Microtubule forces drive nuclear damage in LMNA cardiomyopathy.

Nuclear homeostasis requires a balance of forces between the cytoskeleton and nucleus. Variants in LMNA disrupt this balance by weakening the nuclear lamina, resulting in nuclear damage in contractile tissues and ultimately muscle disease. Intriguingly, disrupting the LINC complex that connects the cytoskeleton to the nucleus has emerged as a promising strategy to ameliorate LMNA cardiomyopathy. Yet how LINC disruption protects the cardiomyocyte nucleus remains unclear. To address this, we developed an assay to quantify the coupling of cardiomyocyte contraction to nuclear deformation and interrogated its dependence on the lamina and LINC complex. We found that the LINC complex was surprisingly dispensable for transferring the majority of contractile strain into the nucleus, and that increased nuclear strain in Lmna-deficient myocytes was not rescued by LINC disruption. However, LINC disruption eliminated the cage of microtubules encircling the nucleus, and disrupting microtubules was sufficient to prevent nuclear damage induced by LMNA deficiency. Through computational modeling we simulated the mechanical stress fields surrounding cardiomyocyte nuclei and show how microtubule compression exploits local vulnerabilities to damage LMNA-deficient nuclei. Our work pinpoints localized, microtubule-dependent force transmission through the LINC complex as a pathological driver and therapeutic target for LMNA cardiomyopathy.

Multi-level transcriptomic analysis of LMNA -related dilated cardiomyopathy identifies disease-driving processes.

LMNA- related dilated cardiomyopathy ( LMNA -DCM) is one of the most severe forms of DCM. The incomplete understanding of the molecular disease mechanisms results in lacking treatment options, leading to high mortality amongst patients. Here, using an inducible, cardiomyocyte-specific lamin A/C depletion mouse model, we conducted a comprehensive transcriptomic study, combining both bulk and single nucleus RNA sequencing, and spanning LMNA -DCM disease progression, to identify potential disease drivers. Our refined analysis pipeline identified 496 genes already misregulated early in disease. The expression of these genes was largely driven by disease specific cardiomyocyte sub-populations and involved biological processes mediating cellular response to DNA damage, cytosolic pattern recognition, and innate immunity. Indeed, DNA damage in LMNA -DCM hearts was significantly increased early in disease and correlated with reduced cardiomyocyte lamin A levels. Activation of cytosolic pattern recognition in cardiomyocytes was independent of cGAS, which is rarely expressed in cardiomyocytes, but likely occurred downstream of other pattern recognition sensors such as IFI16. Altered gene expression in cardiac fibroblasts and immune cell infiltration further contributed to tissue-wide changes in gene expression. Our transcriptomic analysis further predicted significant alterations in cell-cell communication between cardiomyocytes, fibroblasts, and immune cells, mediated through early changes in the extracellular matrix (ECM) in the LMNA -DCM hearts. Taken together, our work suggests a model in which nuclear damage in cardiomyocytes leads to activation of DNA damage responses, cytosolic pattern recognition pathway, and other signaling pathways that activate inflammation, immune cell recruitment, and transcriptional changes in cardiac fibroblasts, which collectively drive LMNA -DCM pathogenesis.

Rear cortex contraction aids in nuclear transit during confined migration by increasing pressure in the cell posterior.

As cells migrate through biological tissues, they must frequently squeeze through micron-sized constrictions in the form of interstitial pores between extracellular matrix fibers and/or other cells. Although it is now well recognized that such confined migration is limited by the nucleus, which is the largest and stiffest organelle, it remains incompletely understood how cells apply sufficient force to move their nucleus through small constrictions. Here, we report a mechanism by which contraction of the cell rear cortex pushes the nucleus forward to mediate nuclear transit through constrictions. Laser ablation of the rear cortex reveals that pushing forces behind the nucleus are the result of increased intracellular pressure in the rear compartment of the cell. The pushing forces behind the nucleus depend on accumulation of actomyosin in the rear cortex and require Rho kinase (ROCK) activity. Collectively, our results suggest a mechanism by which cells generate elevated intracellular pressure in the posterior compartment to facilitate nuclear transit through three-dimensional (3D) constrictions. This mechanism might supplement or even substitute for other mechanisms supporting nuclear transit, ensuring robust cell migrations in confined 3D environments.

Eliminating elevated p53 signaling fails to rescue skeletal muscle defects or extend survival in lamin A/C-deficient mice.

