Lamins are intermediate filament proteins that contribute to numerous cellular functions, including nuclear morphology and mechanical stability. The N-terminal head domain of lamin is critical for higher order filament assembly and function, yet the effects of commonly used N-terminal tags on lamin function remain largely unexplored. Here, we systematically studied the effect of two differently sized tags on Lamin A (LaA) function in a mammalian cell model engineered to allow for precise control of expression of tagged lamin proteins. Untagged, FLAG-tagged, and GFP-tagged LaA completely rescued nuclear shape defects when expressed at similar levels in lamin A/C-deficient (Lmna-/-) MEFs, and all LaA constructs prevented increased nuclear envelope (NE) ruptures in these cells. N-terminal tags, however, altered the nuclear localization of LaA and impaired the ability of LaA to restore nuclear deformability and to recruit Emerin to the nuclear membrane in Lmna-/- MEFs. Our finding that tags impede some LaA functions but not others may explain the partial loss of function phenotypes when tagged lamins are expressed in model organisms and should caution researchers using tagged lamins to study the nucleus.

Most of the single point mutations of the LMNA gene are associated with distinct muscular dystrophies, marked by heterogenous phenotypes but primarily the loss and symmetric weakness of skeletal muscle tissue. The molecular mechanism and phenotype-genotype relationships in these muscular dystrophies are poorly understood. An effort has been here to delineating the adaptation of mechanical inputs into biological response by mutant cells of lamin A associated muscular dystrophy. In this study, we implement engineered smooth and pattern surfaces of particular young modulus to mimic muscle physiological range. Using fluorescence and atomic force microscopy, we present distinct architecture of the actin filament along with abnormally distorted cell and nuclear shape in mutants, which showed a tendency to deviate from wild type cells. Topographic features of pattern surface antagonize the binding of the cell with it. Correspondingly, from the analysis of genome wide expression data in wild type and mutant cells, we report differential expression of the gene products of the structural components of cell adhesion as well as LINC (linkers of nucleoskeleton and cytoskeleton) protein complexes. This study also reveals mis expressed downstream signaling processes in mutant cells, which could potentially lead to onset of the disease upon the application of engineered materials to substitute the role of conventional cues in instilling cellular behaviors in muscular dystrophies. Collectively, these data support the notion that lamin A is essential for proper cellular mechanotransduction from extracellular environment to the genome and impairment of the muscle cell differentiation in the pathogenic mechanism for lamin A associated muscular dystrophy.

Globozoospermia is a form of male infertility characterized by spermatozoa with spherical heads lacking acrosomes. The aim of this study was to evaluate ultrastructural and molecular defects in different types of globozoospermia. Semen samples from 12 infertile patients (9 with complete globozoospermia and 3 with partial globozoospermia) and 10 normozoospermic men (control) were examined by transmission electron microscopy and immunocytochemistry with antibodies against lamin B1. The presence of lamin A and progerin was assessed by reverse transcription-PCR. Whole exome sequencing was performed in three patients. In semen samples with complete and partial globozoospermia, lamin B1 was observed at the periphery of sperm nuclei, whereas lamin A and progerin were absent. Nuclear envelope pores were found in spermatozoa from both patient groups, regardless of morphology and chromatin condensation, in contrast to the control group. Non-condensed chromatin was present in 51%-81% of cases of complete globozoospermia and in 36%-79% of cases of partial globozoospermia. Homozygous DPY19L2 and SPATA16 variants were identified in two patients with partial globozoospermia and one patient with complete globozoospermia. An atypical nuclear membrane with abnormal nuclear pore distribution and lamin B1 localization was observed in spermatozoa from patients with both complete and partial globozoospermia. The genetic defects in the DPY19L2 and SPATA16 genes detected in patients from both globozoospermic groups suggest a generalized disruption of nuclear structure in globozoospermia, highlighting the genetic and phenotypic similarities between complete and partial globozoospermia.

