Condensate interfacial forces reposition DNA loci and probe chromatin viscoelasticity.

Biomolecular condensates assemble in living cells through phase separation and related phase transitions. An underappreciated feature of these dynamic molecular assemblies is that they form interfaces with other cellular structures, including membranes, cytoskeleton, DNA and RNA, and other membraneless compartments. These interfaces are expected to give rise to capillary forces, but there are few ways of quantifying and harnessing these forces in living cells. Here, we introduce viscoelastic chromatin tethering and organization (VECTOR), which uses light-inducible biomolecular condensates to generate capillary forces at targeted DNA loci. VECTOR can be utilized to programmably reposition genomic loci on a timescale of seconds to minutes, quantitatively revealing local heterogeneity in the viscoelastic material properties of chromatin. These synthetic condensates are built from components that naturally form liquid-like structures in living cells, highlighting the potential role for native condensates to generate forces and do work to reorganize the genome and impact chromatin architecture.

Engineering the Cellular Microenvironment: Integrating Three-Dimensional Nontopographical and Two-Dimensional Biochemical Cues for Precise Control of Cellular Behavior.

The development of biomaterials capable of regulating cellular processes and guiding cell fate decisions has broad implications in tissue engineering, regenerative medicine, and cell-based assays for drug development and disease modeling. Recent studies have shown that three-dimensional (3D) nanoscale physical cues such as nanotopography can modulate various cellular processes like adhesion and endocytosis by inducing nanoscale curvature on the plasma and nuclear membranes. Two-dimensional (2D) biochemical cues such as protein micropatterns can also regulate cell function and fate by controlling cellular geometries. Development of biomaterials with precise control over nanoscale physical and biochemical cues can significantly influence programming cell function and fate. In this study, we utilized a laser-assisted micropatterning technique to manipulate the 2D architectures of cells on 3D nanopillar platforms. We performed a comprehensive analysis of cellular and nuclear morphology and deformation on both nanopillar and flat substrates. Our findings demonstrate the precise engineering of single cell architectures through 2D micropatterning on nanopillar platforms. We show that the coupling between the nuclear and cell shape is disrupted on nanopillar surfaces compared to flat surfaces. Furthermore, our results suggest that cell elongation on nanopillars enhances nanopillar-induced endocytosis. We believe our platform serves as a versatile tool for further explorations into programming cell function and fate through combined physical cues that create nanoscale curvature on cell membranes and biochemical cues that control the geometry of the cell.

The condensation of HP1α/Swi6 imparts nuclear stiffness.

Biomolecular condensates have emerged as major drivers of cellular organization. It remains largely unexplored, however, whether these condensates can impart mechanical function(s) to the cell. The heterochromatin protein HP1α (Swi6 in Schizosaccharomyces pombe) crosslinks histone H3K9 methylated nucleosomes and has been proposed to undergo condensation to drive the liquid-like clustering of heterochromatin domains. Here, we leverage the genetically tractable S. pombe model and a separation-of-function allele to elucidate a mechanical function imparted by Swi6 condensation. Using single-molecule imaging, force spectroscopy, and high-resolution live-cell imaging, we show that Swi6 is critical for nuclear resistance to external force. Strikingly, it is the condensed yet dynamic pool of Swi6, rather than the chromatin-bound molecules, that is essential to imparting mechanical stiffness. Our findings suggest that Swi6 condensates embedded in the chromatin meshwork establish the emergent mechanical behavior of the nucleus as a whole, revealing that biomolecular condensation can influence organelle and cell mechanics.

Inhibition of PDIs Downregulates Core LINC Complex Proteins, Promoting the Invasiveness of MDA-MB-231 Breast Cancer Cells in Confined Spaces In Vitro.

