ABSTRACT
Familial hypercholesterolemia (FH) is the most common inherited life-threatening disorder of lipid metabolism. Early diagnosis and treatment are the key to reduce the cumulative life-long cardiovascular burden of patients with FH. The high number of LDLR variants described as variants of unknown significance is the largest obstacle to achieve a definitive FH diagnosis. This study established a time- and cost-effective high-throughput cell-based assay to functionally profile LDLR variants, which allowed us to discriminate disruptive rare variants from silent ones. This work generated a valuable resource for systematic functional characterization of LDLR variants solving 1 of the major issues to achieve a definitive FH diagnosis.
ABSTRACT
An elevated frequency of DNA replication defects is associated with diabetes and cancer. However, data linking these nuclear perturbations to the onset or progression of organ complications remained unexplored. Here, we report that RAGE (Receptor for Advanced Glycated Endproducts), previously believed to be an extracellular receptor, upon metabolic stress localizes to the damaged forks. There it interacts and stabilizes the minichromosome-maintenance (Mcm2-7) complex. Accordingly, RAGE deficiency leads to slowed fork progression, premature fork collapse, hypersensitivity to replication stress agents and reduction of viability, which was reversed by the reconstitution of RAGE. This was marked by the 53BP1/OPT-domain expression and the presence of micronuclei, premature loss-of-ciliated zones, increased incidences of tubular-karyomegaly, and finally, interstitial fibrosis. More importantly, the RAGE-Mcm2 axis was selectively compromised in cells expressing micronuclei in human biopsies and mouse models of diabetic nephropathy and cancer. Thus, the functional RAGE-Mcm2/7 axis is critical in handling replication stress in vitro and human disease.
Subject(s)
Diabetes Mellitus , Minichromosome Maintenance Complex Component 2 , Neoplasms , Receptor for Advanced Glycation End Products , Animals , Humans , Mice , Cell Cycle Proteins/metabolism , DNA Replication/genetics , Minichromosome Maintenance Complex Component 2/genetics , Minichromosome Maintenance Complex Component 2/metabolism , Minichromosome Maintenance Proteins/metabolism , Receptor for Advanced Glycation End Products/metabolismABSTRACT
In recent years, Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) has emerged as a flexible method that enables semi-automated volume ultrastructural imaging. We present a toolset for adherent cells that enables tracking and finding cells, previously identified in light microscopy (LM), in the FIB-SEM, along with the automatic acquisition of high-resolution volume datasets. We detect the underlying grid pattern in both modalities (LM and EM), to identify common reference points. A combination of computer vision techniques enables complete automation of the workflow. This includes setting the coincidence point of both ion and electron beams, automated evaluation of the image quality and constantly tracking the sample position with the microscope's field of view reducing or even eliminating operator supervision. We show the ability to target the regions of interest in EM within 5 µm accuracy while iterating between different targets and implementing unattended data acquisition. Our results demonstrate that executing volume acquisition in multiple locations autonomously is possible in EM.
Subject(s)
Imaging, Three-Dimensional , Volume Electron Microscopy , Microscopy, Electron, Scanning , Imaging, Three-Dimensional/methods , SoftwareABSTRACT
OBJECTIVE: Hyperferremia and hyperferritinemia are observed in patients and disease models of type 2 diabetes mellitus (T2DM). Likewise, patients with genetic iron overload diseases develop diabetes, suggesting a tight link between iron metabolism and diabetes. The liver controls systemic iron homeostasis and is a central organ for T2DM. Here, we investigate how the control of iron metabolism in hepatocytes is affected by T2DM. METHODS: Perls Prussian blue staining was applied to analyze iron distribution in liver biopsies of T2DM patients. To identify molecular mechanisms underlying hepatocyte iron accumulation we established cellular models of insulin resistance by treatment with palmitate and insulin. RESULTS: We show that a subset of T2DM patients accumulates iron in hepatocytes, a finding mirrored in a hepatocyte model of insulin resistance. Iron accumulation can be explained by the repression of the iron exporter ferroportin upon palmitate and/or insulin treatment. While during palmitate treatment the activation of the iron regulatory hormone hepcidin may contribute to reducing ferroportin protein levels in a cell-autonomous manner, insulin treatment decreases ferroportin transcription via the PI3K/AKT and Ras/Raf/MEK/ERK signaling pathways. CONCLUSION: Repression of ferroportin at the transcriptional and post-transcriptional level may contribute to iron accumulation in hepatocytes observed in a subset of patients with T2DM.
