CCCTC-binding factor (CTCF) is a conserved architectural protein that plays crucial roles in gene regulation and three-dimensional (3D) chromatin organization. To better understand mechanisms and evolution of vertebrate genome organization, we analyzed genome occupancy of CTCF in zebrafish utilizing an endogenously epitope-tagged CTCF knock-in allele. Zebrafish CTCF shares similar facets with its mammalian counterparts, including binding to enhancers, active promoters and repeat elements, and bipartite sequence motifs of its binding sites. However, we found that in vivo CTCF binding is not enriched at boundaries of topologically associating domains (TADs) in developing zebrafish, whereas TAD demarcation by chromatin marks did not differ from mammals. Our data suggest that general mechanisms underlying 3D chromatin organization, and in particular the involvement of CTCF in this process, differ between distant vertebrate species.
Salinomycin (1) exhibits a large spectrum of biological activities including the capacity to selectively eradicate cancer stem cells (CSC), making it and its derivatives promising candidates for the development of drug leads against CSC. It has been previously shown that salinomycin and its C20-propargylamine derivative (Ironomycin (2)) accumulate in lysosomes and sequester iron in this organelle. Herein, a library of salinomycin derivatives is reported, including products of C20-amination, C1-esterification, C9-oxidation, and C28-dehydration. The biological activity of these compounds is evaluated against transformed human mammary epithelial HMLER CD24 /CD44 cells, a well-established model of breast CSC, and HMLER CD24 /CD44 cells deprived of CSC properties. Unlike other structural alterations, derivative 4, which displays a cyclopropylamine at position C20, showed a strikingly low IC value of 23 nm against HMLER CD24 /CD44 cells. This study provides highly selective molecules to target the CSC niche, a potential interesting advance for drug development to prevent cancer resistance.
Cancer stem cells (CSC) constitute a subpopulation of cells in solid tumors that is responsible for resistance to conventional chemotherapy, metastasis and cancer relapse. The natural product Salinomycin can selectively target this cell niche by directly interacting with lysosomal iron, taking advantage of upregulated iron homeostasis in CSC. Here, we identify inhibitors of the divalent metal transporter 1 (DMT1) that selectively target CSC by blocking lysosomal iron translocation. This leads to lysosomal iron accumulation, production of reactive oxygen species and cell death with features of ferroptosis. DMT1 inhibitors selectively target CSC in primary cancer cells and circulating tumor cells, demonstrating the physiological relevance of this strategy. Taken together, this opens up opportunities to tackle unmet needs in anti-cancer therapy.
The consequences of alterations in the distribution of intracellular organelles, observed in many diseases, are often not clear. Intracellular organelles alter their morphology and positioning to regulate cell homeostasis and function. We outline how organelle positioning can be studied employing a density-based analysis of 3D images applied to cells that show similar cellular geometries. Quantification is facilitated by the use of single cells seeded on micropatterned substrates that provide cues for controlled cell spreading. This minimal system mimics the reproducible distribution of organelles typically observed in tissues, simplifying image analysis and minimizing the number of cells required for the observation of robust phenotypes. Here we provide guidelines for how the majority of organelles can be efficiently analyzed in cells seeded on adhesive micropatterns. We exemplify how alterations in the positioning of different organelles as a result of the perturbation of the cytoskeleton or associated motor proteins can be efficiently quantified. © 2018 by John Wiley & Sons, Inc.
Distinct histone variants mark chromatin domains in the nucleus. To understand how these marks are established and maintained, one has to decipher how the dynamic distribution of these variants is orchestrated. These dynamics are associated with all DNA-based processes such as DNA replication, repair, transcription, heterochromatin formation and chromosome segregation. Key factors, known as histone chaperones, have been involved in escorting histones, thereby contributing to the chromatin landscape of given cell types. SNAP-tag-based imaging system enables the distinction between old and newly deposited histones, and has proved to be a powerful method for the visualization of histone variant dynamics on a cell-by-cell basis. This approach enables the tracking of specific variants in vivo and defining their timing and mode of deposition throughout the cell cycle and in different nuclear territories. Here, we provide a detailed protocol to exploit the SNAP-tag technology to assess the dynamics of newly synthesized and old histones. We then show that combining the SNAP-tagging of histones with the knockdown of candidate factors, represents an effective approach to decipher the role of key actors in guiding histone dynamics. Here, we specifically illustrate how this strategy was used to identify the essential role of the chaperone HIRA in deposition of newly synthesized histone variant H3.3.
