Biology subjects

Misteli, T.

Publications and source records attributed to Misteli, T..

33 records · Page 2Linked to original sources

bioRxiv2021

HiIDDD: A high-throughput imaging pipeline for the quantitative detection of DNA damage in primary human immune cells

DNA damage is a prominent biomarker for numerous diseases, including cancer and aging. Detection of DNA damage routinely relies on traditional microscopy or cytometric methods. However, these techniques are typically of limited throughput and are not ideally suited for large-scale longitudinal and population studies that require analysis of large sample sets. We have developed HiIDDD (High-throughput Immune cell DNA Damage Detection), a robust, semiquantitative and single-cell assay that measures DNA damage by high-throughput imaging using the two major DNA damage markers 53BP1 and {gamma}-H2AX. We demonstrate sensitive detection of DNA damage in a wide set of freshly isolated and cryopreserved primary human immune cells, including CD4+ and CD8+ T cells, B cells and monocytes with low inter-assay variability. As proof of principle, we demonstrate parallel batch processing of several immune cell types from multiple donors. We find common patterns of DNA damage in multiple immune cell types of donors of varying ages, suggesting that immune cell properties are specific to individuals. These results establish a novel high-throughput assay for the evaluation of DNA damage in large-scale studies.

cell biology↗

bioRxiv2021

Large-scale mapping of positional changes of hypoxia-responsive genes upon activation

Chromosome structure and nuclear organization are important factors in the regulation of gene expression. Transcription of a gene is influenced by local and global chromosome features such as condensation status and histone modifications. The relationship between the position of a gene in the cell nucleus and its activity is less clear. Here, we used high-throughput imaging to perform a large-scale analysis of the spatial location of a set of nearly 100 hypoxia-inducible genes to determine whether their location within the nucleus is correlated with their activity state upon stimulation. Radial distance analysis demonstrated that the majority of HIF- and CREB-inducible hypoxia responsive genes are located in the intermediate region of the nucleus. Radial position of numerous responsive genes changed upon hypoxic treatment. Analysis of the relative distances amongst a subset of HIF target gene groups revealed that some gene pairs also altered their relative location to each other upon hypoxic treatment, suggesting higher order chromatin rearrangements. While these changes in location occurred in response to hypoxic activation of the target genes, they did not correlate with the extent of their activation. These results suggest that induction of the hypoxia-responsive gene expression program is accompanied by spatial alterations of the genome, but that radial and relative gene positions are not directly related to gene activity.

cell biology↗

bioRxiv2021

Activation of endoplasmic reticulum stress via clustering of inner nuclear membrane proteins

One of the major cellular mechanisms to ensure protein homeostasis is the endoplasmic reticulum (ER) stress response. This pathway is typically triggered by accumulation of misfolded proteins in the ER lumen. Here we describe activation of ER stress via protein aggregation in the cell nucleus. We find in the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) activation of ER stress due to the aggregation of the diseases-causing progerin protein at the nuclear envelope. The presence of nucleoplasmic protein aggregates is sensed and signaled to the ER lumen via immobilization and clustering of the inner nuclear membrane protein SUN2, leading to activation of the Unfolded Protein Response (UPR). These results identify a nuclear trigger of ER stress and they provide insight into the molecular disease mechanisms of HGPS.

cell biology↗

bioRxiv2020

A Deep Learning Pipeline for Nucleus Segmentation

Deep learning is rapidly becoming the technique of choice for automated segmentation of nuclei in biological image analysis workflows. In order to evaluate the feasibility of training nuclear segmentation models on small, custom annotated image datasets that have been augmented, we have designed a computational pipeline to systematically compare different nuclear segmentation model architectures and model training strategies. Using this approach, we demonstrate that transfer learning and tuning of training parameters, such as the composition, size and pre-processing of the training image dataset, can lead to robust nuclear segmentation models, which match, and often exceed, the performance of existing, off-the-shelf deep learning models pre-trained on large image datasets. We envision a practical scenario where deep learning nuclear segmentation models trained in this way can be shared across a laboratory, facility, or institution, and continuously improved by training them on progressively larger and varied image datasets. Our work provides computational tools and a practical framework for deep learning-based biological image segmentation using small annotated image datasets.