Subject(s)
Diabetes Mellitus, Type 2 , Insulin Resistance , Insulins , Iron Overload , Humans , Iron/metabolism , Diabetes Mellitus, Type 2/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Iron Overload/metabolism , Hepatocytes/metabolism , Palmitates/metabolism , Insulins/metabolismABSTRACT
Proteins that enter the secretory pathway are transported from their place of synthesis in the endoplasmic reticulum to the Golgi complex by COPII-coated carriers. The networks of proteins that regulate these components in response to extracellular cues have remained largely elusive. Using high-throughput microscopy, we comprehensively screened 378 cytoskeleton-associated and related proteins for their functional interaction with the coat protein complex II (COPII) components SEC23A and SEC23B. Among these, we identified a group of proteins associated with focal adhesions (FERMT2, MACF1, MAPK8IP2, NGEF, PIK3CA, and ROCK1) that led to the downregulation of SEC23A when depleted by siRNA. Changes in focal adhesions induced by plating cells on ECM also led to the downregulation of SEC23A and decreases in VSVG transport from ER to Golgi. Both the expression of SEC23A and the transport defect could be rescued by treatment with a focal adhesion kinase inhibitor. Altogether, our results identify a network of cytoskeleton-associated proteins connecting focal adhesions and ECM-related signaling with the gene expression of the COPII secretory machinery and trafficking.
Subject(s)
COP-Coated Vesicles , Extracellular Matrix , Focal Adhesions , Golgi Apparatus , Vesicular Transport Proteins , COP-Coated Vesicles/genetics , COP-Coated Vesicles/metabolism , Extracellular Matrix/metabolism , Focal Adhesions/genetics , Focal Adhesions/metabolism , Golgi Apparatus/genetics , Golgi Apparatus/metabolism , Protein Transport , Secretory Pathway , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolismABSTRACT
BACKGROUND: Familial Hypercholesterolemia (FH) is a semidominant disorder of the lipid metabolism associated with premature atherosclerosis and coronary heart disease. So far, about 3,000 unique LDLR variants have been described, most of which lack functional evidence proving their effect on LDLR function, despite the important role that functional studies play in variant classification. OBJECTIVE: In this work, we aimed to functionally characterize 13 rare missense variants, identified worldwide and in Portugal, in clinical FH patients. METHODS: LDLR-deficient CHO-ldlA7 cells were transfected with plasmids carrying different LDLR variants generated by site-directed mutagenesis. LDLR activity and expression were assessed by FACS. RESULTS: 11/13 variants affect LDLR function (p.Cys109Phe; p.Cys143Arg; p.Glu267Lys; p.Cys352Ser; p.Ile451Thr; p.His485Gln; p.Asp492Asn; p.Val500Ala; p.Gly529Arg; p.Phe614Ile; p.Glu626Lys) and 2/13 are inconclusive (p.Arg81Cys; p.Gly98Arg;). CONCLUSION: Of the 13 variants studied, 8 were classified as VUS by ACMG criteria, but for 7 of these 8, our functional studies were able to reassign them as Likely pathogenic or Pathogenic. For an accurate diagnosis, an effort must be made to improve functional characterization of putative disease-causing variants.
