ABSTRACT
Restricting the localization of evolutionarily conserved histone H3 variant CENP-A to the centromere is essential to prevent chromosomal instability (CIN), an important hallmark of cancers. Overexpressed CENP-A mislocalizes to non-centromeric regions and contributes to CIN in yeast, flies, and human cells. Centromeric localization of CENP-A is facilitated by the interaction of Mis18ß with CENP-A specific chaperone HJURP. Cellular levels of Mis18ß are regulated by ß-transducin repeat containing protein (ß-TrCP), an F-box protein of SCF (Skp1, Cullin, F-box) E3-ubiquitin ligase complex. Here, we show that defects in ß-TrCP-mediated proteolysis of Mis18ß contributes to the mislocalization of endogenous CENP-A and CIN in a triple-negative breast cancer (TNBC) cell line, MDA-MB-231. CENP-A mislocalization in ß-TrCP depleted cells is dependent on high levels of Mis18ß as depletion of Mis18ß suppresses mislocalization of CENP-A in these cells. Consistent with these results, endogenous CENP-A is mislocalized in cells overexpressing Mis18ß alone. In summary, our results show that ß-TrCP-mediated degradation of Mis18ß prevents mislocalization of CENP-A and CIN. We propose that deregulated expression of Mis18ß may be one of the key mechanisms that contributes to chromosome segregation defects in cancers.
ABSTRACT
The nucleolus is the largest membraneless organelle and nuclear body in mammalian cells. It is primarily involved in the biogenesis of ribosomes, essential macromolecular machines responsible for synthesizing all proteins required by the cell. The assembly of ribosomes is evolutionarily conserved and accounts for the most energy-consuming cellular process needed for cell growth, proliferation, and homeostasis. Despite the significance of this process, the substructural mechanistic principles of the nucleolar function in preribosome biogenesis have only recently begun to emerge. Here, we provide a new perspective using advanced super-resolution microscopy and single-molecule MINFLUX nanoscopy on the mechanistic principles governing ribosomal RNA-seeded nucleolar formation and the resulting tripartite suborganization of the nucleolus driven, in part, by liquid-liquid phase separation. With recent advances in the cryogenic electron microscopy (cryoEM) structural analysis of ribosome biogenesis intermediates, we highlight the current understanding of the step-wise assembly of preribosomal subunits in the nucleolus. Finally, we address how novel anticancer drug candidates target early steps in ribosome biogenesis to exploit these essential dependencies for growth arrest and tumor control.
Subject(s)
Cell Nucleolus , Animals , Humans , Cell Nucleolus/metabolism , Cell Nucleolus/chemistry , Microscopy , Ribosomes/metabolism , Ribosomes/chemistryABSTRACT
The centromeric histone H3 variant CENP-A is overexpressed in many cancers. The mislocalization of CENP-A to noncentromeric regions contributes to chromosomal instability (CIN), a hallmark of cancer. However, pathways that promote or prevent CENP-A mislocalization remain poorly defined. Here, we performed a genome-wide RNAi screen for regulators of CENP-A localization which identified DNAJC9, a J-domain protein implicated in histone H3-H4 protein folding, as a factor restricting CENP-A mislocalization. Cells lacking DNAJC9 exhibit mislocalization of CENP-A throughout the genome, and CIN phenotypes. Global interactome analysis showed that DNAJC9 depletion promotes the interaction of CENP-A with the DNA-replication-associated histone chaperone MCM2. CENP-A mislocalization upon DNAJC9 depletion was dependent on MCM2, defining MCM2 as a driver of CENP-A deposition at ectopic sites when H3-H4 supply chains are disrupted. Cells depleted for histone H3.3, also exhibit CENP-A mislocalization. In summary, we have defined novel factors that prevent mislocalization of CENP-A, and demonstrated that the integrity of H3-H4 supply chains regulated by histone chaperones such as DNAJC9 restrict CENP-A mislocalization and CIN.