Eukaryotic cells tether the nucleoskeleton to the cytoskeleton via a conserved molecular bridge, called the LINC complex. The core of the LINC complex comprises SUN-domain and KASH-domain proteins that directly associate within the nuclear envelope lumen. Intra- and inter-chain disulphide bonds, along with KASH-domain protein interactions, both contribute to the tertiary and quaternary structure of vertebrate SUN-domain proteins. The significance of these bonds and the role of PDIs (protein disulphide isomerases) in LINC complex biology remains unclear. Reducing and non-reducing SDS-PAGE analyses revealed a prevalence of SUN2 homodimers in non-tumorigenic breast epithelia MCF10A cells, but not in the invasive triple-negative breast cancer MDA-MB-231 cell line. Furthermore, super-resolution microscopy revealed SUN2 staining alterations in MCF10A, but not in MDA-MB-231 nuclei, upon reducing agent exposure. While PDIA1 levels were similar in both cell lines, pharmacological inhibition of PDI activity in MDA-MB-231 cells led to SUN-domain protein down-regulation, as well as Nesprin-2 displacement from the nucleus. This inhibition also caused changes in perinuclear cytoskeletal architecture and lamin downregulation, and increased the invasiveness of PDI-inhibited MDA-MB-231 cells in space-restrictive in vitro environments, compared to untreated cells. These results emphasise the key roles of PDIs in regulating LINC complex biology, cellular architecture, biomechanics, and invasion.

The nexus of nuclear envelope dynamics, circular economy and cancer cell pathophysiology.

The nuclear envelope (NE) is a critical component in maintaining the function and structure of the eukaryotic nucleus. The NE and lamina are disassembled during each cell cycle to enable an open mitosis. Nuclear architecture construction and deconstruction is a prime example of a circular economy, as it fulfills a highly efficient recycling program bound to continuous assessment of the quality and functionality of the building blocks. Alterations in the nuclear dynamics and lamina structure have emerged as important contributors to both oncogenic transformation and cancer progression. However, the knowledge of the NE breakdown and reassembly is still limited to a fraction of participating proteins and complexes. As cancer cells contain highly diverse nuclei in terms of DNA content, but also in terms of nuclear number, size, and shape, it is of great interest to understand the intricate relationship between these nuclear features in cancer cell pathophysiology. In this review, we provide insights into how those NE dynamics are regulated, and how lamina destabilization processes may alter the NE circular economy. Moreover, we expand the knowledge of the lamina-associated domain region by using strategic algorithms, including Artificial Intelligence, to infer protein associations, assess their function and location, and predict cancer-type specificity with implications for the future of cancer diagnosis, prognosis and treatment. Using this approach we identified NUP98 and MECP2 as potential proteins that exhibit upregulation in Acute Myeloid Leukemia (LAML) patients with implications for early diagnosis.

Mechanoresponse of Curved Epithelial Monolayers Lining Bowl-Shaped 3D Microwells.

The optimal functioning of many organs relies on the curved architecture of their epithelial tissues. However, the mechanoresponse of epithelia to changes in curvature remains misunderstood. Here, bowl-shaped microwells in hydrogels are designed via photopolymerization to faithfully replicate the shape and dimensions of lobular structures. Leveraging these hydrogel-based microwells, curved epithelial monolayers are engineered, and how in-plane and Gaussian curvatures at the microwell entrance influence epithelial behavior is investigated. Cells and nuclei around the microwell edge display a more pronounced centripetal orientation as the in-plane curvature decreases, and enhanced cell straightness and speed. Moreover, cells reorganize their actin cytoskeleton by forming a supracellular actin cable at the microwell edge, with its size becoming more pronounced as the in-plane curvature decreases. The Gaussian curvature at the microwell entrance enhances the maturation of the supracellular actin cable architecture and leads to a vertical orientation of nuclei toward the bottom of the microwell. Increasing Gaussian curvature results in flattened and elongated nuclear morphologies characterized by highly compacted chromatin states. This approach provides better understanding of the mechanoresponse of curved epithelial monolayers curvatures lining lobular structures. In addition, bowl-shaped microwells offer a powerful platform to study curvature-dependent mechanotransduction pathways in anatomically relevant 3D structures.

Nuclear mechanosensing of the aortic endothelium in health and disease.

The endothelium, the monolayer of endothelial cells that line blood vessels, is exposed to a number of mechanical forces, including frictional shear flow, pulsatile stretching and changes in stiffness influenced by extracellular matrix composition. These forces are sensed by mechanosensors that facilitate their transduction to drive appropriate adaptation of the endothelium to maintain vascular homeostasis. In the aorta, the unique architecture of the vessel gives rise to changes in the fluid dynamics, which, in turn, shape cellular morphology, nuclear architecture, chromatin dynamics and gene regulation. In this Review, we discuss recent work focusing on how differential mechanical forces exerted on endothelial cells are sensed and transduced to influence their form and function in giving rise to spatial variation to the endothelium of the aorta. We will also discuss recent developments in understanding how nuclear mechanosensing is implicated in diseases of the aorta.

