Nuclear Mechanotransduction Research Articles

The Nuclear Option: Evidence Implicating the Cell Nucleus in Mechanotransduction.

Biophysical stimuli presented to cells via microenvironmental properties (e.g., alignment and stiffness) or external forces have a significant impact on cell function and behavior. Recently, the cell nucleus has been identified as a mechanosensitive organelle that contributes to the perception and response to mechanical stimuli. However, the specific mechanotransduction mechanisms that mediate these effects have not been clearly established. Here, we offer a comprehensive review of the evidence supporting (and refuting) three hypothetical nuclear mechanotransduction mechanisms: physical reorganization of chromatin, signaling at the nuclear envelope, and altered cytoskeletal structure/tension due to nuclear remodeling. Our goal is to provide a reference detailing the progress that has been made and the areas that still require investigation regarding the role of nuclear mechanotransduction in cell biology. Additionally, we will briefly discuss the role that mathematical models of cell mechanics can play in testing these hypotheses and in elucidating how biophysical stimulation of the nucleus drives changes in cell behavior. While force-induced alterations in signaling pathways involving lamina-associated polypeptides (LAPs) (e.g., emerin and histone deacetylase 3 (HDAC3)) and transcription factors (TFs) located at the nuclear envelope currently appear to be the most clearly supported mechanism of nuclear mechanotransduction, additional work is required to examine this process in detail and to more fully test alternative mechanisms. The combination of sophisticated experimental techniques and advanced mathematical models is necessary to enhance our understanding of the role of the nucleus in the mechanotransduction processes driving numerous critical cell functions.

Vertical Nanopillars as Probes for in Situ Nuclear Mechanotransduction

The stability and deformability of the cell nucleus are important to many biological processes like migration, proliferation, polarization. When cells are exposed to mechanical force, the force will be transmitted via cytoskeleton to the nucleus, induce shape deformation of the nuclear envelopes, and even change the configurations of nucleoskeletons. However, current techniques for studying nuclear mechanics are limited for studying inducing the effects of subcellular force perturbation in live cells. Here we developed a novel assay of using vertical nanopillar arrays to study the mechanical coupling between cell nucleus and cytoskeleton in live cells. Our results showed that nanopillars can induce deformation of nuclear envelope. By changing the geometry of the nanopillars or the stiffness of the nucleus, we can control the degree of nuclear deformation. Also, cytoskeletons such as actin and intermediate filaments were showed to play important roles in inducing and preventing nuclear deformation, respectively. Furthermore, we showed that mechanical perturbation of the nuclear envelope can cause the reorganization of nuclear lamina, which give the clue that cell nucleus itself may be able to sense and respond to mechanical signals. Overall, vertical nanopillars provide a long-term and non-invasive force to create a subcellular nuclear perturbation, and can be used as a tool for studying nuclear mechanotransduction in live cells.

Nuclear mechanotransduction: Forcing the nucleus to respond

Nuclear mechanotransduction: Forcing the nucleus to respond

The cytolinker plectin regulates nuclear mechanotransduction in keratinocytes.

The transmission of mechanical forces to the nucleus is important for intracellular positioning, mitosis and cell motility, yet the contribution of specific components of the cytoskeleton to nuclear mechanotransduction remains unclear. In this study, we examine how crosstalk between the cytolinker plectin and F-actin controls keratin network organisation and the 3D nuclear morphology of keratinocytes. Using micro-patterned surfaces to precisely manipulate cell shape, we find that cell adhesion and spreading regulate the size and shape of the nucleus. Disruption of the keratin cytoskeleton through loss of plectin facilitated greater nuclear deformation, which depended on acto-myosin contractility. Nuclear morphology did not depend on direct linkage of the keratin cytoskeleton with the nuclear membrane, rather loss of plectin reduced keratin filament density around the nucleus. We further demonstrate that keratinocytes have abnormal nuclear morphologies in the epidermis of plectin-deficient, epidermolysis bullosa simplex patients. Taken together, our data demonstrate that plectin is an essential regulator of nuclear morphology in vitro and in vivo and protects the nucleus from mechanical deformation.

