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Figure S2. Covariance matrix (right) and hierarchical clustering plot (left) of 15 measures of nuclear shape and lamin A/C fluorescence intensity. Each box in the covariance matrix indicates the amount of correlation between two measures. High covariance is indicated in red (see color bar).
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The premature aging disorder, Hutchinson-Gilford progeria syndrome (HGPS), is caused by mutant lamin A, which affects the nuclear scaffolding. The phenotypic hallmark of HGPS is nuclear blebbing. Interestingly, similar nuclear blebbing has also been observed in aged cells from healthy individuals. Recent work has shown that treatment with rapamycin...
Citations
... Even so, the approach may have limitations in clarifying relevant biophysical interpretations due to the multiple shape parameters. In addition, some scalar descriptors including roughness, shape factor, and curvature, are often used to characterize the morphology of cells and nuclei [33][34][35] , and the corresponding analysis aims to capture more information to study and distinguish biological states closely related to morphological dynamics 36 . Among these methods, the widely used CellProfiler 37 and MorpholibJ 38 (a plugin for ImageJ) allow us to extract a large number of features and further reveal the differences across varying cell populations. ...
... Taken together, there is increasing evidence that the study of cell morphology and its relationship to cell functions and migration modes can provide more insight into the mechanisms underlying cell migration. However, the approaches or tools used still have three shortcomings: i) the approaches are mainly used to analyze the morphological changes of single cells or nuclei, and it is unknown how they behave when used to analyze other objects such as cell spheroids [33][34][35] ; ii) the approaches involve many features that allow us to capture enough information of the shape, but these numerous descriptive features could limit the biophysical interpretation of shape changes 32,37,38 ; iii) the approaches are mainly developed based on machine learning algorithms and large amounts of morphological data, which may be limited in analyzing time-varying morphological features 27,29,30 . Therefore, quantifying morphological features of different objects in real-time using a simple and efficient approach becomes a major challenge. ...
Cell migration is crucial for numerous physiological and pathological processes. A cell adapts its morphology, including the overall and nuclear morphology, in response to various cues in complex microenvironments, such as topotaxis and chemotaxis during migration. Thus, the dynamics of cellular morphology can encode migration strategies, from which diverse migration mechanisms can be inferred. However, deciphering the mechanisms behind cell migration encoded in morphology dynamics remains a challenging problem. Here, we present a powerful universal metric, the Cell Morphological Entropy (CME), developed by combining parametric morphological analysis with Shannon entropy. The utility of CME, which accurately quantifies the complex cellular morphology at multiple length scales through the deviation from a perfectly circular shape, is illustrated using a variety of normal and tumor cell lines in different in vitro microenvironments. Our results show how geometric constraints affect the MDA-MB-231 cell nucleus, the emerging interactions of MCF-10A cells migrating on collagen gel, and the critical transition from proliferation to invasion in tumor spheroids. The analysis demonstrates that the CME-based approach provides an effective and physically interpretable tool to measure morphology in real-time across multiple length scales. It provides deeper insight into cell migration and contributes to the understanding of different behavioral modes and collective cell motility in more complex microenvironments.
... 4 To further quantify heterogeneity of cell populations, several image-processing algorithms have been developed. 2,[5][6][7][8] In particular, a machine-learning algorithm termed Visually Aided Morpho-Phenotyping Image Recognition (VAMPIRE), can evaluate the morphology of individual cells or nuclei in a population and subsequently classifies them into representative shape modes. 9 Thus, VAMPIRE provides an important tool to investigate changes in cell and nuclei morphology during tissue maturation and regeneration. ...