Indentation induces instantaneous nuclear stiffening and unfolding of nuclear envelope wrinkles.

The nuclear envelope (NE) separates genomic DNA from the cytoplasm and regulates transport between the cytosol and the nucleus in eukaryotes. Nuclear stiffening enables the cell nucleus to protect itself from extensive deformation, loss of NE integrity, and genome instability. It is known that the reorganization of actin, lamin, and chromatin can contribute to nuclear stiffening. In this work, we show that structural alteration of NE also contributes to instantaneous nuclear stiffening under indentation. In situ mechanical characterization of cell nuclei in intact cells shows that nuclear stiffening and unfolding of NE wrinkles occur simultaneously at the indentation site. A positive correlation between the initial state of NE wrinkles, the unfolding of NE wrinkles, and the stiffening ratio (stiffness fold-change) is found. Additionally, NE wrinkles unfold throughout the nucleus outside the indentation site. Finite element simulation, which involves the purely passive process of structural unfolding, shows that unfolding of NE wrinkles alone can lead to an increase in nuclear stiffness and a reduction in stress and strain levels. Together, these results provide a perspective on how cell nucleus adapts to mechanical stimuli through structural alteration of the NE.

Deleterious Mechanical Deformation Selects Mechanoresilient Cancer Cells with Enhanced Proliferation and Chemoresistance.

Cancer cells in secondary tumors are found to form metastases more efficiently as compared to their primary tumor counterparts. This is partially due to the unfavorable microenvironments encountered by metastasizing cancer cells that result in the survival of a more metastatic phenotype from the original population. However, the role of deleterious mechanical stresses in this change of metastatic potential is unclear. Here, by forcing cancer cells to flow through small capillary-sized constrictions, it is demonstrated that mechanical deformation can select a tumor cell subpopulation that exhibits resilience to mechanical squeezing-induced cell death. Transcriptomic profiling reveals up-regulated proliferation and DNA damage response pathways in this subpopulation, which are further translated into a more proliferative and chemotherapy-resistant phenotype. These results highlight a potential link between the microenvironmental physical stresses and the enhanced malignancy of metastasizing cancer cells which may be utilized as a therapeutic strategy in preventing the metastatic spread of cancer cells.

Mechanotherapy in oncology: Targeting nuclear mechanics and mechanotransduction.

Mechanotherapy is proposed as a new option for cancer treatment. Increasing evidence suggests that characteristic differences are present in the nuclear mechanics and mechanotransduction of cancer cells compared with those of normal cells. Recent advances in understanding nuclear mechanics and mechanotransduction provide not only further insights into the process of malignant transformation but also useful references for developing new therapeutic approaches. Herein, we present an overview of the alterations of nuclear mechanics and mechanotransduction in cancer cells and highlight their implications in cancer mechanotherapy.

Chromatin epigenetics and nuclear lamina keep the nucleus in shape: Examples from natural and accelerated aging.

As the repository of genetic information, the cell nucleus must protect DNA integrity from mechanical stresses. The nuclear lamina, which resides within the nuclear envelope (NE), is made up of lamins, intermediate filaments bound to DNA. The nuclear lamina provides the nucleus with the ability to deal with inward as well as outward mechanical stimuli. Chromatin, in turn, through its degrees of compaction, shares this role with the nuclear lamina, thus, ensuring the plasticity of the nucleus. Perturbation of chromatin condensation or the nuclear lamina has been linked to a plethora of biological conditions, that range from cancer and genetic diseases (laminopathies) to aging, both natural and accelerated, such as the case of Hutchinson-Gilford Progeria Syndrome (HGPS). From the experimental results accumulated so far on the topic, a direct link between variations of the epigenetic pattern and nuclear lamina structure would be suggested, however, it has never been clarified thoroughly. This relationship, instead, has a downstream important implication on nucleus shape, genome preservation, force sensing, and, ultimately, aging-related disease onset. With this review, we aim to collect recent studies on the importance of both nuclear lamina components and chromatin status in nuclear mechanics. We also aim to bring to light evidence of the link between DNA methylation and nuclear lamina in natural and accelerated aging.

Monitoring Mechano-Regulation of Gene Expression by RNA Sequencing.