Modularity in nuclear mechanotransduction and genome regulation (479.3)

Mechanical signals from the extracellular matrix impinge on cellular geometry resulting in altered functional nuclear landscape and gene expression programs. These alterations regulate diverse biological processes including stem‐cell differentiation, developmental genetic programs and cellular homeostasis. How such signals are integrated to the 3D spatio‐temporal organization of the cell nucleus to elicit differential gene expression patterns are poorly understood. To investigate the biophysical principles underlying these processes, we use a multi‐disciplinary approach, combining high resolution imaging of live‐cells cultured on micro‐patterned substrates, single‐cell mechanics and genomics. I will describe some of our ongoing work that is beginning to provide quantitative and modular links between cell geometry and nuclear mechanics in regulating 3D chromosome organization and genetic information.

The regulation of dynamic mechanical coupling between actin cytoskeleton and nucleus by matrix geometry

Cells sense their physical microenvironment and transduce these signals through actin-nuclear links to regulate nuclear functions including gene expression. However, the spatio-temporal coupling between perinuclear actin and nucleus and their functional importance are still unclear. Using micropatterned substrates to control cell geometry, we show that perinuclear actin organization at the apical plane remodels from mesh-like structure to stress fibers. The formation of these apical stress fibers (ASFs) correlated with significant reduction in nuclear height and was found to exert an active compressive load on the nucleus via direct contact with mature focal adhesion sites. Interestingly, the dynamic nature of ASFs was found to transduce forces to chromatin assembly. In addition, geometric perturbations or using pharmacological drugs to inhibit actomyosin contractility of ASFs resulted in nuclear instability. Taken together, our work provides direct evidence of physical links between the nucleus and focal adhesion sites via ASFs, which modulate nuclear homeostatic balance and internal chromatin structure. We suggest that such direct links may underlie nuclear mechanotransduction to regulate genomic programs.

Cell geometric constraints induce modular gene-expression patterns via redistribution of HDAC3 regulated by actomyosin contractility

Physical forces in the form of substrate rigidity or geometrical constraints have been shown to alter gene expression profile and differentiation programs. However, the underlying mechanism of gene regulation by these mechanical cues is largely unknown. In this work, we use micropatterned substrates to alter cellular geometry (shape, aspect ratio, and size) and study the nuclear mechanotransduction to regulate gene expression. Genome-wide transcriptome analysis revealed cell geometry-dependent alterations in actin-related gene expression. Increase in cell size reinforced expression of matrix-related genes, whereas reduced cell-substrate contact resulted in up-regulation of genes involved in cellular homeostasis. We also show that large-scale changes in gene-expression profile mapped onto differential modulation of nuclear morphology, actomyosin contractility and histone acetylation. Interestingly, cytoplasmic-to-nuclear redistribution of histone deacetylase 3 modulated histone acetylation in an actomyosin-dependent manner. In addition, we show that geometric constraints altered the nuclear fraction of myocardin-related transcription factor. These fractions exhibited hindered diffusion time scale within the nucleus, correlated with enhanced serum-response element promoter activity. Furthermore, nuclear accumulation of myocardin-related transcription factor also modulated NF-κB activity. Taken together, our work provides modularity in switching gene-expression patterns by cell geometric constraints via actomyosin contractility.

Endothelial nuclear lamina is not required for glucocorticoid receptor nuclear import but does affect receptor-mediated transcription activation