Subject(s)
Centromere Protein A , Chromosomal Instability , Histones , Humans , Centromere Protein A/metabolism , Centromere Protein A/genetics , Histones/metabolism , Histones/genetics , Minichromosome Maintenance Complex Component 2/metabolism , Minichromosome Maintenance Complex Component 2/genetics , HeLa Cells , HSP40 Heat-Shock Proteins/metabolism , HSP40 Heat-Shock Proteins/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/genetics , Centromere/metabolismABSTRACT
Labelling of nascent stem loops with fluorescent proteins has fostered the visualization of transcription in living cells. Quantitative analysis of recorded fluorescence traces can shed light on kinetic transcription parameters and regulatory mechanisms. However, existing methods typically focus on steady state dynamics. Here, we combine a stochastic process transcription model with a hierarchical Bayesian method to infer global as well locally shared parameters for groups of cells and recover unobserved quantities such as initiation times and polymerase loading of the gene. We apply our approach to the cyclic response of the yeast CUP1 locus to heavy metal stress. Within the previously described slow cycle of transcriptional activity on the scale of minutes, we discover fast time-modulated bursting on the scale of seconds. Model comparison suggests that slow oscillations of transcriptional output are regulated by the amplitude of the bursts. Several polymerases may initiate during a burst.
ABSTRACT
Restricting the localization of the evolutionarily conserved centromeric histone H3 variant CENP-A to centromeres prevents chromosomal instability (CIN). The mislocalization of CENP-A to non-centromeric regions contributes to CIN in yeasts, flies and human cells. Even though overexpression and mislocalization of CENP-A have been reported in cancers, the mechanisms responsible for its mislocalization remain poorly understood. Here, we used an imaging-based high-throughput RNAi screen to identify factors that prevent mislocalization of overexpressed YFP-tagged CENP-A (YFP-CENP-A) in HeLa cells. Among the top five candidates in the screen - the depletion of which showed increased nuclear YFP-CENP-A fluorescence - were the histone chaperones CHAF1B (or p60) and CHAF1A (or p150). Follow-up validation and characterization experiments showed that CHAF1B-depleted cells exhibited CENP-A mislocalization, CIN phenotypes and increased enrichment of CENP-A in chromatin fractions. The depletion of DAXX, a histone H3.3 chaperone, suppressed CENP-A mislocalization and CIN in CHAF1B-depleted cells. We propose that in CHAF1B-depleted cells, DAXX promotes mislocalization of the overexpressed CENP-A to non-centromeric regions, resulting in CIN. In summary, we identified regulators of CENP-A localization and defined a role for CHAF1B in preventing DAXX-dependent CENP-A mislocalization and CIN.
Subject(s)
Chromosomal Proteins, Non-Histone , Histones , Humans , Histones/genetics , Centromere Protein A/genetics , HeLa Cells , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromatin , Centromere/metabolism , Molecular Chaperones/metabolism , Chromosomal Instability , Autoantigens/genetics , Chromatin Assembly Factor-1/geneticsABSTRACT
Cellular functions depend on the dynamic assembly of protein regulator complexes at specific cellular locations. Single Molecule Tracking (SMT) is a method of choice for the biochemical characterization of protein dynamics in vitro and in vivo. SMT follows individual molecules in live cells and provides direct information about their behavior. SMT was successfully applied to mammalian models. However, mammalian cells provide a complex environment where protein mobility depends on numerous factors that are difficult to control experimentally. Therefore, yeast cells, which are unicellular and well-studied with a small and completely sequenced genome, provide an attractive alternative for SMT. The simplicity of organization, ease of genetic manipulation, and tolerance to gene fusions all make yeast a great model for quantifying the kinetics of major enzymes, membrane proteins, and nuclear and cellular bodies. However, very few researchers apply SMT techniques to yeast. Our goal is to promote SMT in yeast to a wider research community. Our review serves a dual purpose. We explain how SMT is conducted in yeast cells, and we discuss the latest insights from yeast SMT while putting them in perspective with SMT of higher eukaryotes.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Animals , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Cell Nucleus/metabolism , Base Sequence , Biophysics , Mammals/metabolismABSTRACT
The MYC oncogene has been studied for decades, yet there is still intense debate over how this transcription factor controls gene expression. Here, we seek to answer these questions with an in vivo readout of discrete events of gene expression in single cells. We engineered an optogenetic variant of MYC (Pi-MYC) and combined this tool with single-molecule RNA and protein imaging techniques to investigate the role of MYC in modulating transcriptional bursting and transcription factor binding dynamics in human cells. We find that the immediate consequence of MYC overexpression is an increase in the duration rather than in the frequency of bursts, a functional role that is different from the majority of human transcription factors. We further propose that the mechanism by which MYC exerts global effects on the active period of genes is by altering the binding dynamics of transcription factors involved in RNA polymerase II complex assembly and productive elongation.