Several studies for the clinical validity of circulating tumor cells (CTCs) in metastatic breast cancer were conducted showing that it is a prognostic biomarker of overall survival. In this work, we consider an individual patient data meta-analysis for nonmetastatic breast cancer to assess the discrimination of CTCs regarding the risk of death. Data are collected in several centers and present correlated failure times for subjects of the same center. However, although the covariate-specific time-dependent receiver operating characteristic (ROC) curve has been widely used for assessing the performance of a biomarker, there is no methodology yet that can handle this specific setting with clustered censored failure times. We propose an estimator for the covariate-specific time-dependent ROC curves and area under the ROC curve when clustered failure times are detected. We discuss the assumptions under which the estimators are consistent and their interpretations. We assume a shared frailty model for modeling the effect of the covariates and the biomarker on the outcome in order to account for the cluster effect. A simulation study was conducted and it shows negligible bias for the proposed estimator and a nonparametric one based on inverse probability censoring weighting, while a semiparametric estimator, ignoring the clustering, is markedly biased. Finally, in our application to breast cancer data, the estimation of the covariate-specific area under the curves illustrates that the CTCs discriminate better patients with inflammatory tumor than patients with noninflammatory tumor, with respect to their risk of death.
The BRCA2 tumor suppressor protein is involved in the maintenance of genome integrity through its role in homologous recombination. In mitosis, BRCA2 is phosphorylated by Polo-like kinase 1 (PLK1). Here we describe how this phosphorylation contributes to the control of mitosis. We identify a conserved phosphorylation site at T207 of BRCA2 that constitutes a bona fide docking site for PLK1 and is phosphorylated in mitotic cells. We show that BRCA2 bound to PLK1 forms a complex with the phosphatase PP2A and phosphorylated-BUBR1. Reducing BRCA2 binding to PLK1, as observed in BRCA2 breast cancer variants S206C and T207A, alters the tetrameric complex resulting in unstable kinetochore-microtubule interactions, misaligned chromosomes, faulty chromosome segregation and aneuploidy. We thus reveal a role of BRCA2 in the alignment of chromosomes, distinct from its DNA repair function, with important consequences on chromosome stability. These findings may explain in part the aneuploidy observed in BRCA2-mutated tumors.
The metabolism of PI(3,5)P2 is regulated by the PIKfyve, VAC14 and FIG4 complex, mutations in which are associated with hypopigmentation in mice. These pigmentation defects indicate a key, but as yet unexplored, physiological relevance of this complex in the biogenesis of melanosomes. Here, we show that PIKfyve activity regulates formation of amyloid matrix composed of PMEL protein within the early endosomes in melanocytes, called stage I melanosomes. PIKfyve activity controls the membrane remodeling of stage I melanosomes, which regulates PMEL abundance, sorting and processing. PIKfyve activity also affects stage I melanosome kiss-and-run interactions with lysosomes, which are required for PMEL amyloidogenesis and the establishment of melanosome identity. Mechanistically, PIKfyve activity promotes both the formation of membrane tubules from stage I melanosomes and their release by modulating endosomal actin branching. Taken together, our data indicate that PIKfyve activity is a key regulator of the melanosomal import-export machinery that fine tunes the formation of functional amyloid fibrils in melanosomes and the maintenance of melanosome identity.This article has an associated First Person interview with the first author of the paper.
Once thought to be a remnant of cell division, the midbody (MB) has recently been shown to have roles beyond its primary function of orchestrating abscission. Despite the emerging roles of post-abscission MBs, how MBs accumulate in the cytoplasm and signal to regulate cellular functions remains unknown. Here, we show that extracellular post-abscission MBs can be internalized by interphase cells, where they reside in the cytoplasm as a membrane-bound signaling structure that we have named the MBsome. We demonstrate that MBsomes stimulate cell proliferation and that MBsome formation is a phagocytosis-like process that depends on a phosphatidylserine/integrin complex, driven by actin-rich membrane protrusions. Finally, we show that MBsomes rely on dynamic actin coats to slow lysosomal degradation and propagate their signaling function. In summary, MBsomes may sometimes serve as intracellular organelles that signal via integrin and EGFR-dependent pathways to promote cell proliferation and anchorage-independent growth and survival.