Epigenetics is the study of heritable changes to the genome and gene expression patterns that are not caused by direct changes to the DNA sequence. Examples of these changes include posttranslational modifications to DNA-bound histone proteins, DNA methylation, and remodeling of nuclear architecture. Collectively, epigenetic changes provide a layer of regulation that affects transcriptional activity of genes while leaving DNA sequences unaltered. Sequence variants or mutations affecting enzymes responsible for modifying or sensing epigenetic marks have been identified in patients with congenital heart disease (CHD), and small-molecule inhibitors of epigenetic complexes have shown promise as therapies for adult heart diseases. Additionally, transgenic mice harboring mutations or deletions of genes encoding epigenetic enzymes recapitulate aspects of human cardiac disease. Taken together, these findings suggest that the evolving field of epigenetics will inform our understanding of congenital and adult cardiac disease and offer new therapeutic opportunities.

Mutations in the LMNA gene-encoding A-type lamins can cause Limb-Girdle muscular dystrophy Type 1B (LGMD1B). This disease presents with weakness and wasting of the proximal skeletal muscles and has a variable age of onset and disease severity. This variability has been attributed to genetic background differences among individuals; however, such variants have not been well characterized. To identify such variants, we investigated a multigeneration family in which affected individuals are diagnosed with LGMD1B. The primary genetic cause of LGMD1B in this family is a dominant mutation that activates a cryptic splice site, leading to a five-nucleotide deletion in the mature mRNA. This results in a frame shift and a premature stop in translation. Skeletal muscle biopsies from the family members showed dystrophic features of variable severity, with the muscle fibers of some family members possessing cores, regions of sarcomeric disruption, and a paucity of mitochondria, not commonly associated with LGMD1B. Using whole genome sequencing (WGS), we identified 21 DNA sequence variants that segregate with the family members possessing more profound dystrophic features and muscle cores. These include a relatively common variant in coiled-coil domain containing protein 78 (CCDC78). This variant was given priority because another mutation in CCDC78 causes autosomal dominant centronuclear myopathy-4, which causes cores in addition to centrally positioned nuclei. Therefore, we analyzed muscle biopsies from family members and discovered that those with both the LMNA mutation and the CCDC78 variant contain muscle cores that accumulated both CCDC78 and RyR1. Muscle cores containing mislocalized CCDC78 and RyR1 were absent in the less profoundly affected family members possessing only the LMNA mutation. Taken together, our findings suggest that a relatively common variant in CCDC78 can impart profound muscle pathology in combination with a LMNA mutation and accounts for variability in skeletal muscle disease phenotypes.

BACKGROUND: The Keap1-Nrf2 pathway serves as a central regulator that mediates transcriptional responses to xenobiotic and oxidative stimuli. Recent studies have shown that Keap1 and Nrf2 can regulate transcripts beyond antioxidant and detoxifying genes, yet the underlying mechanisms remain unclear. Our research has uncovered that Drosophila Keap1 (dKeap1) and Nrf2 (CncC) proteins can control high-order chromatin structure, including heterochromatin.
RESULTS: In this study, we identified the molecular interaction between dKeap1 and lamin Dm0, the Drosophila B-type lamin responsible for the architecture of nuclear lamina and chromatin. Ectopic expression of dKeap1 led to an ectopic localization of lamin to the intra-nuclear area, corelated with the spreading of the heterochromatin marker H3K9me2 into euchromatin regions. Additionally, mis-regulated dKeap1 disrupted the morphology of the nuclear lamina. Knocking down of dKeap1 partially rescued the lethality induced by lamin overexpression, suggesting their genetic interaction during development.
CONCLUSIONS: The discovered dKeap1-lamin interaction suggests a novel role for the Keap1 oxidative/xenobiotic response factor in regulating chromatin architecture.