Subject(s)
Gene Expression Regulation/genetics , Genes, myc/physiology , Transcription, Genetic/physiology , Animals , Cell Line , Humans , Mice , Transcription Factors/metabolismABSTRACT
Regulation of transcription by RNA Polymerase II (RNAPII) is a rapidly evolving area of research. Technological developments in microscopy have revealed insight into the dynamics, structure, and localization of transcription components within single cells. A frequent observation in many studies is the appearance of 'spots' in cell nuclei associated with the transcription process. In this review we highlight studies that characterize the temporal and spatial characteristics of these spots, examine possible pitfalls in interpreting these kind of imaging data, and outline directions where single-cell imaging may advance in ways to further our understanding of transcription regulation.
Subject(s)
Gene Expression Regulation , Transcription, Genetic , Cell Nucleus/genetics , Cell Nucleus/metabolism , Microscopy, Fluorescence/methods , Molecular Imaging/methods , RNA Polymerase II/metabolism , Single-Cell Analysis/methodsABSTRACT
Chromosomal instability (CIN) is a hallmark of many cancers. Restricting the localization of centromeric histone H3 variant CENP-A to centromeres prevents CIN. CENP-A overexpression (OE) and mislocalization have been observed in cancers and correlate with poor prognosis; however, the molecular consequences of CENP-A OE on CIN and aneuploidy have not been defined. Here, we show that CENP-A OE leads to its mislocalization and CIN with lagging chromosomes and micronuclei in pseudodiploid DLD1 cells and xenograft mouse model. CIN is due to reduced localization of proteins to the kinetochore, resulting in defects in kinetochore integrity and unstable kinetochore-microtubule attachments. CENP-A OE contributes to reduced expression of cell adhesion genes and higher invasion of DLD1 cells. We show that CENP-A OE contributes to aneuploidy with karyotypic heterogeneity in human cells and xenograft mouse model. In summary, our results provide a molecular link between CENP-A OE and aneuploidy, and suggest that karyotypic heterogeneity may contribute to the aggressive phenotype of CENP-A-overexpressing cancers.
Subject(s)
Aneuploidy , Centromere Protein A/biosynthesis , Chromosomal Instability , Kinetochores/metabolism , Micronuclei, Chromosome-Defective , Neoplasm Proteins/metabolism , Neoplasms/metabolism , Animals , Cell Line, Tumor , Centromere Protein A/genetics , Heterografts , Humans , Kinetochores/pathology , Mice , Neoplasm Proteins/genetics , Neoplasm Transplantation , Neoplasms/genetics , Neoplasms/pathologyABSTRACT
Yeast prions and mnemons are respectively transmissible and non-transmissible self-perpetuating protein assemblies, frequently based on cross-ß ordered detergent-resistant aggregates (amyloids). Prions cause devastating diseases in mammals and control heritable traits in yeast. It was shown that the de novo formation of the prion form [PSI+] of yeast release factor Sup35 is facilitated by aggregates of other proteins. Here we explore the mechanism of the promotion of [PSI+] formation by Ste18, an evolutionarily conserved gamma subunit of a G-protein coupled receptor, a key player in responses to extracellular stimuli. Ste18 forms detergent-resistant aggregates, some of which are colocalized with de novo generated Sup35 aggregates. Membrane association of Ste18 is required for both Ste18 aggregation and [PSI+] induction, while functional interactions involved in signal transduction are not essential for these processes. This emphasizes the significance of a specific location for the nucleation of protein aggregation. In contrast to typical prions, Ste18 aggregates do not show a pattern of heritability. Our finding that Ste18 levels are regulated by the ubiquitin-proteasome system, in conjunction with the previously reported increase in Ste18 levels upon the exposure to mating pheromone, suggests that the concentration-dependent Ste18 aggregation may mediate a mnemon-like response to physiological stimuli.