Lamins A and C, encoded by the LMNA gene, are nuclear intermediate filaments that provide structural support to the nucleus and contribute to chromatin organization and transcriptional regulation. LMNA mutations cause muscular dystrophies, dilated cardiomyopathy, and other diseases. The mechanisms by which many LMNA mutations result in muscle-specific diseases have remained elusive, presenting a major hurdle in the development of effective treatments. Previous studies using striated muscle laminopathy mouse models found that cytoskeletal forces acting on mechanically fragile Lmna-mutant nuclei led to transient nuclear envelope rupture, extensive DNA damage, and activation of DNA damage response (DDR) pathways in skeletal muscle cells in vitro and in vivo. Furthermore, hearts of Lmna mutant mice have elevated activation of the tumor suppressor protein p53, a central regulator of DDR signaling. We hypothesized that elevated p53 activation could present a pathogenic mechanism in striated muscle laminopathies, and that eliminating p53 activation could improve muscle function and survival in laminopathy mouse models. Supporting a pathogenic function of p53 activation in muscle, stabilization of p53 was sufficient to reduce contractility and viability in wild-type muscle cells in vitro. Using three laminopathy models, we found that increased p53 activity in Lmna-mutant muscle cells primarily resulted from mechanically induced damage to the myonuclei, and not from altered transcriptional regulation due to loss of lamin A/C expression. However, global deletion of p53 in a severe muscle laminopathy model did not reduce the disease phenotype or increase survival, indicating that additional drivers of disease must contribute to the disease pathogenesis.

Immunoengineering can overcome the glycocalyx armour of cancer cells.

Cancer cell glycocalyx is a major line of defence against immune surveillance. However, how specific physical properties of the glycocalyx are regulated on a molecular level, contribute to immune evasion and may be overcome through immunoengineering must be resolved. Here we report how cancer-associated mucins and their glycosylation contribute to the nanoscale material thickness of the glycocalyx and consequently modulate the functional interactions with cytotoxic immune cells. Natural-killer-cell-mediated cytotoxicity is inversely correlated with the glycocalyx thickness of the target cells. Changes in glycocalyx thickness of approximately 10 nm can alter the susceptibility to immune cell attack. Enhanced stimulation of natural killer and T cells through equipment with chimeric antigen receptors can improve the cytotoxicity against mucin-bearing target cells. Alternatively, cytotoxicity can be enhanced through engineering effector cells to display glycocalyx-editing enzymes, including mucinases and sialidases. Together, our results motivate the development of immunoengineering strategies that overcome the glycocalyx armour of cancer cells.

Heterologous expression of Dictyostelium discoideum NE81 in mouse embryo fibroblasts reveals conserved mechanoprotective roles of lamins.

Lamins are nuclear intermediate filament proteins that are ubiquitously found in metazoan cells, where they contribute to nuclear morphology, stability, and gene expression. Lamin-like sequences have recently been identified in distantly related eukaryotes, but it remains unclear whether these proteins share conserved functions with the lamins found in metazoans. Here, we investigate conserved features between metazoan and amoebozoan lamins using a genetic complementation system to express the Dictyostelium discoideum lamin-like protein NE81 in mammalian cells lacking either specific lamins or all endogenous lamins. We report that NE81 localizes to the nucleus in cells lacking Lamin A/C, and that NE81 expression improves nuclear circularity, reduces nuclear deformability, and prevents nuclear envelope rupture in these cells. However, NE81 did not completely rescue loss of Lamin A/C, and was unable to restore normal distribution of metazoan lamin interactors, such as emerin and nuclear pore complexes, which are frequently displaced in Lamin A/C deficient cells. Collectively, our results indicate that the ability of lamins to modulate the morphology and mechanical properties of nuclei may have been a feature present in the common ancestor of Dictyostelium and animals, whereas other, more specialized interactions may have evolved more recently in metazoan lineages.

Endothelial cells metabolically regulate breast cancer invasion toward a microvessel.

Breast cancer metastasis is initiated by invasion of tumor cells into the collagen type I-rich stroma to reach adjacent blood vessels. Prior work has identified that metabolic plasticity is a key requirement of tumor cell invasion into collagen. However, it remains largely unclear how blood vessels affect this relationship. Here, we developed a microfluidic platform to analyze how tumor cells invade collagen in the presence and absence of a microvascular channel. We demonstrate that endothelial cells secrete pro-migratory factors that direct tumor cell invasion toward the microvessel. Analysis of tumor cell metabolism using metabolic imaging, metabolomics, and computational flux balance analysis revealed that these changes are accompanied by increased rates of glycolysis and oxygen consumption caused by broad alterations of glucose metabolism. Indeed, restricting glucose availability decreased endothelial cell-induced tumor cell invasion. Our results suggest that endothelial cells promote tumor invasion into the stroma due, in part, to reprogramming tumor cell metabolism.