UNASSIGNED: Mutations in LMNA encoding nuclear envelope proteins lamin A/C cause dilated cardiomyopathy. Activation of the AKT/mTOR (RAC-α serine/threonine-protein kinase/mammalian target of rapamycin) pathway is implicated as a potential pathophysiologic mechanism. The aim of this study was to assess whether pharmacological inhibition of mTOR signaling has beneficial effects on heart function and prolongs survival in a mouse model of the disease, after onset of heart failure.
UNASSIGNED: We treated male LmnaH222P/H222P mice, after the onset of heart failure, with placebo or either of 2 orally bioavailable mTOR inhibitors: everolimus or NV-20494, a rapamycin analog highly selective against mTORC1. We examined left ventricular remodeling, and the cell biological, biochemical, and histopathologic features of cardiomyopathy, potential drug toxicity, and survival.
UNASSIGNED: Everolimus treatment (n=17) significantly reduced left ventricular dilatation and increased contractility on echocardiography, with a 7% (P=0.018) reduction in left ventricular end-diastolic diameter and a 39% (P=0.0159) increase fractional shortening compared with placebo (n=17) after 6 weeks of treatment. NV-20494 treatment (n=15) yielded similar but more modest and nonsignificant changes. Neither drug prevented the development of cardiac fibrosis. Drug treatment reactivated suppressed autophagy and inhibited mTORC1 signaling in the heart, although everolimus was more potent. With regards to drug toxicity, everolimus alone led to a modest degree of glucose intolerance during glucose challenge. Everolimus (n=20) and NV-20494 (n=20) significantly prolonged median survival in LmnaH222P/H222P mice, by 9% (P=0.0348) and 11% (P=0.0206), respectively, compared with placebo (n=20).
UNASSIGNED: These results suggest that mTOR inhibitors may be beneficial in patients with cardiomyopathy caused by LMNA mutations and that further study is warranted.

B-type lamins are critical nuclear envelope proteins that interact with the three-dimensional genomic architecture. However, identifying the direct roles of B-lamins on dynamic genome organization has been challenging as their joint depletion severely impacts cell viability. To overcome this, we engineered mammalian cells to rapidly and completely degrade endogenous B-type lamins using Auxin-inducible degron technology.
Using live-cell Dual Partial Wave Spectroscopic (Dual-PWS) microscopy, Stochastic Optical Reconstruction Microscopy (STORM), in situ Hi-C, CRISPR-Sirius, and fluorescence in situ hybridization (FISH), we demonstrate that lamin B1 and lamin B2 are critical structural components of the nuclear periphery that create a repressive compartment for peripheral-associated genes. Lamin B1 and lamin B2 depletion minimally alters higher-order chromatin folding but disrupts cell morphology, significantly increases chromatin mobility, redistributes both constitutive and facultative heterochromatin, and induces differential gene expression both within and near lamin-associated domain (LAD) boundaries. Critically, we demonstrate that chromatin territories expand as upregulated genes within LADs radially shift inwards. Our results indicate that the mechanism of action of B-type lamins comes from their role in constraining chromatin motion and spatial positioning of gene-specific loci, heterochromatin, and chromatin domains.
Our findings suggest that, while B-type lamin degradation does not significantly change genome topology, it has major implications for three-dimensional chromatin conformation at the single-cell level both at the lamina-associated periphery and the non-LAD-associated nuclear interior with concomitant genome-wide transcriptional changes. This raises intriguing questions about the individual and overlapping roles of lamin B1 and lamin B2 in cellular function and disease.

The nuclear lamina serves important functions in the nucleus, providing structural support to the nuclear envelope and contributing to chromatin organization. The primary proteins that constitute the lamina are nuclear lamins whose functions are impacted by post-translational modifications, including phosphorylation by protein kinase C (PKC). While PKC-mediated lamin phosphorylation is important for nuclear envelope breakdown during mitosis, less is known about interphase roles for PKC in regulating nuclear structure. Here we show that overexpression of PKC ß, but not PKC α, increases the Lamin A/C mobile fraction in the nuclear envelope in HeLa cells without changing the overall structure of Lamin A/C and Lamin B1 within the nuclear lamina. Conversely, knockdown of PKC ß, but not PKC α, reduces the Lamin A/C mobile fraction. Thus, we demonstrate an isoform-specific role for PKC in regulating interphase Lamin A/C dynamics outside of mitosis.

Here, we report the first characterization of the effects resulting from the manipulation of Soluble-Lamin Associated Protein (SLAP) expression during mammalian brain development. We found that SLAP localizes to the nuclear envelope and when overexpressed causes changes in nuclear morphology and lengthening of mitosis. SLAP overexpression in apical progenitors of the developing mouse brain altered asymmetric cell division, neurogenic commitment and neuronal migration ultimately resulting in unbalance in the proportion of upper, relative to deeper, neuronal layers. Several of these effects were also recapitulated upon Cas9-mediated knockdown. Ultimately, SLAP overexpression during development resulted in a reduction in subcortical projections of young mice and, notably, reduced their exploratory behavior. Our study shows the potential relevance of the previously uncharacterized nuclear envelope protein SLAP in neurodevelopmental disorders.