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1.
Sci Rep ; 11(1): 10625, 2021 05 19.
Article in English | MEDLINE | ID: mdl-34012019

ABSTRACT

Children with Hutchinson-Gilford Progeria Syndrome (HGPS) suffer from multiple cardiovascular pathologies due to the expression of progerin, a mutant form of the nuclear envelope protein Lamin A. Progerin expression has a dramatic effect on arterial smooth muscle cells (SMCs) and results in decreased viability and increased arterial stiffness. However, very little is known about how progerin affects SMC contractility. Here, we studied the LaminAG609G/G609G mouse model of HGPS and found reduced arterial contractility at an early age that correlates with a decrease in smooth muscle myosin heavy chain (SM-MHC) mRNA and protein expression. Traction force microscopy on isolated SMCs from these mice revealed reduced force generation compared to wild-type controls; this effect was phenocopied by depletion of SM-MHC in WT SMCs and overcome by ectopic expression of SM-MHC in HGPS SMCs. Arterial SM-MHC levels are also reduced with age in wild-type mice and humans, suggesting a common defect in arterial contractility in HGPS and normal aging.


Subject(s)
Gene Expression Regulation , Muscle Contraction/physiology , Muscle, Smooth, Vascular/physiopathology , Myosin Heavy Chains/genetics , Progeria/genetics , Progeria/physiopathology , Smooth Muscle Myosins/genetics , Adult , Aged , Aged, 80 and over , Aging/pathology , Animals , Aorta/pathology , Aorta/physiopathology , Humans , Mice, Inbred C57BL , Middle Aged , Myosin Heavy Chains/metabolism , Smooth Muscle Myosins/metabolism
2.
Cell Rep ; 35(3): 109019, 2021 04 20.
Article in English | MEDLINE | ID: mdl-33882318

ABSTRACT

Reversible differentiation of vascular smooth muscle cells (VSMCs) plays a critical role in vascular biology and disease. Changes in VSMC differentiation correlate with stiffness of the arterial extracellular matrix (ECM), but causal relationships remain unclear. We show that VSMC plasticity is mechanosensitive and that both the de-differentiated and differentiated fates are promoted by the same ECM stiffness. Differential equations developed to model this behavior predicted that a null VSMC state generates the dual fates in response to ECM stiffness. Direct measurements of cellular forces, proliferation, and contractile gene expression validated these predictions and showed that fate outcome is mediated by Rac-Rho homeostasis. Rac, through distinct effects on YAP and TAZ, is required for both fates. Rho drives the contractile state alone, so its level of activity, relative to Rac, drives phenotypic choice. Our results show how the cellular response to a single ECM stiffness generates bi-stability and VSMC plasticity.


Subject(s)
Adaptation, Physiological , Mechanotransduction, Cellular/genetics , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Neuropeptides/genetics , rac1 GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/genetics , Actins/genetics , Actins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Differentiation , Cell Proliferation , Gene Expression Regulation , Male , Mice , Mice, Inbred C57BL , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/cytology , Neuropeptides/metabolism , Phenotype , Primary Cell Culture , Promoter Regions, Genetic , Single-Cell Analysis , Transcription, Genetic , YAP-Signaling Proteins/genetics , YAP-Signaling Proteins/metabolism , rac1 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/metabolism
3.
J Cell Biol ; 216(9): 2877-2889, 2017 09 04.
Article in English | MEDLINE | ID: mdl-28701425

ABSTRACT

Nonmuscle myosin II (NMII) is uniquely responsible for cell contractility and thus defines multiple aspects of cell behavior. To generate contraction, NMII molecules polymerize into bipolar minifilaments. Different NMII paralogs are often coexpressed in cells and can copolymerize, suggesting that they may cooperate to facilitate cell motility. However, whether such cooperation exists and how it may work remain unknown. We show that copolymerization of NMIIA and NMIIB followed by their differential turnover leads to self-sorting of NMIIA and NMIIB along the front-rear axis, thus producing a polarized actin-NMII cytoskeleton. Stress fibers newly formed near the leading edge are enriched in NMIIA, but over time, they become progressively enriched with NMIIB because of faster NMIIA turnover. In combination with retrograde flow, this process results in posterior accumulation of more stable NMIIB-rich stress fibers, thus strengthening cell polarity. By copolymerizing with NMIIB, NMIIA accelerates the intrinsically slow NMIIB dynamics, thus increasing cell motility and traction and enabling chemotaxis.