ATR takes a crack at the nuclear envelope.

In this issue, Joo et al.1 and Kovacs et al.2 report that the ATR kinase promotes nuclear envelope rupture through the phosphorylation of Lamin A/C, inducing processes such as cGAS-STING pathway activation, micronuclei clearance, and potentially cell death.

Lamins as structural nuclear elements through evolution.

Lamins are nuclear intermediate filament proteins with important, well-established roles in humans and other vertebrates. Lamins interact with DNA and numerous proteins at the nuclear envelope to determine the mechanical properties of the nucleus, coordinate chromatin organization, and modulate gene expression. Many of these functions are conserved in the lamin homologs found in basal metazoan organisms, including Drosophila and Caenorhabditis elegans. Lamin homologs have also been recently identified in non-metazoans, like the amoeba Dictyostelium discoideum, yet how these proteins compare functionally to the metazoan isoforms is only beginning to emerge. A better understanding of these distantly related lamins is not only valuable for a more complete picture of eukaryotic evolution, but may also provide new insights into the function of vertebrate lamins.

Nuclear damage in LMNA mutant iPSC-derived cardiomyocytes is associated with impaired lamin localization to the nuclear envelope.

The LMNA gene encodes the nuclear envelope proteins Lamins A and C, which comprise a major part of the nuclear lamina, provide mechanical support to the nucleus, and participate in diverse intracellular signaling. LMNA mutations give rise to a collection of diseases called laminopathies, including dilated cardiomyopathy (LMNA-DCM) and muscular dystrophies. Although nuclear deformities are a hallmark of LMNA-DCM, the role of nuclear abnormalities in the pathogenesis of LMNA-DCM remains incompletely understood. Using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from LMNA mutant patients and healthy controls, we show that LMNA mutant iPSC-CM nuclei have altered shape or increased size compared to healthy control iPSC-CM nuclei. The LMNA mutation exhibiting the most severe nuclear deformities, R249Q, additionally caused reduced nuclear stiffness and increased nuclear fragility. Importantly, for all cell lines, the degree of nuclear abnormalities corresponded to the degree of Lamin A/C and Lamin B1 mislocalization from the nuclear envelope. The mislocalization was likely due to altered assembly of Lamin A/C. Collectively, these results point to the importance of correct lamin assembly at the nuclear envelope in providing mechanical stability to the nucleus and suggest that defects in nuclear lamina organization may contribute to the nuclear and cellular dysfunction in LMNA-DCM.

Heterologous expression of Dictyostelium discoideum NE81 in mouse embryo fibroblasts reveals conserved mechanoprotective roles of lamins.

Lamins are nuclear intermediate filament proteins that are ubiquitously found in metazoan cells, where they contribute to nuclear morphology, stability, and gene expression. Lamin-like sequences have recently been identified in distantly related eukaryotes, but it remains unclear if these proteins share conserved functions with the lamins found in metazoans. Here, we investigate conserved features between metazoan and amoebozoan lamins using a genetic complementation system to express the Dictyostelium discoideum lamin-like protein NE81 in mammalian cells lacking either specific lamins or all endogenous lamins. We report that NE81 localizes to the nucleus in cells lacking Lamin A/C, and that NE81 expression improves nuclear circularity, reduces nuclear deformability, and prevents nuclear envelope rupture in these cells. However, NE81 did not completely rescue loss of Lamin A/C, and was unable to restore normal distribution of metazoan lamin interactors, such as emerin and nuclear pore complexes, which are frequently displaced in Lamin A/C deficient cells. Collectively, our results indicate that the ability of lamins to modulate the morphology and mechanical properties of nuclei may have been a feature present in the common ancestor of Dictyostelium and animals, whereas other, more specialized interactions may have evolved more recently in metazoan lineages.

Agarose-based 3D Cell Confinement Assay to Study Nuclear Mechanobiology.

