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Altered microtubule structure, hemichannel localization and beating activity in cardiomyocytes expressing pathologic nuclear lamin A/C.

ABSTRACT: Given the clinical effect of laminopathies, understanding lamin mechanical properties will benefit the treatment of heart failure. Here we report a mechano-dynamic study of LMNA mutations in neonatal rat ventricular myocytes (NRVM) using single cell spectroscopy with Atomic Force Microscopy (AFM) and measured changes in beating force, frequency and contractile amplitude of selected mutant-expressing cells within cell clusters. Furthermore, since beat-to-beat variations can provide clues on the origin of arrhythmias, we analyzed the beating rate variability using a time-domain method which provides a Poincaré plot. Data were further correlated to cell phenotypes. Immunofluorescence and calcium imaging analysis showed that mutant lamin changed NRVMs beating force and frequency. Additionally, we noted an altered microtubule network organization with shorter filament length, and defective hemichannel membrane localization (Connexin 43). These data highlight the interconnection between nucleoskeleton, cytoskeleton and sarcolemmal structures, and the transcellular consequences of mutant lamin protein in the pathogenesis of the cardiac laminopathies.

SUBMITTER: Borin D

PROVIDER: S-EPMC6992992 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Biomechanical defects and rescue of cardiomyocytes expressing pathologic nuclear lamins.

Project description:AimsGiven the clinical impact of LMNA cardiomyopathies, understanding lamin function will fulfill a clinical need and will lead to advancement in the treatment of heart failure. A multidisciplinary approach combining cell biology, atomic force microscopy (AFM), and molecular modeling was used to analyse the biomechanical properties of human lamin A/C gene (LMNA) mutations (E161K, D192G, N195K) using an in vitro neonatal rat ventricular myocyte model.Methods and resultsThe severity of biomechanical defects due to the three LMNA mutations correlated with the severity of the clinical phenotype. AFM and molecular modeling identified distinctive biomechanical and structural changes, with increasing severity from E161K to N195K and D192G, respectively. Additionally, the biomechanical defects were rescued with a p38 MAPK inhibitor.ConclusionsAFM and molecular modeling were able to quantify distinct biomechanical and structural defects in LMNA mutations E161K, D192G, and N195K and correlate the defects with clinical phenotypic severity. Improvements in cellular biomechanical phenotype was demonstrated and may represent a mechanism of action for p38 MAPK inhibition therapy that is now being used in human clinical trials to treat laminopathies.

| S-EPMC5909658 | biostudies-literature

Lamin microaggregates lead to altered mechanotransmission in progerin-expressing cells.

Project description:The nuclear lamina is a meshwork of intermediate filament proteins, and lamin A is the primary mechanical protein. An altered splicing of lamin A, known as progerin, causes the disease Hutchinson-Gilford progeria syndrome. Progerin-expressing cells have altered nuclear shapes and stiffened nuclear lamina with microaggregates of progerin. Here, progerin microaggregate inclusions in the lamina are shown to lead to cellular and multicellular dysfunction. We show with Comsol simulations that stiffened inclusions causes redistribution of normally homogeneous forces, and this redistribution is dependent on the stiffness difference and relatively independent of inclusion size. We also show mechanotransmission changes associated with progerin expression in cells under confinement and cells under external forces. Endothelial cells expressing progerin do not align properly with patterning. Fibroblasts expressing progerin do not align properly to applied cyclic force. Combined, these studies show that altered nuclear lamina mechanics and microstructure impacts cytoskeletal force transmission through the cell.

| S-EPMC7529416 | biostudies-literature

Nuclear localization signal deletion mutants of lamin A and progerin reveal insights into lamin A processing and emerin targeting.

Project description:Lamin A is a major component of the lamina, which creates a dynamic network underneath the nuclear envelope. Mutations in the lamin A gene (LMNA) cause severe genetic disorders, one of which is Hutchinson-Gilford progeria syndrome (HGPS), a disease triggered by a dominant mutant named progerin. Unlike the wild-type lamin A, whose farnesylated C-terminus is excised during post-translational processing, progerin retains its farnesyl tail and accumulates on the nuclear membrane, resulting in abnormal nuclear morphology during interphase. In addition, membrane-associated progerin forms visible cytoplasmic aggregates in mitosis. To examine the potential effects of cytoplasmic progerin, nuclear localization signal (NLS) deleted progerin and lamin A (PGΔNLS and LAΔNLS, respectively) have been constructed. We find that both ΔNLS mutants are farnesylated in the cytosol and associate with a sub-domain of the ER via their farnesyl tails. While the farnesylation on LAΔNLS can be gradually removed, which leads to its subsequent release from the ER into the cytoplasm, PGΔNLS remains permanently farnesylated and membrane-bounded. Moreover, both ΔNLS mutants dominantly affect emerin's nuclear localization. These results reveal new insights into lamin A biogenesis and lamin A-emerin interaction.

| S-EPMC4028357 | biostudies-literature

Concentration-dependent lamin assembly and its roles in the localization of other nuclear proteins.