Subject(s)
Cell Polarity , Chemotaxis , Cytoskeleton/metabolism , Nonmuscle Myosin Type IIA/metabolism , Nonmuscle Myosin Type IIB/metabolism , Animals , COS Cells , Chlorocebus aethiops , Cytoskeleton/genetics , Microscopy, Fluorescence , Microscopy, Video , Nonmuscle Myosin Type IIA/genetics , Nonmuscle Myosin Type IIB/genetics , Protein Multimerization , Protein Stability , RNA Interference , Rats , Signal Transduction , Stress Fibers/metabolism , Time Factors , Transfection
4.
Methods ; 123: 66-75, 2017 07 01.
Article in English | MEDLINE | ID: mdl-28554525

ABSTRACT

In this article, we summarize current findings for the emergence of biophysical properties such as nuclear stiffness, chromatin compaction, chromosome positioning, and chromosome intermingling during stem cell differentiation, which eventually correlated with the changes of gene expression profiles during cellular differentiation. An overview is first provided to link stem cell differentiation with alterations in nuclear architecture, chromatin compaction, along with nuclear and chromatin dynamics. Further, we highlight the recent biophysical and molecular approaches, imaging methods and computational developments in characterizing transcription-related chromosome organization especially chromosome intermingling and nano-scale chromosomal contacts. Finally, the article ends with an outlook towards the emergence of a functional roadmap in setting up chromosome positioning and intermingling in a cell type specific manner during cellular differentiation.


Subject(s)
Cell Nucleus/metabolism , Chromosomes/chemistry , Embryonic Stem Cells/metabolism , Fibroblasts/metabolism , Genome , In Situ Hybridization, Fluorescence/methods , Animals , Cell Differentiation , Cell Nucleus/ultrastructure , Chromosomes/ultrastructure , DNA/genetics , DNA/metabolism , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Embryonic Stem Cells/ultrastructure , Fibroblasts/ultrastructure , Gene Expression Regulation , Histones/genetics , Histones/metabolism , Humans , Mice , Organ Specificity , Transcription, Genetic
5.
Biomaterials ; 35(8): 2411-9, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24388387

ABSTRACT

Embryonic stem (ES) cells exhibit plasticity in nuclear organization as well as variability in gene expression. Although such physicochemical features are important in lineage commitment, mechanistic insights coupling nuclear plasticity and gene expression have not been elucidated. To probe this, we developed single cell micro-patterned assay to map nuclear deformation and its correlation with gene expression. We found an inherent heterogeneity in nuclear pliability of ES cells. Softer nuclei deformed to the underlying substrate geometry while the stiffer ones remained spherical. Stiffer nuclei were strongly correlated with decreased global histone (H3) acetylation and an increase in Lamin A/C expression. Interestingly, these cells also have higher nuclear accumulation of the transcription cofactor MRTF-A (myocardin-related transcription factor A) and an upregulation of its downstream target genes. Taken together, our results provide compelling evidence to show that the mechanical heterogeneity of stem cell nucleus can regulate transcriptional programs during onset of cellular differentiation.


Subject(s)
Cell Differentiation , Cell Nucleus/metabolism , Embryonic Stem Cells/metabolism , Gene Expression Regulation, Developmental , Gene Expression , Acetylation , Animals , Cell Lineage/genetics , Cells, Cultured , Embryonic Stem Cells/cytology , Fibronectins/chemistry , Histones/genetics , Histones/metabolism , Humans , Mice , Microarray Analysis , Microscopy, Confocal , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Transcriptome , Up-Regulation
6.
Biophys J ; 104(3): 553-64, 2013 Feb 05.
Article in English | MEDLINE | ID: mdl-23442906