The advent of high-throughput sequencing techniques has revolutionized biological research. One such method is RNA sequencing, which has become a relatively affordable and routine method for quantifying and comparing gene expression changes over desired experimental conditions. Along with the popularity of the method, a myriad of user-friendly, open-source computational tools have also emerged for differential gene expression analyses. Correspondingly, decades of mechanobiology research have established that mechanical cues, both alone and/or in combination with biochemical signals, can be powerful regulators of transcriptional programs and consequently cell state/fate transitions. Thus, it has become possible to investigate both universal and specific temporally resolved transcriptional responses upon mechanical stimulation genome-wide. This chapter will describe methods to analyze transcriptional changes in response to extrinsic mechanical stretch.

Nuclear transport of STAT6 determines the matrix rigidity dependent M2 activation of macrophages.

Alternatively activated or M2 macrophages, as opposed to the well characterized pro-inflammatory or M1 macrophages, vitally regulate anti-inflammation, wound healing, and tissue repair to maintain tissue homeostasis. Although ubiquitous presence of macrophages in diverse tissues, exposed to different physical environments, infers distinct immune responses of M2 macrophages with high phenotypic heterogeneity, the underlying mechanism of how the varying extracellular mechanical conditions alter their immunological activation remains unclear. Here, we demonstrate that M2 activation requires a threshold mechanical cue from the extracellular microenvironment, and matrix rigidity-dependent macrophage spreading is mediated by the F-actin formation that is essential to regulate mechanosensitive M2 activation of macrophages. We identified a new mechanosensing function of STAT6 (signal transducer and activator of transcription 6), a key transcription factor for M2 activation, whose intranuclear transportation is promoted by the rigid matrix that facilitates the F-actin formation. Our findings further highlight the critical role of mechanosensitive M2 activation of macrophages in long-term adaptation to the extracellular microenvironment by bridging nuclear mechanosensation and immune responses.

Changes in intra-nuclear mechanics in response to DNA damaging agents revealed by time-domain Brillouin micro-spectroscopy.

How DNA damage and repair processes affect the biomechanical properties of the nucleus interior remains unknown. Here, an opto-acoustic microscope based on time-domain Brillouin spectroscopy (TDBS) was used to investigate the induced regulation of intra-nuclear mechanics. With this ultrafast pump-probe technique, coherent acoustic phonons were tracked along their propagation in the intra-nucleus nanostructure and the complex stiffness moduli and thicknesses were measured with an optical resolution. Osteosarcoma cells were exposed to methyl methanesulfonate (MMS) and the presence of DNA damage was tested using immunodetection targeted against damage signaling proteins. TDBS revealed that the intra-nuclear storage modulus decreased significantly upon exposure to MMS, as a result of the chromatin decondensation and reorganization that favors molecular diffusion within the organelle. When the damaging agent was removed and cells incubated for 2 h in the buffer solution before fixation the intra-nuclear reorganization led to an inverse evolution of the storage modulus, the nucleus stiffened. The same tendency was measured when DNA double-strand breaks were caused by cell exposure to ionizing radiation. TDBS microscopy also revealed changes in acoustic dissipation, another mechanical probe of the intra-nucleus organization at the nano-scale, and changes in nucleus thickness during exposure to MMS and after recovery.

The role of cellular traction forces in deciphering nuclear mechanics.

Cellular forces exerted on the extracellular matrix (ECM) during adhesion and migration under physiological and pathological conditions regulate not only the overall cell morphology but also nuclear deformation. Nuclear deformation can alter gene expression, integrity of the nuclear envelope, nucleus-cytoskeletal connection, chromatin architecture, and, in some cases, DNA damage responses. Although nuclear deformation is caused by the transfer of forces from the ECM to the nucleus, the role of intracellular organelles in force transfer remains unclear and a challenging area of study. To elucidate nuclear mechanics, various factors such as appropriate biomaterial properties, processing route, cellular force measurement technique, and micromanipulation of nuclear forces must be understood. In the initial phase of this review, we focused on various engineered biomaterials (natural and synthetic extracellular matrices) and their manufacturing routes along with the properties required to mimic the tumor microenvironment. Furthermore, we discussed the principle of tools used to measure the cellular traction force generated during cell adhesion and migration, followed by recently developed techniques to gauge nuclear mechanics. In the last phase of this review, we outlined the principle of traction force microscopy (TFM), challenges in the remodeling of traction forces, microbead displacement tracking algorithm, data transformation from bead movement, and extension of 2-dimensional TFM to multiscale TFM.