Tissue level properties are commonly studied using histological stains assessed with qualitative scoring methods. As qualitative evaluation is typically insensitive, quantitative analysis provides additional information about pathological mechanisms, but cannot capture structural heterogeneity across cell subpopulations. However, molecular analyses of cell and nuclear behavior have identified that cell and more recently also nuclear shape are highly associated with cell function and mal-function. This study combined a Visually Aided Morpho-Phenotyping Image Recognition (VAMPIRE) analysis that automatically segments cells based on their shape with an added capacity to further discriminate between cells in certain protein-rich extracellular matrix regions. We used tendon as a model system given the enormous changes in organization and cell and nuclear shape they undergo during aging and injury. Our results uncover that multiple shape modes of nuclei exist during maturity and aging in rat tendon and that distinct subgroups of cell nuclei shapes exist in proteoglycan-rich regions during aging. With injury, several immunomarkers (αSMA, CD31, CD146) were associated with more rounded shape modes. In human tendons, the cell nuclei at sites of injury were found to be more rounded relative to in uninjured tissues. To conclude, the tendon tissue changes occurring during aging and injury could be associated with a variation in cell nuclear morphology and the appearance of various region-specific subpopulations. Thus, the methodologies developed allow for a deeper understanding of cell heterogeneity during tendon aging and injury and may be extended to study further clinical applications. This article is protected by copyright. All rights reserved.
... [109][110][111][112][113][114] A. Additional metrics: Nuclear shape descriptors Defining which geometric properties should be used to characterize striated myocyte nuclei, requires further investigation into the mechanisms by which their morphology impacts their function, as well as understanding these mechanisms in the context of changes to the cytoskeleton. 49,90,115 Further, expanding nuclear morphology assessment to better characterize malformations through the use of additional shape descriptors [105][106][107]116,117 could yield more insight into the morphological changes in cardiomyocytes from patients with disease-causing mutations. Additionally, to more fully characterize nuclear morphology, it may be beneficial to evaluate the cytoskeletal architecture surrounding each nucleus and determine the cell type or cell state to which a nucleus belongs. ...
Unbiased evaluation of morphology is crucial to understanding development, mechanics, and pathology of striated muscle tissues. Indeed, the ability of striated muscles to contract and the strength of their contraction is dependent on their tissue-, cellular-, and cytoskeletal-level organization. Accordingly, the study of striated muscles often requires imaging and assessing aspects of their architecture at multiple different spatial scales. While an expert may be able to qualitatively appraise tissues, it is imperative to have robust, repeatable tools to quantify striated myocyte morphology and behavior that can be used to compare across different labs and experiments. There has been a recent effort to define the criteria used by experts to evaluate striated myocyte architecture. In this review, we will describe metrics that have been developed to summarize distinct aspects of striated muscle architecture in multiple different tissues, imaged with various modalities. Additionally, we will provide an overview of metrics and image processing software that needs to be developed. Importantly to any lab working on striated muscle platforms, characterization of striated myocyte morphology using the image processing pipelines discussed in this review can be used to quantitatively evaluate striated muscle tissues and contribute to a robust understanding of the development and mechanics of striated muscles.
... Another easy to compute ratio indicative of nuclear roundness is the nuclear length-to-width ratio or axis ratio. More frequently a related nuclear eccentricity parameter is calculated instead [31,32]. Eccentricity is a measure of the shape of the bounding ellipse; a circle has an eccentricity of 0, and a more elongated shape is associated with a higher eccentricity ( Figure 2a). ...
... This is a very good method to quantify nuclear shape as curvature provides a complete description of nuclear shape. Indeed, defining the mean negative curvature as the average of all the concave curvatures on the boundary of a nucleus, could accurately distinguish the highly-blebbed nuclei in fibroblasts from HGPS patients from the similarly-sized nuclei in healthy donor fibroblasts [31]. In addition, the same paper showed that the mean negative curvature was not dependent on imaging intensity or cell density. ...
Any given cell type has an associated “normal” nuclear morphology, which is important to maintain proper cellular functioning and safeguard genomic integrity. Deviations from this can be indicative of diseases such as cancer or premature aging syndrome. To accurately assess nuclear abnormalities, it is important to use quantitative measures of nuclear morphology. Here, we give an overview of several nuclear abnormalities, including micronuclei, nuclear envelope invaginations, blebs and ruptures, and review the current methods used for image-based quantification of these abnormalities. We discuss several parameters that can be used to quantify nuclear shape and compare their outputs using example images. In addition, we present new pipelines for quantitative analysis of nuclear blebs and invaginations. Quantitative analyses of nuclear aberrations and shape will be important in a wide range of applications, from assessments of cancer cell anomalies to studies of nucleus deformability under mechanical or other types of stress.