ABSTRACT

Stem cells integrate signals from the microenvironment to generate lineage-specific gene expression programs upon differentiation. Undifferentiated cell nuclei are easily deformable, with an active transcriptome, whereas differentiated cells have stiffer nuclei and condensed chromatin. Chromatin organization in the stem cell state is known to be highly dynamic but quantitative characterizations of its plasticity are lacking. Using fluorescence imaging, we study the spatio-temporal dynamics of nuclear architecture and chromatin compaction in mouse embryonic stem (ES) cells and differentiated states. Individual ES cells exhibit a relatively narrow variation in chromatin compaction, whereas primary mouse embryonic fibroblasts (PMEF) show broad distributions. However, spatial correlations in chromatin compaction exhibit an emergent length scale in PMEFs, although they are unstructured and longer ranged in ES cells. We provide evidence for correlated fluctuations with large amplitude and long intrinsic timescales, including an oscillatory component, in both chromatin compaction and nuclear area in ES cells. Such fluctuations are largely frozen in PMEF. The role of actin and Lamin A/C in modulating these fluctuations is described. A simple theoretical formulation reproduces the observed dynamics. Our results suggest that, in addition to nuclear plasticity, correlated spatio-temporal structural fluctuations of chromatin in undifferentiated cells characterize the stem cell state.


Subject(s)
Chromatin/chemistry , Embryonic Stem Cells/chemistry , Actins/metabolism , Animals , Cell Differentiation , Cell Nucleus/chemistry , Cell Nucleus/ultrastructure , Chromatin/metabolism , Cytoskeleton/metabolism , Cytoskeleton/ultrastructure , Embryonic Stem Cells/cytology , Embryonic Stem Cells/ultrastructure , Fibroblasts/chemistry , Fibroblasts/cytology , Lamins/metabolism , Mice
7.
PLoS One ; 7(9): e43718, 2012.
Article in English | MEDLINE | ID: mdl-22957031

ABSTRACT

Cellular differentiation programs are accompanied by large-scale changes in nuclear organization and gene expression. In this context, accompanying transitions in chromatin assembly that facilitates changes in gene expression and cell behavior in a developmental system are poorly understood. Here, we address this gap and map structural changes in chromatin organization during murine T-cell development, to describe an unusual heterogeneity in chromatin organization and associated functional correlates in T-cell lineage. Confocal imaging of DNA assembly in cells isolated from bone marrow, thymus and spleen reveal the emergence of heterogeneous patterns in DNA organization in mature T-cells following their exit from the thymus. The central DNA pattern dominated in immature precursor cells in the thymus whereas both central and peripheral DNA patterns were observed in naïve and memory cells in circulation. Naïve T-cells with central DNA patterns exhibited higher mechanical pliability in response to compressive loads in vitro and transmigration assays in vivo, and demonstrated accelerated expression of activation-induced marker CD69. T-cell activation was characterized by marked redistribution of DNA assembly to a central DNA pattern and increased nuclear size. Notably, heterogeneity in DNA patterns recovered in cells induced into quiescence in culture, suggesting an internal regulatory mechanism for chromatin reorganization. Taken together, our results uncover an important component of plasticity in nuclear organization, reflected in chromatin assembly, during T-cell development, differentiation and transmigration.


Subject(s)
Cell Movement , DNA/metabolism , Lymphocyte Activation , T-Lymphocytes/immunology , Animals , Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/metabolism , Bone Marrow Cells/cytology , Cell Lineage , Cell Nucleus/metabolism , Chromatin/metabolism , Hematopoietic Stem Cells/cytology , Lectins, C-Type/metabolism , Mice , Microscopy, Confocal/methods , Models, Biological , Models, Statistical , Sequence Analysis, DNA , Spleen/cytology , T-Lymphocytes/cytology
8.
Methods Cell Biol ; 98: 57-78, 2010.
Article in English | MEDLINE | ID: mdl-20816230

ABSTRACT

The interphase nucleus is an active organelle involved in processing genetic information. In higher order eukaryotes, information control is compartmentalized - for example at the scale of inter-chromosome territories and nuclear bodies. Regulatory proteins, nuclear bodies and chromatin assembly are found to be highly dynamic within the nucleus of primary cells and through cellular differentiation programs. In this chapter we describe live-cell fluorescence based techniques and single particle tracking analysis, to probe the spatio-temporal dimension in nuclear function.