Project description:The nuclear lamina (NL) consists of lamin polymers and proteins that bind to the polymers. Disruption of NL proteins such as lamin and emerin leads to developmental defects and human diseases. However, the expression of multiple lamins, including lamin-A/C, lamin-B1, and lamin-B2, in mammals has made it difficult to study the assembly and function of the NL. Consequently, it has been unclear whether different lamins depend on one another for proper NL assembly and which NL functions are shared by all lamins or are specific to one lamin. Using mouse cells deleted of all or different combinations of lamins, we demonstrate that the assembly of each lamin into the NL depends primarily on the lamin concentration present in the nucleus. When expressed at sufficiently high levels, each lamin alone can assemble into an evenly organized NL, which is in turn sufficient to ensure the even distribution of the nuclear pore complexes. By contrast, only lamin-A can ensure the localization of emerin within the NL. Thus, when investigating the role of the NL in development and disease, it is critical to determine the protein levels of relevant lamins and the intricate shared or specific lamin functions in the tissue of interest.

| S-EPMC3982994 | biostudies-literature

Cilia-like beating of active microtubule bundles.

Project description:The mechanism that drives the regular beating of individual cilia and flagella, as well as dense ciliary fields, remains unclear. We describe a minimal model system, composed of microtubules and molecular motors, which self-assemble into active bundles exhibiting beating patterns reminiscent of those found in eukaryotic cilia and flagella. These observations suggest that hundreds of molecular motors, acting within an elastic microtubule bundle, spontaneously synchronize their activity to generate large-scale oscillations. Furthermore, we also demonstrate that densely packed, actively bending bundles spontaneously synchronize their beating patterns to produce collective behavior similar to metachronal waves observed in ciliary fields. The simple in vitro system described here could provide insights into beating of isolated eukaryotic cilia and flagella, as well as their synchronization in dense ciliary fields.

| S-EPMC3172966 | biostudies-literature

Flagella-like Beating of a Single Microtubule.

Project description:Kinesin motors can induce a buckling instability in a microtubule with a fixed minus end. Here we show that by modifying the surface with a protein-repellent functionalization and using clusters of kinesin motors, the microtubule can exhibit persistent oscillatory motion resembling the beating of sperm flagella. The observed period is of the order of 1 min. From the experimental images we theoretically determine a distribution of motor forces that explains the observed shapes using a maximum likelihood approach. A good agreement is achieved with a small number of motor clusters acting simultaneously on a microtubule. The tangential forces exerted by a cluster are mostly in the range 0-8 pN toward the microtubule minus end, indicating the action of 1 or 2 kinesin motors. The lateral forces are distributed symmetrically and mainly below 10 pN, while the lateral velocity has a strong peak around zero. Unlike well-known models for flapping filaments, kinesins are found to have a strong "pinning" effect on the beating filaments. Our results suggest new strategies to utilize molecular motors in dynamic roles that depend sensitively on the stress built-up in the system.

| S-EPMC6727605 | biostudies-literature

Altered stability of nuclear lamin-B marks the onset of aging in male Drosophila.

Project description:Epigenetic alterations occur during aging, but it remains unclear what epigenetic features are associated with the onset of physiological decline in animals. Nuclear lamin-B forms the filamentous meshwork underneath the nuclear envelope, providing the structural scaffold necessary for genome organization and gene regulation. We found that reduced level of nuclear lamin-B protein coincides with the decline in locomotor activity and stress resistance in young adult male Drosophila. Ubiquitous lamin-B expression improves locomotor activity of the male flies at the expense of lower stress resistance and shorten lifespan. This observation suggests that tissue-specific expression of lamin-B may regulate different aspects of animal physiology during aging. To test this hypothesis, specific GAL-4 lines were used to drive the expression of lamin-B in specific neuronal populations and muscle tissues in male flies. Ectopic expression of lamin-B in the dopaminergic neurons within the protocerebral anterior medial region of the brain improves the locomotor activity of the male flies with little impact on their stress responses and lifespan. Interestingly, age-dependent decrease in the level of lamin-B protein is independent of its mRNA expression. Instead, cellular thermal shift assay showed that lamin-B and CP190 insulator protein undergo significant change in their solubility during aging. This suggests that the increased solubility of lamin-B protein may contribute to its reduced stability and degradation during aging.

| S-EPMC8947137 | biostudies-literature

Spontaneously beating cardiomyocytes derived from white mature adipocytes.