cell biology↗

bioRxiv2019

Self-assembly of multi-component mitochondrial nucleoids via phase separation

Mitochondria contain an autonomous and spatially segregated genome. The organizational unit of their genome is the nucleoid, which consists of mitochondrial DNA (mtDNA) and associated architectural proteins. Here, we show that phase separation is the primary physical mechanism for assembly and size-control of the mitochondrial nucleoid. The major mtDNA-binding protein TFAM spontaneously phase separates in vitro via weak, multivalent interactions into viscoelastic droplets with slow internal dynamics. In combination, TFAM and mtDNA form multiphase, gel-like structures in vitro, which recapitulate the in vivo dynamic behavior of mt-nucleoids. Enlarged, phase-separated, yet transcriptionally active, nucleoids are present in mitochondria from patients with the premature aging disorder Hutchinson-Gilford Progeria Syndrome (HGPS) and are associated with mitochondrial dysfunction. These results point to phase separation as an evolutionarily conserved mechanism of genome organization. HighlightsO_LIMitochondrial genomes are organized by phase separation. C_LIO_LIThe main packaging protein TFAM and mtDNA combine to form viscoelastic, multiphase droplets in vitro. C_LIO_LIMitochondrial nucleoids exhibit phase behavior in vivo, including dynamic rearrangements and heterogenous organization. C_LIO_LICoalescence and enlargement of mt-nucleoids occur upon loss of mitochondrial homeostasis as well as in prematurely aged cells and are associated with mitochondrial dysfunction. C_LI

cell biology↗

bioRxiv2019

Assessment of the Utility of Gene Positioning Biomarkers in the Stratification of Prostate Cancers

In eukaryotic cells, the genome is spatially organized in a non-random fashion within the confines of the interphase nucleus, and most genes occupy preferred nuclear positions. For some genomic loci these positioning patterns are context specific, reflected in the distinct location of certain genes and chromosomes in different cell types and in disease. Disease-related differential spatial positioning of genes has led to the hypothesis that the spatial reorganization of the genome may be utilized as a diagnostic biomarker. In keeping with this possibility, the positioning patterns of specific genes can be used to reproducibly discriminate benign tissues from cancerous ones. In addition to the use of spatial genome organization for diagnostic purposes, we explore here the potential use of spatial genome organization as a prognostic tool. This is a pressing need since in many cancer types there is a lack of accurate markers to predict the aggressiveness of individual tumors. We find that directional repositioning of SP100 and TGFB3 gene loci stratifies prostate cancers of differing Gleason scores. A more peripheral position of SP100 and TGFB3 in the nucleus, compared to benign tissues, is associated with low Gleason score cancers, whereas more internal positioning correlates with higher Gleason scores. Conversely, LMNA is more internally positioned in many non-metastatic prostate cancers, while its position is indistinguishable from benign tissue in metastatic cancer. Our findings of subtype-specific gene positioning patterns in prostate cancer provides a proof-of-concept for the potential usefulness of spatial gene positioning as a prognostic biomarker.

cancer biology↗

bioRxiv2019

The nucleoporin ELYS regulates nuclear size by controlling NPC number and nuclear import capacity

How intracellular organelles acquire their characteristic sizes is a fundamental cell biological question. Given the stereotypical changes in nuclear size in cancer, it is particularly important to understand the mechanisms that control nuclear size in human cells. Here we use a high-throughput imaging RNAi screen to identify and mechanistically characterize ELYS, a nucleoporin required for postmitotic nuclear pore complex (NPC) assembly, as a determinant of nuclear size in mammalian cells. We show that ELYS knockdown results in small nuclei, the accumulation of cytoplasmic lamin aggregates, reduced nuclear lamin B2 localization, lower NPC density, and decreased nuclear import. Increasing nuclear import by importin overexpression rescues nuclear size and lamin B2 import, while inhibiting importin /{beta} nuclear import decreases nuclear size. Conversely, ELYS overexpression leads to increased nuclear size, enrichment of nuclear lamin B2 staining at the nuclear periphery, and elevated NPC density and nuclear import. Consistent with these observations, knockdown or inhibition of exportin 1 increases nuclear size. In summary, we identify ELYS and NPC density as novel positive effectors of mammalian nuclear size and propose that nuclear size is controlled by nuclear import capacity.