Phenotypic cell-based assays have proven to be efficient at discovering first-in-class therapeutic drugs mainly because they allow for scanning a wide spectrum of possible targets at once. However, despite compelling methodological advances, posterior identification of a compound’s mechanism of action (MOA) has remained difficult and highly refractory to automated analyses. Methods such as the cell painting assay and multiplexing fluorescent dyes to reveal broadly relevant cellular components were recently suggested for MOA prediction. We demonstrated that adding fluorescent dyes to a single assay has limited impact on MOA prediction accuracy, as monitoring only the nuclei stain could reach compelling levels of accuracy. This observation suggested that multiplexed measurements are highly correlated and nuclei stain could possibly reflect the general state of the cell. We then hypothesized that combining unrelated and possibly simple cell-based assays could bring a solution that would be biologically and technically more relevant to predict a drug target than using a single assay multiplexing dyes. We show that such a combination of past screen data could rationally be reused in screening facilities to train an ensemble classifier to predict drug targets and prioritize a possibly large list of unknown compound hits at once.
High-content screening is an important tool in drug discovery and characterization. Often, high-content drug screens are performed on one single-cell line. Yet, a single-cell line cannot be thought of as a perfect disease model. Many diseases feature an important molecular heterogeneity. Consequently, a drug may be effective against one molecular subtype of a disease, but less so against another. To characterize drugs with respect to their effect not only on one cell line but on a panel of cell lines is therefore a promising strategy to streamline the drug discovery process.
Using a cell-based assay monitoring differential protein transport in the secretory pathway coupled to high-content screening, we have identified three molecules that specifically reduce the delivery of the major co-receptor for HIV-1, CCR5, to the plasma membrane. They have no effect on the closely related receptors CCR1 and CXCR4. These molecules are also potent in primary macrophages as they markedly decrease HIV entry. At the molecular level, two of these molecules inhibit the critical palmitoylation of CCR5 and thereby block CCR5 in the early secretory pathway. Our results open a clear therapeutics avenue based on trafficking control and demonstrate that preventing HIV infection can be performed at the level of its receptor delivery.
The steady-state localization of Golgi-resident glycosylation enzymes in the Golgi apparatus depends on a balance between anterograde and retrograde transport. Using the Retention Using Selective Hooks (RUSH) assay and high-content screening, we identified small molecules that perturb the localization of Mannosidase II (ManII) used as a model cargo for Golgi resident enzymes. In particular, we found that two compounds known as EGFR tyrosine kinase inhibitors, namely BML-265 and Tyrphostin AG1478 disrupt Golgi integrity and abolish secretory protein transport of diverse cargos, thus inducing brefeldin A-like effects. Interestingly, BML-265 and Tyrphostin AG1478 affect Golgi integrity and transport in human cells but not in rodent cells. The effects of BML-265 are reversible since Golgi integrity and protein transport are quickly restored upon washout of the compounds. BML-265 and Tyrphostin AG1478 do not lead to endosomal tubulation suggesting that, contrary to brefeldin A, they do not target the -Golgi ARF GEF BIG1 and BIG2. They quickly induce COPI dissociation from Golgi membranes suggesting that, in addition to EGFR kinase, the -Golgi ARF GEF GBF1 might also be a target of these molecules. Accordingly, overexpression of GBF1 prevents the effects of BML-265 and Tyrphostin AG1478 on Golgi integrity.
Monolayered epithelia are composed of tight cell assemblies that ensure polarized exchanges. EpCAM, an unconventional epithelial-specific cell adhesion molecule, is assumed to modulate epithelial morphogenesis in animal models, but little is known regarding its cellular functions. Inspired by the characterization of cellular defects in a rare EpCAM-related human intestinal disease, we find that the absence of EpCAM in enterocytes results in an aberrant apical domain. In the course of this pathological state, apical translocation towards tricellular contacts (TCs) occurs with striking tight junction belt displacement. These unusual cell organization and intestinal tissue defects are driven by the loss of actomyosin network homoeostasis and contractile activity clustering at TCs, yet is reversed by myosin-II inhibitor treatment. This study reveals that adequate distribution of cortical tension is crucial for individual cell organization, but also for epithelial monolayer maintenance. Our data suggest that EpCAM modulation protects against epithelial dysplasia and stabilizes human tissue architecture.