol distribution in the PM, ruling out that natamycin binds to pre-assembled sterol domains. Depletion of sphingolipids using myriocin increased natamycin binding to yeast cells, likely by increasing the ergosterol fraction in the outer PM leaflet. We conclude that ergosterol-specific aggregation of natamycin in the yeast PM underlies its antifungal activity, which can be synergistically enhanced by inhibitors of sphingolipid synthesis. SignificanceErgosterol is the major sterol in the membranes of fungi and a major target for antifungal treatments. Polyene macrolides, such as natamycin, are known to target ergosterol but the underlying mechanisms for their preference for this yeast sterol compared to mammalian cholesterol is not understood. This study shows that natamycin forms aggregates when associated with yeast S. cerevisiae in an ergosterol-dependent manner. Cholesterol can only partially substitute for ergosterol with respect to natamycin binding and aggregation. Membrane-associated aggregation of natamycin is not the result of pre-formed sterol domains in the cell membrane, as we show by direct visualization of minimally modified ergosterol and cholesterol analogs. Inhibiting sphingolipid synthesis increased membrane association and antifungal activity of natamycin, suggesting that targeting sphingolipids in combination with polyene macrolides could lead to novel drug treatment approaches against fungal infections.

biophysics↗

bioRxiv2023

Synthesis and characterization of novel intrinsically fluorescent analogs of cholesterol with improved photophysical properties.

Live-cell imaging of cholesterol trafficking depends on suitable cholesterol analogs. However, existing fluorescent analogs of cholesterol either show very different physico-chemical properties compared to cholesterol or demand excitation in the ultraviolet spectral region. We present novel intrinsically fluorescent sterols containing four conjugated double bonds in the ring system and either a hydroxy or a keto group in the C3 position. Synthesis of these probes involves dehydrogenation of 7-dehydrocholesterol using mercury(II) acetate, Swern oxidation/dehydrogenation, and stereoselective Luche reduction. Molecular dynamics simulations and nuclear magnetic resonance spectroscopy reveal that the analog with a 3-hydroxy-group like cholesterol can condense fatty acyl chains and form hydrogen bonds to water molecules at the bilayer interface. The emission of both probes is red-shifted by 80-120 nm compared to the widely used sterol analogs dehydroergosterol or cholestatrienol. This allows for their imaging on conventional microscopes, as we here show in giant unilamellar vesicles. These experiments reveal a preferred partitioning of both sterol probes into the biologically relevant liquid-ordered phase. In conclusion, we present a synthesis strategy leading to novel intrinsically fluorescent sterol probes with close resemblance of cholesterol. Their improved photophysical properties will allow for live-cell imaging of sterol transport in the future.

biophysics↗

bioRxiv2023

Degradation of Fatty Acid Export Protein1 by Rhomboid-Like Protease11 Contributes to Cold Tolerance in Arabidopsis

Plants need to adapt to different stresses to optimize growth under unfavorable conditions. The abundance of the chloroplast envelope located Fatty Acid Export Protein1 (FAX1) decreases after the onset of low temperatures. However, it was unclear how FAX1 degradation occurs and whether altered FAX1 abundance contributes to cold tolerance in plants. The rapid cold-induced increase in rhomboid-like protease11 (RBL11) transcript, the physical interaction of RBL11 with FAX1, the specific FAX1 degradation after RBL11 expression, and the absence of cold-induced FAX1 degradation in rbl11 loss-of-function mutants suggest that this enzyme is responsible for FAX1 degradation. Proteomic analyses showed that rbl11 mutants have higher levels of FAX1 and other proteins involved in membrane lipid homeostasis, suggesting that RBL11 is a key element in the remodeling of membrane properties during cold. Consequently, in the cold, rbl11 mutants show a shift in lipid biosynthesis towards the eukaryotic pathway, which coincides with impaired cold tolerance. To demonstrate that cold sensitivity is due to increased FAX1 levels, FAX1 overexpressors were analyzed. rbl11 and FAX1 overexpressor mutants show superimposable phenotypic defects upon exposure to cold temperatures. Our results show that the cold-induced degradation of FAX1 by RBL11 is critical for Arabidopsis to survive cold and freezing periods. One sentence summaryDegradation of the inner envelope protein Fatty Acid Export1 via Rhomboid Like Protease11 represents a critical process to achieve cold and frost tolerance in Arabidopsis