The lamina serves to maintain the nuclear structure and stiffness while acting as a scaffold for heterochromatin and many transcriptional proteins. Its role in endothelial mechanotransduction, specifically how nuclear mechanics impact gene regulation under shear stress, is not fully understood. In this study, we successfully silenced lamin A/C in bovine aortic endothelial cells to determine its role in both glucocorticoid receptor (GR) nuclear translocation and glucocorticoid response element (GRE) transcriptional activation in response to dexamethasone and shear stress. Nuclear translocation of GR, an anti-inflammatory nuclear receptor, in response to dexamethasone or shear stress (5, 10, and 25 dyn/cm(2)) was observed via time-lapse cell imaging and quantified using a Bayesian image analysis algorithm. Transcriptional activity of the GRE promoter was assessed using a dual-luciferase reporter plasmid. We found no dependence on nuclear lamina for GR translocation from the cytoplasm into the nucleus. However, the absence of lamin A/C led to significantly increased expression of luciferase under dexamethasone and shear stress induction as well as changes in histone protein function. PCR results for NF-κB inhibitor alpha (NF-κBIA) and dual specificity phosphatase 1 (DUSP1) genes further supported our luciferase data with increased expression in the absence of lamin. Our results suggest that absence of lamin A/C does not hinder passage of GR into the nucleus, but nuclear lamina is important to properly regulate GRE transcription. Nuclear lamina, rather than histone deacetylase (HDAC), is a more significant mediator of shear stress-induced transcriptional activity, while dexamethasone-initiated transcription is more HDAC dependent. Our findings provide more insights into the molecular pathways involved in nuclear mechanotransduction.

Mesenchymal Stem Cell and Chondrocyte Fates in a Multishear Microdevice Are Regulated by Yes-Associated Protein

Mechanical cues exert considerable influence on the fates of stem cells and terminally differentiated chondrocytes. The elucidation of the interactions between cell fate and mechanical cues in nuclear mechanotransduction will provide new clues to modulate tissue homeostasis and regeneration. In this study, we used an integrated microfluidic perfusion device to simultaneously generate multiple-parameter fluid shear stresses to investigate the role of fluid flow stimuli in the regulation of Yes-associated protein (YAP) expression and the fates of mesenchymal stem cells (MSCs) and primary chondrocytes. YAP expression was regulated by the level of fluid flow stimulus in both MSCs and chondrocytes. An increase in the magnitude of stimulation enhanced the expression of YAP, ultimately resulting in an increase in osteogenesis and a decrease in adipogenesis for MSCs, and initiating dedifferentiation for chondrocytes. Cytochalasin D not only repressed nuclear YAP accumulation in the flow state, but also abrogated flow-induced effects on MSC differentiation and the chondrocyte phenotype, resulting in MSC adipogenesis and the maintenance of the chondrocyte phenotype. Our findings reveal the connection between YAP and MSC/chondrocyte fates in a fluid flow-induced mechanical microenvironment and provide new insights into the mechanisms by which mechanical cues regulate the fates of MSCs and chondrocytes.

Geometric Constraints on Cells Induce Cytoplasmic to Nuclear Redistribution of Transcription Co-Factors to Regulate Gene Expression

Physical forces in the form of substrate rigidity or geometrical constraints have been shown to alter gene expression profile and differentiation programs. However, the underlying mechanism of gene regulation by these mechanical cues is largely unknown. In this work, we use micropatterned substrates to alter cellular geometry (shape, aspect ratio and size) and study the nuclear mechanotransduction to regulate gene expression. We show that geometric constraints result in differential modulation of nuclear morphology, acto-myosin contractility, histone acetylation and the activity of transcription co-factor, MRTF-A. In addition, genome-wide transcriptome analysis revealed cell geometry dependent alterations in chromosomal activity and actin dependent gene expression. Promoter analysis of these differentially regulated genes showed that serum response factor (SRF) was an essential regulatory factor sensitive to geometric cues. Further, we show that geometric constraints resulted in nuclear translocation of MRTF-A and enhanced serum response element (SRE) promoter activity. Interestingly, nuclear accumulation of MRTF-A by geometric constraints also modulated NF-κB activity. Taken together, our work provides mechanistic insights underlying the regulation of gene expression by cellular geometry.