... Although cell division is a tightly regulated process, any damage and subsequent failure in the repair mechanism leads to various genomic modifications such as a change in the shape of the nucleus, formation of nuclear blebs (NBs), and micronuclei [1]. NBs are nuclear protrusions that are round to oval in shape and are attached to the nucleus either directly or with a narrow string, formed due to an imbalance between various proteins of the nuclear lamina [2,3]. NBs are regarded as one of the biomarkers for genomic instability [4]. ...
Introduction:
Tobacco contains several genotoxic agents including N-nitrosamine which has the potential to cause significant nuclear damage. Nuclear blebbing is a form of protrusion on the nuclear membrane and could potentially be caused by tobacco-induced genotoxicity and is closely associated with malignancy. Thus, the present study aimed to assess if tobacco-associated oral potentially malignant disorders including oral submucous fibrosis (OSF) and oral leukoplakia have a higher nuclear blebbing frequency than patients with normal oral mucosa with no history of tobacco use.
Methods:
The sample consisted of patients with OSF (n = 30) and oral leukoplakia (n = 10) and normal oral mucosa (n = 10). Exfoliated cells collected from the study groups were smeared on a clean microscopic slide and stained by May-Grunwald-Giemsa stain. A baseline frequency of nuclear blebbing was evaluated using a bright-field microscope with a ×100 objective. The number of nuclear blebbing per 1,000 epithelial cells was recorded and expressed in percentage. ANOVA, the Mann-Whitney U test, and Spearman's correlation were used to analyze the data.
Results:
The mean rank of distribution of nuclear blebbing showed significant difference between all 3 groups, with the highest frequency noted in leukoplakia, followed by oral submucous and normal oral mucosa. Within OSF, the frequency of nuclear blebbing significantly increased from early stage to advanced stage. In OSF, a statistically significant positive linear correlation was noted between duration (in years), frequency (per day) of tobacco use, clinical grading, and nuclear blebbing.
Discussion/conclusions:
The frequency of nuclear blebbing was significantly higher in oral potentially malignant disorders than normal mucosa. Nuclear blebbing also exhibited a strong dose- and time-dependent correlation with tobacco usage and clinical staging in OSF. The nuclear blebbing frequency could be a noninvasive, economic tool to assess malignant risk in tobacco-induced oral potentially malignant disorders.
... A popular approach to address this shortcoming consists of defining additional geometric and statistical descriptors of cells, some of which are based on the curvature and roughness of the cell and nuclear contours 14,30,40 . This has led to an expansion of morphological descriptors (see Box 1), with the premise that these additional descriptors would help to better define and differentiate cellular subtypes. ...
... Curvature-defined as the degree of deviation from a straight line. It is calculated as the reciprocal of the radius of a circle fitted at each boundary point 40 . ...
... In this section, we briefly describe and compare other methods used to characterize the morphology of cells. The most common approach to quantify cell/nuclear shape morphology is to use scalar descriptors such as shape factor, curvature, and roughness 14,30,40 . This type of analysis is based on discriminative methods that try to capture just enough information to distinguish and investigate biological states 17 . ...