Project description:AimsAdipose stromal cells and dissociated brown adipose tissue have been shown to generate cardiomyocyte-like cells. However, it is not clear whether white mature adipocytes have the same potential, even though a close relationship has been found between adipocytes and vascular endothelial cells, another cardiovascular cell type. The objective of this study was to examine if white adipocytes would be able to supply cardiomyocytes.Methods and resultsWe prepared a highly purified population of lipid-filled adipocytes from mice, 6-7 weeks of age. When allowed to lose lipids, the adipocytes assumed a fibroblast-like morphology, so-called dedifferentiated fat (DFAT) cells. Subsequently, 10-15% of the DFAT cells spontaneously differentiated into cardiomyocyte-like cells, in which the cardiomyocyte phenotype was identified by morphological observations, expression of cardiomyocyte-specific markers, and immunocytochemical staining. In addition, electrophysiological studies revealed pacemaker activity in these cells, and functional studies showed that a beta-adrenergic agonist stimulated the beating rate, whereas a beta-antagonist reduced it. In vitro treatment of newly isolated adipocytes or DFAT cells with inhibitors of bone morphogenetic proteins (BMP) and Wnt signalling promoted the development of the cardiomyocyte phenotype as determined by the number or beating colonies of cardiomyocyte-like cells and expression of troponin I, a cardiomyocyte-specific marker. Inhibition of BMP was most effective in promoting the cardiomyocyte phenotype in adipocytes, whereas Wnt-inhibition was most effective in DFAT cells.ConclusionWhite mature adipocytes can differentiate into cardiomyocyte-like cells, suggesting a link between adipocyte and cardiomyocyte differentiation.

| S-EPMC2791054 | biostudies-literature

Lamin A/C-dependent localization of Nesprin-2, a giant scaffolder at the nuclear envelope.

Project description:The vertebrate proteins Nesprin-1 and Nesprin-2 (also referred to as Enaptin and NUANCE) together with ANC-1 of Caenorhabditis elegans and MSP-300 of Drosophila melanogaster belong to a novel family of alpha-actinin type actin-binding proteins residing at the nuclear membrane. Using biochemical techniques, we demonstrate that Nesprin-2 binds directly to emerin and the C-terminal common region of lamin A/C. Selective disruption of the lamin A/C network in COS7 cells, using a dominant negative lamin B mutant, resulted in the redistribution of Nesprin-2. Furthermore, using lamin A/C knockout fibroblasts we show that lamin A/C is necessary for the nuclear envelope localization of Nesprin-2. In normal skin where lamin A/C is differentially expressed, strong Nesprin-2 expression was found in all epidermal layers, including the basal layer where only lamin C is present. This indicates that lamin C is sufficient for proper Nesprin-2 localization at the nuclear envelope. Expression of dominant negative Nesprin-2 constructs and knockdown studies in COS7 cells revealed that the presence of Nesprin-2 at the nuclear envelope is necessary for the proper localization of emerin. Our data imply a scaffolding function of Nesprin-2 at the nuclear membrane and suggest a potential involvement of this multi-isomeric protein in human disease.

| S-EPMC1165422 | biostudies-literature

Drosophila klarsicht has distinct subcellular localization domains for nuclear envelope and microtubule localization in the eye.

Project description:The Drosophila klarsicht (klar) gene is required for developmentally regulated migrations of photoreceptor cell nuclei in the eye. klar encodes a large ( approximately 250 kD) protein with only one recognizable amino acid sequence motif, a KASH (Klar, Anc-1, Syne-1 homology) domain, at its C terminus. It has been proposed that Klar facilitates nuclear migration by linking the nucleus to the microtubule organizing center (MTOC). Here we perform genetic and immunohistochemical experiments that provide a critical test of this model. We analyze mutants in the endogenous klar gene and also flies that express deleted forms of Klar protein from transgenes. We find that the KASH domain of Klar is critical for perinuclear localization and for function. In addition, we find that the N-terminal portion of Klar is also important for function and contains a domain that localizes the protein to microtubules apical to the nucleus. These results provide strong support for a model in which Klar links the nucleus to the MTOC.

| S-EPMC1448802 | biostudies-literature

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