Cellular Geometry Mediated Apical Stress Fibers Dynamically Couples Nucleus to Focal Adhesion

Cells sense their physical microenvironment and transduce these signals through actin-nuclear links to regulate nuclear functions including gene expression. However, the spatio-temporal coupling between actin cytoskeleton and the nucleus and its modulation by cell geometry are still unclear. using micro-patterned substrates to control cell geometry, we show that perinuclear actin organization at the apical plane remodels from mesh-like structure to stress fibers. The formation of these apical stress fibers (ASF) correlated with significant reduction in nuclear height and was found to exert an active compressive load on the nucleus via direct contact with mature focal adhesion sites. We further show, using quantitative fluorescence spectroscopy experiments, that these ASFs were dynamically coupled to the nucleus via outer nuclear membrane proteins nesprin2G. Taken together, our work provides direct evidence of physical links between the nucleus and focal adhesion sites via ASFs. We suggest that such direct links may underlie nuclear mechanotransduction to regulate genomic programs.

Nuclear mechanotransduction: Response of the lamina to extracellular stress with implications in aging

Mechnotransduction, the phenomenon by which cells respond to applied force, is necessary for normal cell processes and is implicated in the pathology of several diseases including atherosclerosis. The exact mechanisms which govern how forces can affect gene expression have not been determined, but putative direct force effects on the genome would require transduction through the nuclear lamina. In this study we show that nuclei in cells exposed to shear stress significantly change shape, upregulate nuclear lamins and move lamins from the nuclear interior to the nuclear periphery. We hypothesize that the augmentation of the nuclear lamina at the nuclear periphery protects the nuclear interior from the force and allows a nuclear adaptation to shear stress. We also investigate the shear stress response of nuclei in cells that have been transfected with lamin A Δ50, which significantly stiffens nuclei. Lamin A Δ50 causes the premature aging syndrome Hutchinson-Gilford progeria syndrome (HGPS) and models many aspects of normal aging. We find that the presence of lamin A Δ50 in only 30% of cells greatly reduces the response of the nuclear lamina in all cells in the flow field. We suggest that cells expressing lamin A Δ50 lack the ability to adapt to flow and may prevent neighboring cells from adapting as well. These results provide insight into the development of cardiovascular disease both in patients with HGPS and in normal aging.

Nuclear Shape, Mechanics, and Mechanotransduction

In eukaryotic cells, the nucleus contains the genome and is the site of transcriptional regulation. The nucleus is the largest and stiffest organelle and is exposed to mechanical forces transmitted through the cytoskeleton from outside the cell and from force generation within the cell. Here, we discuss the effect of intra- and extracellular forces on nuclear shape and structure and how these force-induced changes could be implicated in nuclear mechanotransduction, ie, force-induced changes in cell signaling and gene transcription. We review mechanical studies of the nucleus and nuclear structural proteins, such as lamins. Dramatic changes in nuclear shape, organization, and stiffness are seen in cells where lamin proteins are mutated or absent, as in genetically engineered mice, RNA interference studies, or human disease. We examine the different mechanical pathways from the force-responsive cytoskeleton to the nucleus. We also highlight studies that link changes in nuclear shape with cell function during developmental, physiological, and pathological modifications. Together, these studies suggest that the nucleus itself may play an important role in the response of the cell to force.

Alpha smooth muscle actin distribution in cytoplasm and nuclear invaginations of connective tissue fibroblasts

Alpha smooth muscle actin (alpha-SMA) was recently shown to be present in mouse subcutaneous tissue fibroblasts in the absence of tissue injury. In this study, we used a combination of immunohistochemistry and correlative confocal scanning laser and electron microscopy to investigate the structural organization of alpha-SMA in relation to the nucleus. Furthermore, we explored colocalization analysis as a method for quantifying the amount of alpha-SMA in close approximation to the nucleic acid marker, 4',6-diamidino-2-phenyl-indole, dihydrochloride. Our findings indicate the presence of alpha-SMA within nuclear invaginations in close proximity to the nuclear membrane, but not in the nucleoplasm. Although the function of these alpha-SMA-rich nuclear invaginations is at present unknown, the morphology of these structures suggests their possible involvement in cellular and nuclear mechanotransduction as well as nuclear transport.