cell biology↗

bioRxiv2018

Interplay of primary sequence and RNA secondary structure in determining 5′ splice site choice

Selective use of 5' splice sites is a common mechanism by which pre-mRNAs are alternatively spliced. Whereas the sequence requirements of 5' splice site choice have been well characterized, other important determinants remain poorly defined. Here we apply a combination of structural mapping by SHAPE-MaP and targeted mutational analysis in a cell-based system to comprehensively probe the interplay of primary sequence, secondary RNA structure, regulatory elements and linear splice site position to determine mechanisms of splice site choice in vivo. Using the disease-causing alternative 5' splice site selection in LMNA in the premature aging disorder Hutchinson-Gilford Progeria Syndrome as a model system, we identify RNA secondary structural elements near the alternative 5' splice sites. We show that splice site choice is significantly influenced by the structural context of the available splice sites. While local structure alone is not sufficient to account for splice site selection, the choice of 5' splice sites depends on the structural stability of the 5' splice site region which is conferred by downstream elements. In addition, relative positioning of the competing sites within the primary sequence of the pre-mRNA is a predictor of 5' splice site usage, with the distal position favored over the proximal, regardless of sequence composition. Together, these results reveal an intricate interplay amongst RNA sequence, secondary structure and splice site position in determining 5' splice site choice.

molecular biology↗

bioRxiv2018

HiPLA: High-throughput imaging Proximity Ligation Assay

Protein-protein interactions are essential for cellular structure and function. To delineate how the intricate assembly of protein interactions contribute to cellular processes in health and disease, new methodologies that are both highly sensitive and can be applied at large scale are needed. Here, we develop HiPLA (high-throughput imaging proximity ligation assay), a method that employs the antibody-based proximity ligation assay in a high-throughput imaging screening format to systematically probe protein interactomes. Using HiPLA, we probe the interaction of 60 proteins and associated PTMs with the nuclear lamina in a model of the premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS). We identify a subset of proteins that differentially interact with the nuclear lamina in HGPS. In combination with quantitative indirect immunofluorescence, we find that the majority of differential interactions were accompanied by corresponding changes in expression of the interacting protein. Taken together, HiPLA offers a novel approach to probe cellular protein-protein interaction at a large scale and reveals mechanistic insights into the assembly of protein complexes.

cell biology↗

bioRxiv2018

Single molecule analysis of lamin dynamics

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 (NETs) 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 A {Delta}50 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 how pathogenic mutations or cellular processes affect protein dynamics at the NE.

cell biology↗

bioRxiv2018

Effects of Human Sex Chromosome Dosage on Spatial Chromosome Organization

Sex chromosome aneuploidies (SCAs) are common genetic syndromes characterized by the presence of an aberrant number of X and Y chromosomes due to meiotic defects. These conditions impact structure and function of diverse tissues, but the proximal effects of SCA on genome organization are unknown. Here, to determine the consequences of SCAs on global genome organization, we have analyzed multiple architectural features of chromosome organization in a comprehensive set of primary cells from SCA patients with various ratios of X and Y chromosomes by use of imaging-based high-throughput Chromosome Territory Mapping (HiCTMap). We find that X chromosome supernumeracy does not affect the size, volume or nuclear position of the Y chromosome or an autosomal chromosome. In contrast, the active X chromosome undergoes architectural changes as a function of increasing X copy number, as measured by a decrease in size and an increase in circularity, which is indicative of chromatin compaction. With Y chromosome supernumeracy, Y chromosome size is reduced suggesting higher chromatin condensation. The radial positioning of chromosomes is unaffected in SCA karyotypes. Taken together, these observations document changes in genome architecture in response to alterations in sex chromosome numbers and point to trans-effects of dosage compensation on chromosome organization.

cell biology↗

bioRxiv2018

RefCell: Multi-dimensional analysis of image-based high-throughput screens based on ‘typical cells’

