Components and Mechanisms of Nuclear Mechanotransduction.

The cell nucleus is best known as the container of the genome. Its envelope provides a barrier for passive macromolecule diffusion, which enhances the control of gene expression. As its largest and stiffest organelle, the nucleus also defines the minimal space requirements of a cell. Internal or external pressures that deform a cell to its physical limits cause a corresponding nuclear deformation. Evidence is consolidating that the nucleus, in addition to its genetic functions, serves as a physical sensing device for critical cell body deformation. Nuclear mechanotransduction allows cells to adapt their acute behaviors, mechanical stability, paracrine signaling, and fate to their physical surroundings. This review summarizes the basic chemical and mechanical properties of nuclear components, and how these properties are thought to be utilized for mechanosensing.

Loss of an H3K9me anchor rescues laminopathy-linked changes in nuclear organization and muscle function in an Emery-Dreifuss muscular dystrophy model.

Mutations in the nuclear structural protein lamin A produce rare, tissue-specific diseases called laminopathies. The introduction of a human Emery-Dreifuss muscular dystrophy (EDMD)-inducing mutation into the C. elegans lamin (LMN-Y59C), recapitulates many muscular dystrophy phenotypes, and correlates with hyper-sequestration of a heterochromatic array at the nuclear periphery in muscle cells. Using muscle-specific emerin Dam-ID in worms, we monitored the effects of the mutation on endogenous chromatin. An increased contact with the nuclear periphery along chromosome arms, and an enhanced release of chromosomal centers, coincided with the disease phenotypes of reduced locomotion and compromised sarcomere integrity. The coupling of the LMN-Y59C mutation with the ablation of CEC-4, a chromodomain protein that anchors H3K9-methylated chromatin at the nuclear envelope (NE), suppressed the muscle-associated disease phenotypes. Deletion of cec-4 also rescued LMN-Y59C-linked alterations in chromatin organization and some changes in transcription. Sequences that changed position in the LMN-Y59C mutant, are enriched for E2F (EFL-2)-binding sites, consistent with previous studies suggesting that altered Rb-E2F interaction with lamin A may contribute to muscle dysfunction. In summary, we were able to counteract the dominant muscle-specific defects provoked by LMNA mutation by the ablation of a lamin-associated H3K9me anchor, suggesting a novel therapeutic pathway for EDMD.

Emerin self-assembly and nucleoskeletal coupling regulate nuclear envelope mechanics against stress.

Emerin is an integral nuclear envelope protein that participates in the maintenance of nuclear shape. When mutated or absent, emerin causes X-linked Emery-Dreifuss muscular dystrophy (EDMD). To understand how emerin takes part in molecular --scaffolding at the nuclear envelope and helps protect the nucleus against mechanical stress, we established its nanoscale organization using single-molecule tracking and super-resolution microscopy. We show that emerin monomers form localized oligomeric nanoclusters stabilized by both lamin A/C and the SUN1-containing linker of nucleoskeleton and cytoskeleton (LINC) complex. Interactions of emerin with nuclear actin and BAF (also known as BANF1) additionally modulate its membrane mobility and its ability to oligomerize. In nuclei subjected to mechanical challenges, the mechanotransduction functions of emerin are coupled to changes in its oligomeric state, and the incremental self-assembly of emerin determines nuclear shape adaptation against mechanical forces. We also show that the abnormal nuclear envelope deformations induced by EDMD emerin mutants stem from improper formation of lamin A/C and LINC complex-stabilized emerin oligomers. These findings place emerin at the center of the molecular processes that regulate nuclear shape remodeling in response to mechanical challenges.

The Nuclear Envelope in Ageing and Progeria.