Cells in living tissues are exposed to substantial mechanical forces and constraints imposed by neighboring cells, the extracellular matrix, and external factors. Mechanical forces and physical confinement can drive various cellular responses, including changes in gene expression, cell growth, differentiation, and migration, all of which have important implications in physiological and pathological processes, such as immune cell migration or cancer metastasis. Previous studies have shown that nuclear deformation induced by 3D confinement promotes cell contractility but can also cause DNA damage and changes in chromatin organization, thereby motivating further studies in nuclear mechanobiology. In this protocol, we present a custom-developed, easy-to-use, robust, and low-cost approach to induce precisely defined physical confinement on cells using agarose pads with micropillars and externally applied weights. We validated the device by confirming nuclear deformation, changes in nuclear area, and cell viability after confinement. The device is suitable for short- and long-term confinement studies and compatible with imaging of both live and fixed samples, thus presenting a versatile approach to studying the impact of 3D cell confinement and nuclear deformation on cellular function. This article contains detailed protocols for the fabrication and use of the confinement device, including live cell imaging and labeling of fixed cells for subsequent analysis. These protocols can be amended for specific applications. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Design and fabrication of the confinement device wafer Basic Protocol 2: Cell confinement assay Support Protocol 1: Fixation and staining of cells after confinement Support protocol 2: Live/dead staining of cells during confinement.

The lamin A/C Ig-fold undergoes cell density-dependent changes that alter epitope binding.

Lamins A/C are nuclear intermediate filament proteins that are involved in diverse cellular mechanical and biochemical functions. Here, we report that recognition of Lamins A/C by a commonly used antibody (JOL-2) that binds the Lamin A/C Ig-fold and other antibodies targeting similar epitopes is highly dependent on cell density, even though Lamin A/Clevels do not change. We propose that the effect is caused by partial unfolding or masking of the C'E and/or EF loops of the Ig-fold in response to cell spreading. Surprisingly, JOL-2 antibody labeling was insensitive to disruption of cytoskeletal filaments or the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Furthermore, neither nuclear stiffness nor nucleo-cytoskeletal force transmission changed with cell density. These findings are important for the interpretation of immunofluorescence data for Lamin A/C and also raise the intriguing prospect that the conformational changes may play a role in Lamin A/C mediated cellular function.

PP2A methylesterase PME-1 suppresses anoikis and is associated with therapy relapse of PTEN-deficient prostate cancers.

While organ-confined prostate cancer (PCa) is mostly therapeutically manageable, metastatic progression of PCa remains an unmet clinical challenge. Resistance to anoikis, a form of cell death initiated by cell detachment from the surrounding extracellular matrix, is one of the cellular processes critical for PCa progression towards aggressive disease. Therefore, further understanding of anoikis regulation in PCa might provide therapeutic opportunities. Here, we discover that PCa tumours with concomitant inhibition of two tumour suppressor phosphatases, PP2A and PTEN, are particularly aggressive, having < 50% 5-year secondary-therapy-free patient survival. Functionally, overexpression of PME-1, a methylesterase for the catalytic PP2A-C subunit, inhibits anoikis in PTEN-deficient PCa cells. In vivo, PME-1 inhibition increased apoptosis in in ovo PCa tumour xenografts, and attenuated PCa cell survival in zebrafish circulation. Molecularly, PME-1-deficient PC3 cells display increased trimethylation at lysines 9 and 27 of histone H3 (H3K9me3 and H3K27me3), a phenotype known to correlate with increased apoptosis sensitivity. In summary, our results demonstrate that PME-1 supports anoikis resistance in PTEN-deficient PCa cells. Clinically, these results identify PME-1 as a candidate biomarker for a subset of particularly aggressive PTEN-deficient PCa.

Blockage of lamin-A/C loss diminishes the pro-inflammatory macrophage response.

Mutations and defects in nuclear lamins can cause major pathologies, including inflammation and inflammatory diseases. Yet, the underlying molecular mechanisms are not known. We now report that the pro-inflammatory activation of macrophages, as induced by LPS or pathogenic E. coli, reduces Lamin-A/C levels thereby augmenting pro-inflammatory gene expression and cytokine secretion. We show that the activation of bone-marrow-derived macrophages (BMDMs) causes the phosphorylation and degradation of Lamin-A/C, as mediated by CDK1 and Caspase-6, respectively, necessary for upregulating IFN-ß expression. Enhanced IFN-ß expression subsequently increases pro-inflammatory gene expression via the IFN-ß-STAT axis. Pro-inflammatory gene expression was also amplified in the complete absence of Lamin-A/C. Alternatively, pharmacological inhibition of either Lamin-A/C phosphorylation or degradation significantly downregulated pro-inflammatory gene expression, as did the targeting of IFN-ß-STAT pathway members, i.e. phospho-STAT1 and phospho-STAT3. As Lamin-A/C is a previously unappreciated regulator of the pro-inflammatory macrophage response, our findings suggest novel opportunities to treat inflammatory diseases.