Subject(s)
Hyperlipoproteinemia Type II , Receptors, LDL , Animals , CHO Cells , Cricetinae , Cricetulus , Humans , Hyperlipoproteinemia Type II/diagnosis , Mutation , Mutation, Missense , Phenotype , Receptors, LDL/genetics , Receptors, LDL/metabolismABSTRACT
An attractive approach to treat people with Cystic Fibrosis (CF), a life-shortening disease caused by mutant CFTR, is to compensate for the absence of this chloride/bicarbonate channel by activating alternative (non-CFTR) chloride channels. One obvious target for such "mutation-agnostic" therapeutic approach is TMEM16A (anoctamin-1/ANO1), a calcium-activated chloride channel (CaCC) which is also expressed in the airways of people with CF, albeit at low levels. To find novel TMEM16A regulators of both traffic and function, with the main goal of identifying candidate CF drug targets, we performed a fluorescence cell-based high-throughput siRNA microscopy screen for TMEM16A trafficking using a double-tagged construct expressed in human airway cells. About 700 genes were screened (2 siRNAs per gene) of which 262 were identified as candidate TMEM16A modulators (179 siRNAs enhanced and 83 decreased TMEM16A traffic), being G-protein coupled receptors (GPCRs) enriched on the primary hit list. Among the 179 TMEM16A traffic enhancer siRNAs subjected to secondary screening 20 were functionally validated. Further hit validation revealed that siRNAs targeting two GPCRs - ADRA2C and CXCR3 - increased TMEM16A-mediated chloride secretion in human airway cells, while their overexpression strongly diminished calcium-activated chloride currents in the same cell model. The knockdown, and likely also the inhibition, of these two TMEM16A modulators is therefore an attractive potential therapeutic strategy to increase chloride secretion in CF.
Subject(s)
Anoctamin-1 , Cystic Fibrosis , Neoplasm Proteins , Anoctamin-1/antagonists & inhibitors , Anoctamin-1/genetics , Calcium/metabolism , Cystic Fibrosis/drug therapy , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Gene Knockdown Techniques , Humans , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , RNA, Small Interfering/geneticsABSTRACT
Complex traits are characterized by multiple genes and variants acting simultaneously on a phenotype. However, studying the contribution of individual pairs of genes to complex traits has been challenging since human genetics necessitates very large population sizes, while findings from model systems do not always translate to humans. Here, we combine genetics with combinatorial RNAi (coRNAi) to systematically test for pairwise additive effects (AEs) and genetic interactions (GIs) between 30 lipid genome-wide association studies (GWAS) genes. Gene-based burden tests from 240,970 exomes show that in carriers with truncating mutations in both, APOB and either PCSK9 or LPL ("human double knock-outs") plasma lipid levels change additively. Genetics and coRNAi identify overlapping AEs for 12 additional gene pairs. Overlapping GIs are observed for TOMM40/APOE with SORT1 and NCAN. Our study identifies distinct gene pairs that modulate plasma and cellular lipid levels primarily via AEs and nominates putative drug target pairs for improved lipid-lowering combination therapies.
Subject(s)
Genome-Wide Association Study/methods , Proprotein Convertase 9/metabolism , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/metabolism , Apolipoproteins B/genetics , Apolipoproteins B/metabolism , Apolipoproteins E/genetics , Apolipoproteins E/metabolism , Humans , Lipoprotein Lipase/genetics , Lipoprotein Lipase/metabolism , Mitochondrial Precursor Protein Import Complex Proteins/genetics , Mitochondrial Precursor Protein Import Complex Proteins/metabolism , Neurocan/genetics , Neurocan/metabolism , Proprotein Convertase 9/geneticsABSTRACT
With the advancement of laser-based microscopy tools, it is now possible to explore mechano-kinetic processes occurring inside the cell. Here, we describe the advanced protocol for studying the DNA repair kinetics in real time using the laser to induce the DNA damage. This protocol can be used for inducing, testing, and studying the repair mechanisms associated with DNA double-strand breaks, interstrand cross-link repair, and single-strand break repair. For complete details on the use and execution of this protocol, please refer to Kumar et al. (2017, 2020).