Development from embryo to adult, organismal homeostasis and ageing are consecutive processes that rely on several functions of the nuclear envelope (NE). The NE compartmentalises the eukaryotic cells and provides physical stability to the genetic material in the nucleus. It provides spatiotemporal regulation of gene expression by controlling nuclear import and hence access of transcription factors to target genes as well as organisation of the genome into open and closed compartments. In addition, positioning of chromatin relative to the NE is important for DNA replication and repair and thereby also for genome stability. We discuss here the relevance of the NE in two classes of age-related human diseases. Firstly, we focus on the progeria syndromes Hutchinson-Gilford (HGPS) and Nestor-Guillermo (NGPS), which are caused by mutations in the LMNA and BANF1 genes, respectively. Both genes encode ubiquitously expressed components of the nuclear lamina that underlines the nuclear membranes. HGPS and NGPS patients manifest symptoms of accelerated ageing and cells from affected individuals show similar defects as cells from healthy old donors, including signs of increased DNA damage and epigenetic alternations. Secondly, we describe how several age-related neurodegenerative diseases, such as amyotrophic lateral sclerosis and Huntington's disease, are related with defects in nucleocytoplasmic transport. A common feature of this class of diseases is the accumulation of nuclear pore proteins and other transport factors in inclusions. Importantly, genetic manipulations of the nucleocytoplasmic transport machinery can alleviate disease-related phenotypes in cell and animal models, paving the way for potential therapeutic interventions.

A case of restrictive dermopathy in a Hutterite newborn: Diagnosis and creative skin-directed management.

Restrictive dermopathy is a lethal autosomal recessive disease characterized by tightly adherent skin, distinctive facial dysmorphisms, arthrogryposis, and pulmonary hypoplasia. While clinical findings are unique, histopathology and genetic analysis are critical for early diagnostic confirmation and to initiate appropriate management for this lethal disease. We report on a preterm Hutterite male neonate with biallelic ZMPSTE24 mutations to highlight the clinical and histopathological features of restrictive dermopathy and share our skin-directed management strategies.

The Influence of a Genetic Variant in CCDC78 on LMNA-Associated Skeletal Muscle Disease.

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.

A common variant that alters SUN1 degradation associates with hepatic steatosis and metabolic traits in multiple cohorts.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD), and its progressive form steatohepatitis (NASH), represent a genetically and phenotypically diverse entity for which there is no approved therapy, making it imperative to define the spectrum of pathways contributing to its pathogenesis. Rare variants in genes encoding nuclear envelope proteins cause lipodystrophy with early-onset NAFLD/NASH; we hypothesized that common variants in nuclear envelope-related genes might also contribute to hepatic steatosis and NAFLD. METHODS: Using hepatic steatosis as the outcome of interest, we performed an association meta-analysis of nuclear envelope-related coding variants in three large discovery cohorts (N >120,000 participants), followed by phenotype association studies in large validation cohorts (N >600,000) and functional testing of the top steatosis-associated variant in cell culture. RESULTS: A common protein-coding variant, rs6461378 (SUN1 H118Y), was the top steatosis-associated variant in our association meta-analysis (p <0.001). In ancestrally distinct validation cohorts, rs6461378 associated with histologic NAFLD and with NAFLD-related metabolic traits including increased serum fatty acids, type 2 diabetes, hypertension, cardiovascular disease, and decreased HDL. SUN1 H118Y was subject to increased proteasomal degradation relative to wild-type SUN1 in cells, and SUN1 H118Y-expressing cells exhibited insulin resistance and increased lipid accumulation. CONCLUSIONS: Collectively, these data support a potential causal role for the common SUN1 variant rs6461378 in NAFLD and metabolic disease. IMPACT AND IMPLICATIONS: Non-alcoholic fatty liver disease (NAFLD), with an estimated global prevalence of nearly 30%, is a growing cause of morbidity and mortality for which there is no approved pharmacologic therapy. Our data provide a rationale for broadening current concepts of NAFLD genetics and pathophysiology to include the nuclear envelope, and particularly Sad1 and UNC84 domain containing 1 (SUN1), as novel contributors to this common liver disease. Furthermore, if future studies confirm causality of the common SUN1 H118Y variant, it has the potential to become a broadly relevant therapeutic target in NAFLD and metabolic disease.

Disease progression rate is a strong predictor of ventricular arrhythmias in patients with cardiac laminopathies: a primary prevention cohort study.