Cell morphology encodes essential information on many underlying biological processes. It is commonly used by clinicians and researchers in the study, diagnosis, prognosis, and treatment of human diseases. Quantification of cell morphology has seen tremendous advances in recent years. However, effectively defining morphological shapes and evaluating the extent of morphological heterogeneity within cell populations remain challenging. Here we present a protocol and software for the analysis of cell and nuclear morphology from fluorescence or bright-field images using the VAMPIRE algorithm (https://github.com/kukionfr/VAMPIRE_open). This algorithm enables the profiling and classification of cells into shape modes based on equidistant points along cell and nuclear contours. Examining the distributions of cell morphologies across automatically identified shape modes provides an effective visualization scheme that relates cell shapes to cellular subtypes based on endogenous and exogenous cellular conditions. In addition, these shape mode distributions offer a direct and quantitative way to measure the extent of morphological heterogeneity within cell populations. This protocol is highly automated and fast, with the ability to quantify the morphologies from 2D projections of cells seeded both on 2D substrates or embedded within 3D microenvironments, such as hydrogels and tissues. The complete analysis pipeline can be completed within 60 minutes for a dataset of ~20,000 cells/2,400 images. This protocol describes VAMPIRE, an unsupervised machine-learning approach that can be used to quantify and categorize cellular morphology from fluorescence or bright-field images of cells grown in 2D, 3D and tissue slices.
... In in vitro analysis, rapamycin or its analog everolimus reduced nuclear blebbing, delayed cellular senescence and enhanced degradation of progerin in HGPS fibroblasts. [254][255][256] A phase I/II trial of everolimus in combination with lonafarnib in patients with HGPS and other progeroid laminopathies was started in 2015; the first results are still outstanding and expected for 2020. 66 Sulforaphane, an antioxidant derived from cruciferous vegetables, also stimulated progerin clearance by autophagy in HGPS fibroblasts. ...
Progeroid disorders make up a heterogeneous group of very rare hereditary diseases characterized by clinical signs that often mimic physiological aging in a premature manner. Apart from Hutchinson‐Gilford progeria syndrome, one of the best‐investigated progeroid disorders, a wide spectrum of other premature aging phenotypes exist, which differ significantly in their clinical presentation and molecular pathogenesis. Next‐generation sequencing (NGS)‐based approaches have made it feasible to determine the molecular diagnosis in the early stages of a disease. Nevertheless, a broad clinical knowledge on these disorders and their associated symptoms is still fundamental for a comprehensive patient management and for the interpretation of variants of unknown significance from NGS data sets. This review provides a detailed overview on characteristic clinical features and underlying molecular genetics of well‐known as well as only recently identified premature aging disorders and also highlights novel findings towards future therapeutic options.
... However, inhibition of farnesylation does not ameliorate the DNA damage phenotypes unless the treatment is combined with other drugs such as rapamycin [98]. Another clinical trial combines farnesylation inhibition with a mTOR pathway inhibitor called everolimus, similar to rapamycin [99,100] (https://clinicaltrials.gov/ct2/show/NCT02579044). A potential concern regarding these protocols is that prelamin A and B accumulate in the nucleoplasm when farnesylation is inhibited, which may have a deleterious effect in terms of incorporation of mature lamin into the nuclear lamina [101]. ...
The eukaryotic nucleus controls most cellular processes. It is isolated from the cytoplasm by the nuclear envelope, which plays a prominent role in the structural organization of the cell, including nucleocytoplasmic communication, chromatin positioning, and gene expression. Alterations in nuclear composition and function are eminently pronounced upon stress and during premature and physiological aging. These alterations are often accompanied by epigenetic changes in histone modifications. We review, here, the role of nuclear envelope proteins and histone modifiers in the 3-dimensional organization of the genome and the implications for gene expression. In particular, we focus on the nuclear lamins and the chromatin-associated protein BAF, which are linked to Hutchinson–Gilford and Nestor–Guillermo progeria syndromes, respectively. We also discuss alterations in nuclear organization and the epigenetic landscapes during normal aging and various stress conditions, ranging from yeast to humans.
... Rapamycin, a mechanistic target of rapamycin (mTOR) inhibitor, has been shown to decrease insoluble progerin aggregates within nuclei of HGPS fibroblasts through autophagic degradation (Cao et al. 2011, Cenni et al. 2011 and have an positive effect on MSC derived from HGPS iPS cells by reducing the number of progerin expressing cells (Blondel et al. 2014). Further, rapamycin has been demonstrated to reduce nuclear blebbing in HGPS fibroblast cells (Driscoll et al. 2012) and improve nuclear shape in MSC derived from HGPS iPS cells in a quarter of the population (Blondel et al. 2014). More clinically relevant rapalogues such as temsirolimus and everolimus generate a range of improvements in HGPS cells; with temsirolimus reducing progerin levels, increasing proliferation, correcting mis-shaped nuclei and improving the fraction of DNA damage foci (Gabriel et al. 2016). ...