Subject(s)
Biomechanical Phenomena/physiology , DNA Repair/physiology , Microscopy, Confocal/methods , DNA/genetics , DNA Breaks, Double-Stranded , DNA Breaks, Single-Stranded , DNA Damage/genetics , DNA Repair/genetics , Kinetics , LasersABSTRACT
Nitrogen fixation has a critical role in marine primary production, yet our understanding of marine nitrogen-fixers (diazotrophs) is hindered by limited observations. Here, we report a quantitative image analysis pipeline combined with mapping of molecular markers for mining >2,000,000 images and >1300 metagenomes from surface, deep chlorophyll maximum and mesopelagic seawater samples across 6 size fractions (<0.2-2000 µm). We use this approach to characterise the diversity, abundance, biovolume and distribution of symbiotic, colony-forming and particle-associated diazotrophs at a global scale. We show that imaging and PCR-free molecular data are congruent. Sequence reads indicate diazotrophs are detected from the ultrasmall bacterioplankton (<0.2 µm) to mesoplankton (180-2000 µm) communities, while images predict numerous symbiotic and colony-forming diazotrophs (>20 µm). Using imaging and molecular data, we estimate that polyploidy can substantially affect gene abundances of symbiotic versus colony-forming diazotrophs. Our results support the canonical view that larger diazotrophs (>10 µm) dominate the tropical belts, while unicellular cyanobacterial and non-cyanobacterial diazotrophs are globally distributed in surface and mesopelagic layers. We describe co-occurring diazotrophic lineages of different lifestyles and identify high-density regions of diazotrophs in the global ocean. Overall, we provide an update of marine diazotroph biogeographical diversity and present a new bioimaging-bioinformatic workflow.
Subject(s)
Molecular Imprinting/methods , Nitrogen Fixation/genetics , Nitrogen/metabolism , Seawater/chemistry , Aquatic Organisms , Bacteria/genetics , Bacteria/metabolism , Cyanobacteria/genetics , Cyanobacteria/metabolism , Nitrogen Fixation/physiology , Oceans and Seas , Phylogeny , Plankton/metabolism , Seawater/microbiology , Symbiosis/genetics , Symbiosis/physiologyABSTRACT
SUMMARY: Modern bioimaging and related areas such as sensor technology have undergone tremendous development over the last few years. As a result, contemporary imaging techniques, particularly electron microscopy (EM) and light sheet microscopy, can frequently generate datasets attaining sizes of several terabytes (TB). As a consequence, even seemingly simple data operations such as cropping, chromatic- and drift-corrections and even visualisation, poses challenges when applied to thousands of time points or tiles. To address this we developed BigDataProcessor2-a Fiji plugin facilitating processing workflows for TB sized image datasets. AVAILABILITY AND IMPLEMENTATION: BigDataProcessor2 is available as a Fiji plugin via the BigDataProcessor update site. The application is implemented in Java and the code is publicly available on GitHub (https://github.com/bigdataprocessor/bigdataprocessor2). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Subject(s)
Microscopy , Software , Fiji , Microscopy/methods , Workflow , Image Processing, Computer-Assisted/methodsABSTRACT
Fibrosis can affect any organ, resulting in the loss of tissue architecture and function with often life-threatening consequences. Pathologically, fibrosis is characterised by the expansion of connective tissue due to excessive deposition of extracellular matrix (ECM) proteins, including the fibrillar forms of collagen. A significant limitation for discovering cures for fibrosis is the availability of suitable human models and techniques to quantify mature fibrillar collagen deposition as close as possible to human physiological conditions.Here we have extensively characterised an ex vivo cultured human lung tissue-derived, precision-cut lung slices (hPCLS) model using label-free second harmonic generation (SHG) light microscopy to quantify fibrillar collagen deposition and mass spectrometry-based techniques to obtain a proteomic and metabolomic fingerprint of hPCLS in ex vivo culture.We demonstrate that hPCLS are viable and metabolically active, with mesenchymal, epithelial, endothelial and immune cell types surviving for at least 2â weeks in ex vivo culture. Analysis of hPCLS-conditioned supernatants showed a strong induction of pulmonary fibrosis-related ECM proteins upon transforming growth factor-ß1 (TGF-ß1) stimulation. This upregulation of ECM proteins was not translated into an increased deposition of fibrillar collagen. In support of this observation, we revealed the presence of a pro-ECM degradation activity in our ex vivo cultures of hPCLS, inhibition of which by a metalloproteinase inhibitor resulted in increased collagen deposition in response to TGF-ß1 stimulation.Together the data show that an integrated approach of measuring soluble pro-fibrotic markers alongside quantitative SHG-based analysis of fibrillar collagen is a valuable tool for studying pro-fibrotic signalling and testing anti-fibrotic agents.