Subject(s)
Cell Physiological Phenomena/genetics , Cells/metabolism , Chromatin Assembly and Disassembly/physiology , Transcription, Genetic/physiology , Animals , Cell Culture Techniques , Cells, Cultured , Fluorescence Recovery After Photobleaching/methods , Humans , Kinetics , Models, Theoretical , Spectrometry, Fluorescence/methods
9.
Biophys J ; 96(9): 3832-9, 2009 May 06.
Article in English | MEDLINE | ID: mdl-19413989

ABSTRACT

Cellular differentiation and developmental programs require changing patterns of gene expression. Recent experiments have revealed that chromatin organization is highly dynamic within living cells, suggesting possible mechanisms to alter gene expression programs, yet the physical basis of this organization is unclear. In this article, we contrast the differences in the dynamic organization of nuclear architecture between undifferentiated mouse embryonic stem cells and terminally differentiated primary mouse embryonic fibroblasts. Live-cell confocal tracking of nuclear lamina evidences highly flexible nuclear architecture within embryonic stem cells as compared to primary mouse embryonic fibroblasts. These cells also exhibit significant changes in histone and heterochromatin binding proteins correlated with their distinct epigenetic signatures as quantified by immunofluorescence analysis. Further, we follow histone dynamics during the development of the Drosophila melanogaster embryo, which gives an insight into spatio-temporal evolution of chromatin plasticity in an organismal context. Core histone dynamics visualized by fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and fluorescence anisotropy within the developing embryo, revealed an intriguing transition from plastic to frozen chromatin assembly synchronous with cellular differentiation. In the embryo, core histone proteins are highly mobile before cellularization, actively exchanging with the pool in the yolk. This hyperdynamic mobility decreases as cellularization and differentiation programs set in. These findings reveal a direct correlation between the dynamic transitions in chromatin assembly with the onset of cellular differentiation and developmental programs.


Subject(s)
Cell Differentiation/physiology , Chromatin/metabolism , Embryonic Development/physiology , Animals , Cells, Cultured , Drosophila melanogaster , Elasticity , Fibroblasts/physiology , Fluorescence Polarization , Fluorescence Recovery After Photobleaching , Fluorescent Antibody Technique , Heterochromatin/metabolism , Histones/metabolism , Kinetics , Lamins/metabolism , Mice , Microscopy, Confocal , Spectrometry, Fluorescence , Stem Cells/physiology
10.
Physiol Genomics ; 25(2): 203-15, 2006 Apr 13.
Article in English | MEDLINE | ID: mdl-16403844

ABSTRACT

To define gene expression profiles that occur during the initial activation of human innate immunity, we administered intravenous endotoxin (n = 8) or saline (n = 4) to healthy subjects and hybridized RNA from blood mononuclear cells (0, 0.5, 6, 24, 168 h) or whole blood (0, 3, 6, 24, 168 h) to oligonucleotide probe arrays. The greatest change in mononuclear cell gene expression occurred at 6 h (439 induced and 428 repressed genes, 1% false discovery rate, and 50% fold change) including increased expression of genes associated with pathogen recognition molecules and signaling cascades linked to receptors associated with cell mobility and activation. Induced defense response genes included cytokines, chemokines, and their respective receptors, acute-phase transcription factors, proteases, arachidonate metabolites, and oxidases. Repressed defense response genes included those associated with co-stimulatory molecules, T and cytotoxic lymphocytes, natural killer (NK) cells, and protein synthesis. Gene expression profiles of whole blood had similar biological themes. Over 100 genes not typically associated with acute inflammation were differentially regulated after endotoxin. By 24 h, gene expression had returned to baseline values. Thus the inflammatory response of circulating leukocytes to endotoxin in humans is characterized by a rapid amplification and subsidence of gene expression. These results indicate that a single intravascular exposure to endotoxin produces a large but temporally short perturbation of the blood transcriptome.


Subject(s)
Endotoxemia/immunology , Endotoxins/toxicity , Gene Expression Regulation , Immunity, Innate , Leukocytes, Mononuclear/metabolism , Adult , Chemokines/genetics , Chemokines/metabolism , Cytokines/genetics , Cytokines/metabolism , Endotoxemia/blood , Female , Humans , Immunity, Innate/genetics , Leukocytes, Mononuclear/drug effects , Male , Middle Aged , Oligonucleotide Array Sequence Analysis/methods , RNA, Messenger/metabolism , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Reproducibility of Results , Time Factors
11.
Blood ; 104(1): 270-80, 2004 Jul 01.
Article in English | MEDLINE | ID: mdl-15031206