Subject(s)
GTP-Binding Protein gamma Subunits/metabolism , Peptide Termination Factors/metabolism , Protein Aggregates , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Cell Membrane/metabolism , GTP-Binding Protein gamma Subunits/analysis , Peptide Termination Factors/analysis , Proteolysis , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/analysis , UbiquitinationABSTRACT
The nuclear envelope (NE) is an essential cellular structure that contributes to nuclear stability, organization, and function. Mutations in NE-associated proteins result in a myriad of pathologies with widely diverse clinical manifestations, ages of onsets, and affected tissues. Notably, several hundred disease-causing mutations have been mapped to the LMNA gene, which encodes the intermediate filament proteins lamin A and C, two of the major architectural components of the nuclear envelope. However, how NE dysfunction leads to the highly variable pathologies observed in patient cells and tissues remains poorly understood. One model suggests alterations in the dynamic properties of the nuclear lamina and its associated proteins contribute to disease phenotype. Here, we describe the application of single molecule tracking (SMT) methodology to characterize the behavior of nuclear envelope transmembrane proteins and nuclear lamins in their native cellular environment at the single molecule level. As proof-of-concept, we demonstrate by SMT that Halo-tagged lamin B1, Samp1, lamin A, and lamin AΔ50 have distinct binding and kinetic properties, and we identify several disease-relevant mutants which exhibit altered binding dynamics. SMT is also able to separately probe the dynamics of the peripheral and the nucleoplasmic populations of lamin A mutants. We suggest that SMT is a robust and sensitive method to investigate the relationship between pathogenic mutations or cellular processes and protein dynamics at the NE.
Subject(s)
Cell Nucleus/genetics , Membrane Proteins/genetics , Nuclear Envelope/genetics , Nuclear Proteins/genetics , Humans , Lamin Type A/genetics , Lamin Type A/metabolism , Lamin Type B/genetics , Mutation/genetics , Nuclear Envelope/metabolism , Nuclear Lamina/genetics , Nuclear Lamina/metabolismABSTRACT
The Dam1 complex is an essential component of the outer kinetochore that mediates attachments between spindle microtubules and chromosomes. Dam1p, a subunit of the Dam1 complex, binds to microtubules and is regulated by Aurora B/Ipl1p phosphorylation. We find that overexpression of cAMP-dependent protein kinase (PKA) catalytic subunits (i.e., TPK1, TPK2, TPK3) is lethal in DAM1 mutants and increases the rate of chromosome loss in wild-type cells. Replacing an evolutionarily conserved PKA site (S31) in Dam1p with a nonphosphorylatable alanine suppressed the high-copy PKA dosage lethality in dam1-1 Consistent with Dam1p as a target of PKA, we find that in vitro PKA can directly phosphorylate S31 in Dam1p and we observed phosphorylation of S31 in Dam1p purified from asynchronously growing yeast cells. Cells carrying high-copy TPK2 or a Dam1p phospho-mimetic S31D mutant displayed a reduction in Dam1p localization at the kinetochore, suggesting that PKA phosphorylation plays a role in assembly and/or stability of the Dam1 complex. Furthermore, we observed spindle defects associated with S31 phosphorylation. Finally, we find that phosphorylation of Dam1p on S31 is reduced when glucose is limiting as well as during α-factor arrest, conditions that inhibit PKA activity. These observations suggest that the PKA site of Dam1p participates in regulating kinetochore activity. While PKA is a well-established effector of glucose signaling, our work shows for the first time that glucose-dependent PKA activity has an important function in chromosome segregation.