plant biology↗

bioRxiv2023

UPP affects chloroplast development by interfering with chloroplast proteostasis

Arabidopsis uracil phosphoribosyltransferase (UPP) is an essential enzyme which appears to have a previously unknown, moonlighting activity. Our analysis of UPP amiRNA mutants has confirmed that this vital function is crucial for chloroplast development and growth. Interestingly, this function appears to be unrelated to nucleotide homeostasis since nucleotide levels were not altered in the studied mutants. Transcriptomics and proteomic analysis suggest that UPP plays a role in chloroplast proteostasis, especially under high light (HL). Immunoblots of mature plants and a de-etiolation experiment with young seedlings revealed PetC, the iron-sulfur protein of the cytochrome b6f complex, as a putative UPP target. In addition, UPP and PetC were identified in a high molecular weight complex. Consistently, we show that PetC is massively reduced in UPP knock-down plants. The block observed in photosynthetic electron transport, as evidenced by reduced high light-induced non-photochemical quenching (NPQ) but increased unregulated energy dissipation (NO), might be therefore a consequence of reduced PetC. After HL treatment, UPP amiRNA mutants showed impaired photosynthesis and reduced carbohydrate contents, resulting in an inability to induce flavonoid biosynthesis. In addition, the levels of the osmoprotectants raffinose, proline and fumarate were found to be reduced. Proteases, including thylakoid filamentation temperature-sensitive 1, 5 (FtsH), caseinolytic protease proteolytic subunit 1 (ClpP1), and processing peptidases, as well as components of the chloroplast protein import machinery, were up-regulated. In sum, our work suggests that UPP assists in stabilizing and protection of PetC during the assembly of the Fe-S cluster and targeting to the thylakoid.

molecular biology↗

bioRxiv2023

Reversal of Biological Age in Multiple Rat Organs by Young Porcine Plasma Fraction

Young blood plasma is known to confer beneficial effects on various organs in mice and rats. However, it was not known whether plasma from young pigs rejuvenates old rat tissues at the epigenetic level; whether it alters the epigenetic clock, which is a highly accurate molecular biomarker of aging. To address this question, we developed and validated six different epigenetic clocks for rat tissues that are based on DNA methylation values derived from n=613 tissue samples. As indicated by their respective names, the rat pan-tissue clock can be applied to DNA methylation profiles from all rat tissues, while the rat brain-, liver-, and blood clocks apply to the corresponding tissue types. We also developed two epigenetic clocks that apply to both human and rat tissues by adding n=1366 human tissue samples to the training data. We employed these six rat clocks to investigate the rejuvenation effects of a porcine plasma fraction treatment in different rat tissues. The treatment more than halved the epigenetic ages of blood, heart, and liver tissue. A less pronounced, but statistically significant, rejuvenation effect could be observed in the hypothalamus. The treatment was accompanied by progressive improvement in the function of these organs as ascertained through numerous biochemical/physiological biomarkers and behavioral responses to assess cognitive functions. An immunoglobulin G (IgG) N-glycosylation pattern shift from pro-to anti-inflammatory also indicated reversal of glycan aging. Overall, this study demonstrates that a young porcine plasma-derived treatment markedly reverses aging in rats according to epigenetic clocks, IgG glycans, and other biomarkers of aging.

developmental biology↗

bioRxiv2023

Primary human lung fibroblasts exhibit trigger- but not disease-specific cellular senescence and impair alveolar epithelial cell progenitor function

Aging is the main risk factor for chronic lung diseases including idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). Accordingly, hallmarks of aging such as cellular senescence are increased in different cell types such as fibroblasts in the lungs of these patients. However, whether the senescent phenotype of fibroblasts derived from IPF or COPD differs is still unknown. Therefore, we characterized senescence at baseline and after exposure to disease-relevant insults (H2O2, bleomycin, and TGF-{beta}1) in cultured primary human lung fibroblasts (phLF) from control donors, IPF, or COPD patients. We found that phLF from different disease-origins have a low baseline senescence. H2O2 and bleomycin treatment induced a senescent phenotype in phLF whereas TGF-{beta}1 only had a pro-fibrotic effect. Interestingly, we did not observe any differences in susceptibility to senescence induction in phLF based on disease origin. However, exposure to different stimuli resulted in different senescent programs in phLF. Moreover, senescent phLF reduced colony formation efficiency of alveolar epithelial progenitor cells. In conclusion, the senescent phenotype of phLF is mainly determined by the senescence inducer and impairs alveolar epithelial progenitor capacity in vitro.