Confined migration induces heterochromatin formation and alters chromatin accessibility.

During migration, cells often squeeze through small constrictions, requiring extensive deformation. We hypothesized that nuclear deformation associated with such confined migration could alter chromatin organization and function. By studying cells migrating through microfluidic devices that mimic interstitial spaces in vivo, we found that confined migration results in increased H3K9me3 and H3K27me3 heterochromatin marks that persist for days. This "confined migration-induced heterochromatin" (CMiH) was distinct from heterochromatin formation during migration initiation. Confined migration decreased chromatin accessibility at intergenic regions near centromeres and telomeres, suggesting heterochromatin spreading from existing sites. Consistent with the overall decrease in accessibility, global transcription was decreased during confined migration. Intriguingly, we also identified increased accessibility at promoter regions of genes linked to chromatin silencing, tumor invasion, and DNA damage response. Inhibiting CMiH reduced migration speed, suggesting that CMiH promotes confined migration. Together, our findings indicate that confined migration induces chromatin changes that regulate cell migration and other functions.

Effects of mutant lamins on nucleo-cytoskeletal coupling in Drosophila models of LMNA muscular dystrophy.

The nuclei of multinucleated skeletal muscles experience substantial external force during development and muscle contraction. Protection from such forces is partly provided by lamins, intermediate filaments that form a scaffold lining the inner nuclear membrane. Lamins play a myriad of roles, including maintenance of nuclear shape and stability, mediation of nuclear mechanoresponses, and nucleo-cytoskeletal coupling. Herein, we investigate how disease-causing mutant lamins alter myonuclear properties in response to mechanical force. This was accomplished via a novel application of a micropipette harpooning assay applied to larval body wall muscles of Drosophila models of lamin-associated muscular dystrophy. The assay enables the measurement of both nuclear deformability and intracellular force transmission between the cytoskeleton and nuclear interior in intact muscle fibers. Our studies revealed that specific mutant lamins increase nuclear deformability while other mutant lamins cause nucleo-cytoskeletal coupling defects, which were associated with loss of microtubular nuclear caging. We found that microtubule caging of the nucleus depended on Msp300, a KASH domain protein that is a component of the linker of nucleoskeleton and cytoskeleton (LINC) complex. Taken together, these findings identified residues in lamins required for connecting the nucleus to the cytoskeleton and suggest that not all muscle disease-causing mutant lamins produce similar defects in subcellular mechanics.

Pitx2 patterns an accelerator-brake mechanical feedback through latent TGFß to rotate the gut.

The vertebrate intestine forms by asymmetric gut rotation and elongation, and errors cause lethal obstructions in human infants. Rotation begins with tissue deformation of the dorsal mesentery, which is dependent on left-sided expression of the Paired-like transcription factor Pitx2. The conserved morphogen Nodal induces asymmetric Pitx2 to govern embryonic laterality, but organ-level regulation of Pitx2 during gut asymmetry remains unknown. We found Nodal to be dispensable for Pitx2 expression during mesentery deformation. Intestinal rotation instead required a mechanosensitive latent transforming growth factor-ß (TGFß), tuning a second wave of Pitx2 that induced reciprocal tissue stiffness in the left mesentery as mechanical feedback with the right side. This signaling regulator, an accelerator (right) and brake (left), combines biochemical and biomechanical inputs to break gut morphological symmetry and direct intestinal rotation.

Low lamin A levels enhance confined cell migration and metastatic capacity in breast cancer.

Aberrations in nuclear size and shape are commonly used to identify cancerous tissue. However, it remains unclear whether the disturbed nuclear structure directly contributes to the cancer pathology or is merely a consequence of other events occurring during tumorigenesis. Here, we show that highly invasive and proliferative breast cancer cells frequently exhibit Akt-driven lower expression of the nuclear envelope proteins lamin A/C, leading to increased nuclear deformability that permits enhanced cell migration through confined environments that mimic interstitial spaces encountered during metastasis. Importantly, increasing lamin A/C expression in highly invasive breast cancer cells reflected gene expression changes characteristic of human breast tumors with higher LMNA expression, and specifically affected pathways related to cell-ECM interactions, cell metabolism, and PI3K/Akt signaling. Further supporting an important role of lamins in breast cancer metastasis, analysis of lamin levels in human breast tumors revealed a significant association between lower lamin A levels, Akt signaling, and decreased disease-free survival. These findings suggest that downregulation of lamin A/C in breast cancer cells may influence both cellular physical properties and biochemical signaling to promote metastatic progression.