Subject(s)
Microscopy , Pulmonary Fibrosis , Fibrosis , Humans , Lung/pathology , Proteomics , Pulmonary Fibrosis/pathology , Transforming Growth Factor beta1ABSTRACT
Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a chloride channel normally expressed at the surface of epithelial cells. The most frequent mutation, resulting in Phe-508 deletion, causes CFTR misfolding and its premature degradation. Low temperature or pharmacological correctors can partly rescue the Phe508del-CFTR processing defect and enhance trafficking of this channel variant to the plasma membrane (PM). Nevertheless, the rescued channels have an increased endocytosis rate, being quickly removed from the PM by the peripheral protein quality-control pathway. We previously reported that rescued Phe508del-CFTR (rPhe508del) can be retained at the cell surface by stimulating signaling pathways that coax the adaptor molecule ezrin (EZR) to tether rPhe508del-Na+/H+-exchange regulatory factor-1 complexes to the actin cytoskeleton, thereby averting the rapid internalization of this channel variant. However, the molecular basis for why rPhe508del fails to recruit active EZR to the PM remains elusive. Here, using a proteomics approach, we characterized and compared the core components of wt-CFTR- or rPhe508del-containing macromolecular complexes at the surface of human bronchial epithelial cells. We identified calpain 1 (CAPN1) as an exclusive rPhe508del interactor that prevents active EZR recruitment, impairs rPhe508del anchoring to actin, and reduces its stability in the PM. We show that either CAPN1 down-regulation or its chemical inhibition dramatically improves the functional rescue of Phe508del-CFTR in airway cells. These observations suggest that CAPN1 constitutes an appealing target for pharmacological intervention, as part of CF combination therapies restoring Phe508del-CFTR function.
Subject(s)
Aminopyridines/pharmacology , Benzodioxoles/pharmacology , Calpain/antagonists & inhibitors , Cell Membrane/drug effects , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Calpain/metabolism , Cell Membrane/metabolism , Cells, Cultured , Computational Biology , Cystic Fibrosis Transmembrane Conductance Regulator/analysis , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Genetic Variation/drug effects , Humans , Proteomics , TemperatureABSTRACT
Skeletal muscle (SKM) differentiation is a highly regulated process leading to the formation of specialised cells with reorganised compartments and organelles, such as those of the early secretory pathway. During SKM differentiation the Golgi complex (GC) redistributes close to the nuclear envelope and in small distinct peripheral structures distributed throughout the myotube. Concurrently, GC elements closely associate with endoplasmic reticulum-exit sites (ERES). The mechanisms underlying this reorganisation and its relevance for SKM differentiation are poorly understood. Here, we show, by time-lapse imaging studies, that the changes in GC organisation involve GC fragmentation and redistribution of ERES with the formation of tightly associated GC-ERES units. We show that knockdown of GM130 (also known as GOLGA2) or p115 (also known as USO1), two regulators of the early secretory pathway, impairs GC and ERES reorganisation. This in turn results in inhibition of myotube fusion and M-cadherin (also known as CDH15) transport to the sarcolemma. Taken together, our data suggest that the correct reorganisation of the early secretory pathway components plays an important role in SKM differentiation and, thus, associated pathologies.