ABSTRACT

In sickle cell disease, deoxygenation of intra-erythrocytic hemoglobin S leads to hemoglobin polymerization, erythrocyte rigidity, hemolysis, and microvascular occlusion. Ischemia-reperfusion injury, plasma hemoglobin-mediated nitric oxide consumption, and free radical generation activate systemic inflammatory responses. To characterize the role of circulating leukocytes in sickle cell pathogenesis we performed global transcriptional analysis of blood mononuclear cells from 27 patients in steady-state sickle cell disease (10 patients treated and 17 patients untreated with hydroxyurea) compared with 13 control subjects. We used gender-specific gene expression to validate human microarray experiments. Patients with sickle cell disease demonstrated differential gene expression of 112 genes involved in heme metabolism, cell-cycle regulation, antioxidant and stress responses, inflammation, and angiogenesis. Inducible heme oxygenase-1 and downstream proteins biliverdin reductase and p21, a cyclin-dependent kinase, were up-regulated, potentially contributing to phenotypic heterogeneity and absence of atherosclerosis in patients with sickle cell disease despite endothelial dysfunction and vascular inflammation. Hydroxyurea therapy did not significantly affect leukocyte gene expression, suggesting that such therapy has limited direct anti-inflammatory activity beyond leukoreduction. Global transcriptional analysis of circulating leukocytes highlights the intense oxidant and inflammatory nature of steady-state sickle cell disease and provides insight into the broad compensatory responses to vascular injury.


Subject(s)
Anemia, Sickle Cell/blood , Leukocytes, Mononuclear/metabolism , Adult , Anemia, Sickle Cell/genetics , Anemia, Sickle Cell/pathology , Case-Control Studies , Cluster Analysis , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , Female , Flow Cytometry , Gene Expression Profiling , Heme Oxygenase (Decyclizing)/genetics , Heme Oxygenase (Decyclizing)/metabolism , Heme Oxygenase-1 , Hemolysis , Humans , Inflammation/blood , Leukocyte Count , Leukocytes, Mononuclear/cytology , Male , Membrane Proteins , Middle Aged , Oligonucleotide Array Sequence Analysis , Oxidative Stress/physiology
12.
Blood ; 101(7): 2652-60, 2003 Apr 01.
Article in English | MEDLINE | ID: mdl-12456506

ABSTRACT

Systemic inflammation because of sepsis results in endothelial cell activation and microvascular injury. High-mobility group protein-1 (HMGB1), a novel inflammatory molecule, is a late mediator of endotoxin shock and is present in the blood of septic patients. The receptor for advanced glycation end products (RAGE) is expressed on endothelium and is a receptor for HMGB1. Here we examine the effects of HMGB1 on human endothelial cell function. Recombinant human HMGB1 (rhHMGB1) was cloned and expressed in Escherichia coli and incubated with human microvascular endothelium. rhHMGB1 caused a dose- and time-dependent increase in the expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and RAGE. rhHMGB1 induced the secretion of tumor necrosis factor-alpha (TNFalpha), interleukin 8 (IL-8), monocyte chemotactic protein-1 (MCP-1), plasminogen activator inhibitor 1 (PAI-1), and tissue plasminogen activator (tPA) (P <.01). rhHMGB1 stimulation resulted in transient phosphorylation of mitogen-activated protein (MAP) kinases, extracellular signal-related kinase (ERK), Jun N-terminal kinase (JNK), and p38, and in nuclear translocation of transcription factors NF-kappaB and Sp1. These effects are partially mediated by TNFalpha autocrine stimulation, as anti-TNFalpha antibodies significantly decrease chemokine and adhesion molecule responses (P

Subject(s)
Endothelium, Vascular/metabolism , HMGB1 Protein/physiology , Inflammation/etiology , Blood Coagulation Factors/metabolism , Cell Adhesion Molecules/metabolism , Cell Line , Chemokines/metabolism , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , HMGB1 Protein/pharmacology , Humans , Inflammation/metabolism , MAP Kinase Signaling System , Microcirculation/cytology , Receptor for Advanced Glycation End Products , Receptors, Immunologic/metabolism , Recombinant Proteins/pharmacology , Sepsis , Transcription Factors/metabolism , Tumor Necrosis Factor-alpha/metabolism
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