Hutchinson–Gilford progeria syndrome (HGPS) is a rare, premature ageing syndrome in children. HGPS is normally caused by a mutation in the LMNA gene, encoding nuclear lamin A. The classical mutation in HGPS leads to the production of a toxic truncated version of lamin A, progerin, which retains a farnesyl group. Farnesyltransferase inhibitors (FTI), pravastatin and zoledronic acid have been used in clinical trials to target the mevalonate pathway in HGPS patients to inhibit farnesylation of progerin, in order to reduce its toxicity. Some other compounds that have been suggested as treatments include rapamycin, IGF1 and N-acetyl cysteine (NAC). We have analysed the distribution of prelamin A, lamin A, lamin A/C, progerin, lamin B1 and B2 in nuclei of HGPS cells before and after treatments with these drugs, an FTI and a geranylgeranyltransferase inhibitor (GGTI) and FTI with pravastatin and zoledronic acid in combination. Confirming other studies prelamin A, lamin A, progerin and lamin B2 staining was different between control and HGPS fibroblasts. The drugs that reduced progerin staining were FTI, pravastatin, zoledronic acid and rapamycin. However, drugs affecting the mevalonate pathway increased prelamin A, with only FTI reducing internal prelamin A foci. The distribution of lamin A in HGPS cells was improved with treatments of FTI, pravastatin and FTI + GGTI. All treatments reduced the number of cells displaying internal speckles of lamin A/C and lamin B2. Drugs targeting the mevalonate pathway worked best for progerin reduction, with zoledronic acid removing internal progerin speckles. Rapamycin and NAC, which impact on the MTOR pathway, both reduced both pools of progerin without increasing prelamin A in HGPS cell nuclei.
... The lateral location of individual nuclei was identified from the maximalintensity projection of the confocal stacks using a two-dimensional (2D) image processing algorithm (29). In this technique, an approximate initial boundary is converged to the actual boundary of the nucleus by an active contour method (30) (Fig. S2, A-C). ...
... A region slightly larger than the 2D boundary, five pixels on each side, was cropped from the confocal stack and processed individually for obtaining the boundary surface of the nucleus. Our algorithm is an extension of the method in (29) for 2D images to 3D. We first binarized the cropped volume using Otsu's threshold (31), estimated the boundary surface of the thresholded region, and further dilated the boundary surface to get a smooth, outer-boundary surface of the nucleus. ...
Morphology of the nucleus is an important regulator of gene expression. Nuclear morphology is in turn a function of the forces acting on it and the mechanical properties of the nuclear envelope. Here, we present a two-parameter, nondimensional mechanical model of the nucleus that reveals a relationship among nuclear shape parameters, such as projected area, surface area, and volume. Our model fits the morphology of individual nuclei and predicts the ratio between forces and modulus in each nucleus. We analyzed the changes in nuclear morphology of liver cells due to hepatitis C virus (HCV) infection using this model. The model predicted a decrease in the elastic modulus of the nuclear envelope and an increase in the pre-tension in cortical actin as the causes for the change in nuclear morphology. These predictions were validated biomechanically by showing that liver cells expressing HCV proteins possessed enhanced cellular stiffness and reduced nuclear stiffness. Concomitantly, cells expressing HCV proteins showed downregulation of lamin-A,C and upregulation of β-actin, corroborating the predictions of the model. Our modeling assumptions are broadly applicable to adherent, monolayer cell cultures, making the model amenable to investigate changes in nuclear mechanics due to other stimuli by merely measuring nuclear morphology. Toward this, we present two techniques, graphical and numerical, to use our model for predicting physical changes in the nucleus.