Subject(s)
Cell Cycle Proteins/genetics , Chromosome Segregation , Glucose/metabolism , Kinetochores/metabolism , Microtubule-Associated Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Signal TransductionABSTRACT
It is unknown how the dynamic binding of transcription factors (TFs) is molecularly linked to chromatin remodeling and transcription. Using single-molecule tracking (SMT), we show that the chromatin remodeler RSC speeds up the search process of the TF Ace1p for its response elements (REs) at the CUP1 promoter. We quantified smFISH mRNA data using a gene bursting model and demonstrated that RSC regulates transcription bursts of CUP1 only by modulating TF occupancy but does not affect initiation and elongation rates. We show by SMT that RSC binds to activated promoters transiently, and based on MNase-seq data, that RSC does not affect the nucleosomal occupancy at CUP1. Therefore, transient binding of Ace1p and rapid bursts of transcription at CUP1 may be dependent on short repetitive cycles of nucleosome mobilization. This type of regulation reduces the transcriptional noise and ensures a homogeneous response of the cell population to heavy metal stress.
Subject(s)
DNA-Binding Proteins/genetics , Gene Expression Regulation, Fungal , Metallothionein/genetics , RNA, Messenger/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Transcription Factors/genetics , Chromatin Assembly and Disassembly , DNA-Binding Proteins/metabolism , Metallothionein/metabolism , Models, Genetic , Nucleosomes/chemistry , Nucleosomes/metabolism , Promoter Regions, Genetic , Protein Binding , RNA, Messenger/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Single Molecule Imaging/methods , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
Follicular CD8+ T (fCD8) cells reside within B cell follicles and are thought to be immune-privileged sites of HIV/SIV infection. We have observed comparable levels of fCD8 cells between chronically SIV-infected rhesus macaques with low viral loads (LVL) and high viral loads (HVL), raising the question concerning their contribution to viremia control. In this study, we sought to clarify the role of SIV-specific fCD8 cells in lymph nodes during the course of SIV infection in rhesus macaques. We observed that fCD8 cells, T follicular helper (Tfh) cells, and T follicular regulatory cells (Tfreg) were all elevated in chronic SIV infection. fCD8 cells of LVL animals tended to express more Gag-specific granzyme B and exhibited significantly greater killing than did HVL animals, and their cell frequencies were negatively correlated with viremia, suggesting a role in viremia control. Env- and Gag-specific IL-21+ Tfh of LVL but not HVL macaques negatively correlated with viral load, suggesting better provision of T cell help to fCD8 cells. Tfreg positively correlated with fCD8 cells in LVL animals and negatively correlated with viremia, suggesting a potential benefit of Tfreg via suppression of chronic inflammation. In contrast, in HVL macaques, Tfreg and fCD8 cell frequencies tended to be negatively correlated, and a positive correlation was seen between Tfreg number and viremia, suggesting possible dysfunction and suppression of an effective fCD8 cell immune response. Our data suggest that control of virus-infected cells in B cell follicles not only depends on fCD8 cell cytotoxicity but also on complex fCD8 cell associations with Tfh cells and Tfreg.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/immunology , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Regulatory/immunology , Viremia/immunology , Animals , B-Lymphocytes/immunology , B-Lymphocytes/virology , CD8-Positive T-Lymphocytes/virology , Female , Inflammation/immunology , Inflammation/virology , Interleukins/immunology , Lymph Nodes/immunology , Lymph Nodes/virology , Macaca mulatta , Male , Simian Acquired Immunodeficiency Syndrome/virology , T-Lymphocytes, Helper-Inducer/virology , T-Lymphocytes, Regulatory/virology , Viral Load/immunology , Viremia/virologyABSTRACT
Population-based assays have been employed extensively to investigate the interactions of transcription factors (TFs) with chromatin and are often interpreted in terms of static and sequential binding. However, fluorescence microscopy techniques reveal a more dynamic binding behaviour of TFs in live cells. Here we analyse the strengths and limitations of in vivo single-molecule tracking and performed a comprehensive analysis on the intranuclear dwell times of four steroid receptors and a number of known cofactors. While the absolute residence times estimates can depend on imaging acquisition parameters due to sampling bias, our results indicate that only a small proportion of factors are specifically bound to chromatin at any given time. Interestingly, the glucocorticoid receptor and its cofactors affect each other's dwell times in an asymmetric manner. Overall, our data indicate transient rather than stable TF-cofactors chromatin interactions at response elements at the single-molecule level.