BackgroundImage-based high-throughput screening (HTS) reveals a high level of heterogeneity in single cells and multiple cellular states may be observed within a single population. Cutting-edge high-dimensional analysis methods are successful in characterizing cellular heterogeneity, but they suffer from the \"curse of dimensionality\" and non-standardized outputs.\n\nResultsHere we introduce RefCell, a multi-dimensional analysis pipeline for image-based HTS that reproducibly captures cells with typical combinations of features in reference states, and uses these \"typical cells\" as a reference for classification and weighting of metrics. RefCell quantitatively assesses the heterogeneous deviations from typical behavior for each analyzed perturbation or sample.\n\nConclusionsWe apply RefCell to the analysis of data from a high-throughput imaging screen of a library of 320 ubiquitin protein targeted siRNAs selected to gain insights into the mechanisms of premature aging (progeria). RefCell yields results comparable to a more complex clustering based single cell analysis method, which both reveal more potential hits than conventional average based analysis.

bioinformatics↗

bioRxiv2017

HiCTMap: Detection and Analysis of Chromosome Territory Structure and Position by High-throughput Imaging

The spatial organization of chromosomes in the nuclear space is an extensively studied field that relies on measurements of structural features and 3D positions of chromosomes with high precision and robustness. However, no tools are currently available to image and analyze chromosome territories in a high-throughput format. Here, we have developed High-throughput Chromosome Territory Mapping (HiCTMap), a method for the robust and rapid analysis of 2D and 3D chromosome territory positioning in mammalian cells. HiCTMap is a high-throughput imaging-based chromosome detection method which enables routine analysis of chromosome structure and nuclear position. Using an optimized FISH staining protocol in a 384-well plate format in conjunction with a bespoke automated image analysis workflow, HiCTMap faithfully detects chromosome territories and their position in 2D and 3D in a large population of cells per experimental condition. We apply this novel technique to visualize chromosomes 18, X, and Y in male and female primary human skin fibroblasts, and show accurate detection of the correct number of chromosomes in the respective genotypes. Given the ability to visualize and quantitatively analyze large numbers of nuclei, we use HiCTMap to measure chromosome territory area and volume with high precision and determine the radial position of chromosome territories using either centroid or equidistant-shell analysis. The HiCTMap protocol is also compatible with RNA FISH as demonstrated by simultaneous labeling of X chromosomes and Xist RNA in female cells. We suggest HiCTMap will be a useful tool for routine precision mapping of chromosome territories in a wide range of cell types and tissues.

cell biology↗

bioRxiv2017

Heterogeneity and Intrinsic Variation in Spatial Genome Organization

The genome is hierarchically organized in 3D space and its architecture is altered in differentiation, development and disease. Some of the general principles that determine global 3D genome organization have been established. However, the extent and nature of cell-to-cell and cell-intrinsic variability in genome architecture are poorly characterized. Here, we systematically probe the heterogeneity in genome organization in human fibroblasts by combining high-resolution Hi-C datasets and high-throughput genome imaging. Optical mapping of several hundred genome interaction pairs at the single cell level demonstrates low steady-state frequencies of colocalization in the population and independent behavior of individual alleles in single nuclei. Association frequencies are determined by genomic distance, higher-order chromatin architecture and chromatin environment. These observations reveal extensive variability and heterogeneity in genome organization at the level of single cells and alleles and they demonstrate the coexistence of a broad spectrum of chromatin and genome conformations in a cell population.

molecular biology↗

bioRxiv2017

Causes and consequences of nuclear gene positioning

The eukaryotic genome is organized in a manner that allows folding of the genetic material in the confined space of the cell nucleus, while at the same time enabling its physiological function. A major principle of spatial genome organization is the non-random position of genomic loci relative to other loci and to nuclear bodies. The mechanisms that determine the spatial position of a locus, and how position affects function, are just beginning to be characterized. Initial results suggest multiple, gene-specific mechanisms and the involvement of a wide range of cellular machineries. In this Commentary, we review recent findings from candidate approaches and unbiased screening methods that provide initial insight into the cellular mechanisms of positioning and their functional consequences. We highlight several specific mechanisms including tethering to the nuclear periphery, passage through replication and histone modifications that contribute to gene positioning in yeast, plants and mammals.

molecular biology↗