cell biology↗

bioRxiv2023

OrgaMapper: A robust and easy-to-use workflow for analyzing organelle positioning

Eukaryotic cells are highly compartmentalized by a variety of organelles that carry out specific cellular processes. The position of these organelles within the cell is elaborately regulated and vital for their function. For instance, the position of lysosomes relative to the nucleus controls their degradative capacity and is altered in pathophysiological conditions. The molecular components orchestrating the precise localization of organelles remain incompletely understood. A confounding factor in these studies is the fact that organelle positioning is surprisingly non-trivial to address. E.g., perturbations that affect the localization of organelles often lead to secondary phenotypes such as changes in cell or organelle size. These phenotypes could potentially mask effects or lead to the identification of false positive hits. To uncover and test potential molecular components at scale, accurate and easy to use analysis tools are required that allow robust measurements of organelle positioning. Here, we present an analysis workflow for the faithful, robust, and quantitative analysis of organelle positioning phenotypes. Our workflow consists of an easy to use Fiji plugin and an R Shiny App. These tools enable users without background in image or data analysis to (1) segment single cells and nuclei and to detect organelles, (2) to measure cell size and the distance between detected organelles and the nucleus, (3) to measure intensities in the organelle channel plus one additional channel, and (4) to plot the results in informative graphs. Using simulated data and immunofluorescent images of cells in which the function of known factors for lysosome positioning has been perturbed, we show that the workflow is robust against common problems for the accurate assessment of organelle positioning such as changes of cell shape and size, organelle size and background.

cell biology↗

bioRxiv2023

COGNITIVE REJUVENATION IN OLD RATS BY HIPPOCAMPAL OSKM GENE THERAPY

Impaired performance in spatial learning and memory during aging in rats is associated with morphological and molecular changes in the brain, particularly in the hippocampus. Here, we assessed the cognitive performance of young (3.5 mo.) untreated rats and old (25.3 mo.) treated and control rats. Treatment was carried out by intrahippocampal injection of an adenovector that carries the GFP reporter gene as well as the 4 Yamanaka genes. Learning and spatial memory performance were assessed by means of the Barnes maze test. The learning performance of the OSKM-treated old rats was significantly improved compared to that of the control old counterparts. A marginal (P=0.06) improvement in the spatial memory was recorded in the treated versus control old rats. OSKM gene expression induced no pathological changes in the brain. The morphology and number of hippocampal cell populations like astrocytes and mature neurons did not show any changes with the treatment in the old rats as compared with the control old counterparts. The rat pan tissue DNAm age marker revealed that old OSKM gene-treated rats show a trend towards a decrease in epigenetic age. The Limma package was used to assess differential methylation by fitting linear models to the methylation data for specific group comparisons. Comparison of differential methylation between old treated and old control hippocampal DNA samples identified 671 differentially methylated CpGs probes (DPMs) in the DNA of OSKM-treated hippocampi (p<0.05). Assessment of the DPMs in old versus young controls revealed the presence of 1,279 hypomethylated CpGs near the promoter regions in young hippocampi (versus old controls) and 914 hypermethylated CpGs near the promoter in young hippocampi compared to old control hippocampi. We found a subset of 174 hypomethylated CpGs in the hippocampal DNA from old OSKM rats and young controls both compared with old control hippocampi. This means that in the hippocampal DNA there is a common set of CpGs which are hypermethylated during aging and are demethylated by the OSKM genes. This observation suggested that in these 174 CpGs the hypermethylation induced by aging is reversed by the demethylation effect of the OSKM genes on the same 174 CpGs. This observation can be interpreted as a rejuvenation effect of the OSKM genes of the old hippocampal methylome. Our results extend to the rat the evidence that viral vector-mediated delivery of the Yamanaka genes in the brain has strong regenerative effects without adverse side effects.

neuroscience↗