Subject(s)
Autoantigens/metabolism , Cell Differentiation , Golgi Matrix Proteins/metabolism , Membrane Proteins/metabolism , Muscle, Skeletal/metabolism , Sarcolemma/metabolism , Secretory Pathway , Vesicular Transport Proteins/metabolism , Animals , Autoantigens/genetics , Cell Line , Golgi Matrix Proteins/genetics , Membrane Proteins/genetics , Mice , Muscle, Skeletal/cytology , Sarcolemma/genetics , Vesicular Transport Proteins/geneticsABSTRACT
An attractive possibility to treat Cystic Fibrosis (CF), a severe condition caused by dysfunctional CFTR, an epithelial anion channel, is through the activation of alternative (non-CFTR) anion channels. Anoctamin 1 (ANO1) was demonstrated to be a Ca2+-activated chloride channel (CaCC) and thus of high potential to replace CFTR. Despite that ANO1 is expressed in human lung CF tissue, it is present at the cell surface at very low levels. In addition, little is known about regulation of ANO1 traffic, namely which factors promote its plasma membrane (PM) localization. Here, we generated a novel cellular model, expressing an inducible 3HA-ANO1-eGFP construct, and validated its usage as a microscopy tool to monitor for ANO1 traffic. We demonstrate the robustness and specificity of this cell-based assay, by the identification of siRNAs acting both as ANO1 traffic enhancer and inhibitor, targeting respectively COPB1 and ESYT1 (extended synaptotagmin-1), the latter involved in coupling of the endoplasmic reticulum to the PM at specific microdomains. We further show that knockdown of ESYT1 (and family members ESYT2 and ESYT3) significantly decreased ANO1 current density. This ANO1 cell-based assay constitutes an important tool to be further used in high-throughput screens and drug discovery of high relevance for CF and cancer.
Subject(s)
Anoctamin-1/metabolism , Cystic Fibrosis/metabolism , Models, Biological , Neoplasm Proteins/metabolism , Synaptotagmins/metabolism , Anoctamin-1/genetics , Cell Line , Cystic Fibrosis/genetics , Cystic Fibrosis/pathology , Humans , Neoplasm Proteins/genetics , Protein Transport , Synaptotagmins/geneticsABSTRACT
We present a 3D-fluorescence imaging and classification tool for high throughput analysis of microbial eukaryotes in environmental samples. It entails high-content feature extraction that permits accurate automated taxonomic classification and quantitative data about organism ultrastructures and interactions. Using plankton samples from the Tara Oceans expeditions, we validate its applicability to taxonomic profiling and ecosystem analyses, and discuss its potential for future integration of eukaryotic cell biology into evolutionary and ecological studies.
Subject(s)
Ecosystem , Environmental Microbiology , Eukaryota/cytology , Eukaryota/physiology , Imaging, Three-Dimensional/methods , Optical Imaging/methods , Eukaryota/classificationABSTRACT
Laser-mediated dissection methods have been used for many years to micro-irradiate biological samples, but recent technological progress has rendered this technique more precise, powerful, and easy to use. Today pulsed lasers can be operated with diffraction limited, sub-micrometer precision to ablate intracellular structures. Here, we discuss laser nanosurgery setups and the instrumentation in our laboratory. We describe how to use this technique to ablate cytoskeletal elements in living cells. We also show how this technique can be used in multicellular organisms, to micropuncture and/or ablate cells of interest and finally how to monitor a successful laser nanosurgery.
Subject(s)
Cytological Techniques/methods , Laser Therapy/methods , Microsurgery/methods , Nanotechnology/methods , Actins/metabolism , Animals , Biomarkers , Calibration , Cytological Techniques/instrumentation , HeLa Cells , Humans , Laser Therapy/instrumentation , Microscopy/instrumentation , Microscopy/methods , Microsurgery/instrumentation , Nanotechnology/instrumentation , ZebrafishABSTRACT
Genome integrity relies on precise coordination between DNA replication and chromosome segregation. Whereas replication stress attracted much attention, the consequences of mitotic perturbations for genome integrity are less understood. Here, we knockdown 47 validated mitotic regulators to show that a broad spectrum of mitotic errors correlates with increased DNA breakage in daughter cells. Unexpectedly, we find that only a subset of these correlations are functionally linked. We identify the genuine mitosis-born DNA damage events and sub-classify them according to penetrance of the observed phenotypes. To demonstrate the potential of this resource, we show that DNA breakage after cytokinesis failure is preceded by replication stress, which mounts during consecutive cell cycles and coincides with decreased proliferation. Together, our results provide a resource to gauge the magnitude and dynamics of DNA breakage associated with mitotic aberrations and suggest that replication stress might limit propagation of cells with abnormal karyotypes.