Subject(s)
Molecular Imaging/methods , Receptors, Steroid/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Chromatin/metabolism , Corticosterone/pharmacology , DNA Helicases/metabolism , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Green Fluorescent Proteins/genetics , Mice , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Protein Interaction Mapping , Rats , Receptors, Glucocorticoid/analysis , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Receptors, Steroid/analysis , Receptors, Steroid/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Single-Cell Analysis/methods , Time Factors , Transcription Factor AP-1/metabolism , Transcription Factors/metabolismABSTRACT
Chromosomal instability (CIN) is a hallmark of many cancers and a major contributor to tumorigenesis. Centromere and kinetochore associated proteins such as the evolutionarily conserved centromeric histone H3 variant CENP-A, associate with centromeric DNA for centromere function and chromosomal stability. Stringent regulation of cellular CENP-A levels prevents its mislocalization in yeast and flies to maintain genome stability. CENP-A overexpression and mislocalization are observed in several cancers and reported to be associated with increased invasiveness and poor prognosis. We examined whether there is a direct relationship between mislocalization of overexpressed CENP-A and CIN using HeLa and chromosomally stable diploid RPE1 cell lines as model systems. Our results show that mislocalization of overexpressed CENP-A to chromosome arms leads to chromosome congression defects, lagging chromosomes, micronuclei formation and a delay in mitotic exit. CENP-A overexpressing cells showed altered localization of centromere and kinetochore associated proteins such as CENP-C, CENP-T and Nuf2 leading to weakened native kinetochores as shown by reduced interkinetochore distance and CIN. Importantly, our results show that mislocalization of CENP-A to chromosome arms is one of the major contributors for CIN as depletion of histone chaperone DAXX prevents CENP-A mislocalization and rescues the reduced interkinetochore distance and CIN phenotype in CENP-A overexpressing cells. In summary, our results establish that CENP-A overexpression and mislocalization result in a CIN phenotype in human cells. This study provides insights into how overexpression of CENP-A may contribute to CIN in cancers and underscore the importance of understanding the pathways that prevent CENP-A mislocalization for genome stability.
Subject(s)
Centromere Protein A/metabolism , Centromere/genetics , Centromere/metabolism , Chromosomal Instability , Histones/metabolism , Cell Line , Centromere Protein A/genetics , Chromosome Segregation , Diploidy , Gene Expression , HeLa Cells , Histones/genetics , Humans , Kinetochores/metabolism , Micronuclei, Chromosome-Defective , Models, Biological , Neoplasms/genetics , Neoplasms/metabolism , Phenotype , Protein BindingABSTRACT
Extracellular vesicles (EVs), including exosomes and microvesicles, are 30-800 nm vesicles that are released by most cell types, as biological packages for intercellular communication. Their importance in cancer and inflammation makes EVs and their cargo promising biomarkers of disease and cell-free therapeutic agents. Emerging high-resolution cytometric methods have created a pressing need for efficient fluorescent labeling procedures to visualize and detect EVs. Suitable labels must be bright enough for one EV to be detected without the generation of label-associated artifacts. To identify a strategy that robustly labels individual EVs, we used nanoFACS, a high-resolution flow cytometric method that utilizes light scattering and fluorescence parameters along with sample enumeration, to evaluate various labels. Specifically, we compared lipid-, protein-, and RNA-based staining methods and developed a robust EV staining strategy, with the amine-reactive fluorescent label, 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester, and size exclusion chromatography to remove unconjugated label. By combining nanoFACS measurements of light scattering and fluorescence, we evaluated the sensitivity and specificity of EV labeling assays in a manner that has not been described for other EV detection methods. Efficient characterization of EVs by nanoFACS paves the way towards further study of EVs and their roles in health and disease.