ATM Modulates Nuclear Mechanics by Regulating Lamin A Levels.

Ataxia-telangiectasia mutated (ATM) is one of the three main apical kinases at the crux of DNA damage response and repair in mammalian cells. ATM activates a cascade of downstream effector proteins to regulate DNA repair and cell cycle checkpoints in response to DNA double-strand breaks. While ATM is predominantly known for its role in DNA damage response and repair, new roles of ATM have recently begun to emerge, such as in regulating oxidative stress or metabolic pathways. Here, we report the surprising discovery that ATM inhibition and deletion lead to reduced expression of the nuclear envelope protein lamin A. Lamins are nuclear intermediate filaments that modulate nuclear shape, structure, and stiffness. Accordingly, inhibition or deletion of ATM resulted in increased nuclear deformability and enhanced cell migration through confined spaces, which requires substantial nuclear deformation. These findings point to a novel connection between ATM and lamin A and may have broad implications for cells with ATM mutations-as found in patients suffering from Ataxia Telangiectasia and many human cancers-which could lead to enhanced cell migration and increased metastatic potential.

Genome-Directed Cell Nucleus Assembly.

The cell nucleus is frequently considered a cage in which the genome is placed to protect it from various external factors. Inside the nucleus, many functional compartments have been identified that are directly or indirectly involved in implementing genomic DNA's genetic functions. For many years, it was assumed that these compartments are assembled on a proteinaceous scaffold (nuclear matrix), which provides a structural milieu for nuclear compartmentalization and genome folding while simultaneously offering some rigidity to the cell nucleus. The results of research in recent years have made it possible to consider the cell nucleus from a different angle. From the "box" in which the genome is placed, the nucleus has become a kind of mobile exoskeleton, which is formed around the packaged genome, under the influence of transcription and other processes directly related to the genome activity. In this review, we summarize the main arguments in favor of this point of view by analyzing the mechanisms that mediate cell nucleus assembly and support its resistance to mechanical stresses.

Modelling Nuclear Morphology and Shape Transformation: A Review.

As one of the most important cellular compartments, the nucleus contains genetic materials and separates them from the cytoplasm with the nuclear envelope (NE), a thin membrane that is susceptible to deformations caused by intracellular forces. Interestingly, accumulating evidence has also indicated that the morphology change of NE is tightly related to nuclear mechanotransduction and the pathogenesis of diseases such as cancer and Hutchinson-Gilford Progeria Syndrome. Theoretically, with the help of well-designed experiments, significant progress has been made in understanding the physical mechanisms behind nuclear shape transformation in different cellular processes as well as its biological implications. Here, we review different continuum-level (i.e., energy minimization, boundary integral and finite element-based) approaches that have been developed to predict the morphology and shape change of the cell nucleus. Essential gradients, relative advantages and limitations of each model will be discussed in detail, with the hope of sparking a greater research interest in this important topic in the future.

Nuclear envelope mechanobiology: linking the nuclear structure and function.

The nucleus, central to cellular activity, relies on both direct mechanical input as well as its molecular transducers to sense external stimuli and respond by regulating intra-nuclear chromatin organization that determines cell function and fate. In mesenchymal stem cells of musculoskeletal tissues, changes in nuclear structures are emerging as a key modulator of their differentiation and proliferation programs. In this review we will first introduce the structural elements of the nucleoskeleton and discuss the current literature on how nuclear structure and signaling are altered in relation to environmental and tissue level mechanical cues. We will focus on state-of-the-art techniques to apply mechanical force and methods to measure nuclear mechanics in conjunction with DNA, RNA, and protein visualization in living cells. Ultimately, combining real-time nuclear deformations and chromatin dynamics can be a powerful tool to study mechanisms of how forces affect the dynamics of genome function.

Molecular models of LINC complex assembly at the nuclear envelope.

Large protein complexes assemble at the nuclear envelope to transmit mechanical signals between the cytoskeleton and nucleoskeleton. These protein complexes are known as the linkers of the nucleoskeleton and cytoskeleton complexes (LINC complexes) and are formed by the interaction of SUN and KASH domain proteins in the nuclear envelope. Ample evidence suggests that SUN-KASH complexes form higher-order assemblies to withstand and transfer forces across the nuclear envelope. Herein, we present a review of recent studies over the past few years that have shed light on the mechanisms of SUN-KASH interactions, their higher order assembly, and the molecular mechanisms of force transfer across these complexes.