Subject(s)
Extracellular Vesicles/metabolism , Flow Cytometry , Staining and Labeling , Amines , Animals , Cell Line , Cell-Derived Microparticles/metabolism , Chromatography, Gel , Flow Cytometry/methods , Fluorescent Dyes , Mice , Micelles , Staining and Labeling/methodsABSTRACT
Progressive, technological achievements in the quantitative fluorescence microscopy field are allowing researches from many different areas to start unraveling the dynamic intricacies of biological processes inside living cells. From super-resolution microscopy techniques to tracking of individual proteins, fluorescence microscopy is changing our perspective on how the cell works. Fortunately, a growing number of research groups are exploring single-molecule studies in living cells. However, no clear consensus exists on several key aspects of the technique such as image acquisition conditions, or analysis of the obtained data. Here, we describe a detailed approach to perform single-molecule tracking (SMT) of transcription factors in living cells to obtain key binding characteristics, namely their residence time and bound fractions. We discuss different types of fluorophores, labeling density, microscope, cameras, data acquisition, and data analysis. Using the glucocorticoid receptor as a model transcription factor, we compared alternate tags (GFP, mEOS, HaloTag, SNAP-tag, CLIP-tag) for potential multicolor applications. We also examine different methods to extract the dissociation rates and compare them with simulated data. Finally, we discuss several challenges that this exciting technique still faces.
Subject(s)
Epithelial Cells/metabolism , Image Processing, Computer-Assisted/statistics & numerical data , Receptors, Glucocorticoid/genetics , Single Molecule Imaging/methods , Animals , Antigens, Differentiation, B-Lymphocyte/genetics , Antigens, Differentiation, B-Lymphocyte/metabolism , Cell Line, Tumor , Epithelial Cells/ultrastructure , Gene Expression Regulation , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hep G2 Cells , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/metabolism , Humans , Kinetics , MCF-7 Cells , Mice , Protein Binding , Receptors, Glucocorticoid/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
Human chromosomes occupy distinct territories in the interphase nucleus. Such chromosome territories (CTs) are positioned according to gene density. Gene-rich CTs are generally located in the center of the nucleus, while gene-poor CTs are positioned more towards the nuclear periphery. However, the association between gene expression levels and the radial positioning of genes within the CT is still under debate. In the present study, we performed three-dimensional fluorescence in situ hybridization experiments in the colorectal cancer cell lines DLD-1 and LoVo using whole chromosome painting probes for chromosomes 8 and 11 and BAC clones targeting four genes with different expression levels assessed by gene expression arrays and RT-PCR. Our results confirmed that the two over-expressed genes, MYC on chromosome 8 and CCND1 on chromosome 11, are located significantly further away from the center of the CT compared to under-expressed genes on the same chromosomes, i.e., DLC1 and SCN3B. When CCND1 expression was reduced after silencing the major transcription factor of the WNT/ß-catenin signaling pathway, TCF7L2, the gene was repositioned and mostly detected in the interior of the CT. Thus, we suggest a non-random distribution in which over-expressed genes are located more towards the periphery of the respective CTs.
Subject(s)
Cell Nucleus/metabolism , Chromosomes, Human/metabolism , Interphase , Signal Transduction , Transcription Factor 7-Like 2 Protein/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Cell Line, Tumor , Cell Nucleus/genetics , Chromosomes, Human/genetics , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Gene Expression Regulation , Humans , In Situ Hybridization, FluorescenceABSTRACT
In vivo single molecule tracking has recently developed into a powerful technique for measuring and understanding the transient interactions of transcription factors (TF) with their chromatin response elements. However, this method still lacks a solid foundation for distinguishing between specific and non-specific interactions. To address this issue, we took advantage of the power of molecular genetics of yeast. Yeast TF Ace1p has only five specific sites in the genome and thus serves as a benchmark to distinguish specific from non-specific binding. Here, we show that the estimated residence time of the short-residence molecules is essentially the same for Hht1p, Ace1p and Hsf1p, equaling 0.12-0.32 s. These three DNA-binding proteins are very different in their structure, function and intracellular concentration. This suggests that (i) short-residence molecules are bound to DNA non-specifically, and (ii) that non-specific binding shares common characteristics between vastly different DNA-bound proteins and thus may have a common underlying mechanism. We develop new and robust procedure for evaluation of adverse effects of labeling, and new quantitative analysis procedures that significantly improve residence time measurements by accounting for fluorophore blinking. Our results provide a framework for the reliable performance and analysis of single molecule TF experiments in yeast.