AIMS: Cardiac disease progression prior to first ventricular arrhythmia (VA) in LMNA genotype-positive patients is not described. METHODS AND RESULTS: We performed a primary prevention cohort study, including consecutive LMNA genotype-positive patients from our centre. Patients underwent repeated clinical, electrocardiographic, and echocardiographic examinations. Electrocardiographic and echocardiographic disease progression as a predictor of first-time VA was evaluated by generalized estimation equation analyses. Threshold values at transition to an arrhythmic phenotype were assessed by threshold regression analyses. We included 94 LMNA genotype-positive patients without previous VA (age 38 ± 15 years, 32% probands, 53% females). Nineteen (20%) patients experienced VA during 4.6 (interquartile range 2.1-7.3) years follow up, at mean age 50 ± 11 years. We analysed 536 echocardiographic and 261 electrocardiogram examinations. Individual patient disease progression was associated with VA [left ventricular ejection fraction (LVEF) odds ratio (OR) 1.4, 95% confidence interval (CI) 1.2-1.6 per 5% reduction, left ventricular end-diastolic volume index (LVEDVi) OR 1.2 (95% CI 1.1-1.3) per 5 mL/m2 increase, PR interval OR 1.2 (95% CI 1.1-1.4) per 10 ms increase]. Threshold values for transition to an arrhythmic phenotype were LVEF 44%, LVEDVi 77 mL/m2, and PR interval 280 ms. CONCLUSIONS: Incidence of first-time VA was 20% during 4.6 years follow up in LMNA genotype-positive patients. Individual patient disease progression by ECG and echocardiography were strong predictors of VA, indicating that disease progression rate may have additional value to absolute measurements when considering primary preventive ICD. Threshold values of LVEF <44%, LVEDVi >77 mL/m2, and PR interval >280 ms indicated transition to a more arrhythmogenic phenotype.

The wide and growing range of lamin B-related diseases: from laminopathies to cancer.

B-type lamins are fundamental components of the nuclear lamina, a complex structure that acts as a scaffold for organization and function of the nucleus. Lamin B1 and B2, the most represented isoforms, are encoded by LMNB1 and LMNB2 gene, respectively. All B-type lamins are synthesized as precursors and undergo sequential post-translational modifications to generate the mature protein. B-type lamins are involved in a wide range of nuclear functions, including DNA replication and repair, regulation of chromatin and nuclear stiffness. Moreover, lamins B1 and B2 regulate several cellular processes, such as tissue development, cell cycle, cellular proliferation, senescence, and DNA damage response. During embryogenesis, B-type lamins are essential for organogenesis, in particular for brain development. As expected from the numerous and pivotal functions of B-type lamins, mutations in their genes or fluctuations in their expression levels are critical for the onset of several diseases. Indeed, a growing range of human disorders have been linked to lamin B1 or B2, increasing the complexity of the group of diseases collectively known as laminopathies. This review highlights the recent findings on the biological role of B-type lamins under physiological or pathological conditions, with a particular emphasis on brain disorders and cancer.

Decreased mechanotransduction prevents nuclear collapse in a Caenorhabditis elegans laminopathy.

The function of the nucleus depends on the integrity of the nuclear lamina, an intermediate filament network associated with the linker of nucleoskeleton and cytoskeleton (LINC) complex. The LINC complex spans the nuclear envelope and mediates nuclear mechanotransduction, the process by which mechanical signals and forces are transmitted across the nuclear envelope. In turn, the AAA+ ATPase torsinA is thought to regulate force transmission from the cytoskeleton to the nucleus. In humans, mutations affecting nuclear envelope-associated proteins cause laminopathies, including progeria, myopathy, and dystonia, though the extent to which endogenous mechanical stresses contribute to these pathologies is unclear. Here, we use the Caenorhabditis elegans germline as a model to investigate mechanisms that maintain nuclear integrity as germ cell nuclei progress through meiotic development and migrate for gametogenesis-processes that require LINC complex function. We report that decreasing the function of the C. elegans torsinA homolog, OOC-5, rescues the sterility and premature aging caused by a null mutation in the single worm lamin homolog. We show that decreasing OOC-5/torsinA activity prevents nuclear collapse in lamin mutants by disrupting the function of the LINC complex. At a mechanistic level, OOC-5/torsinA promotes the assembly or maintenance of the lamin-associated LINC complex and this activity is also important for interphase nuclear pore complex insertion into growing germline nuclei. These results demonstrate that LINC complex-transmitted forces damage nuclei with a compromised nuclear lamina. Thus, the torsinA-LINC complex nexus might comprise a therapeutic target for certain laminopathies by preventing damage from endogenous cellular forces.

InterLINCing Chromatin Organization and Mechanobiology in Laminopathies.

PURPOSE OF REVIEW: In this review, we explore the chromatin-related consequences of laminopathy-linked mutations through the lens of mechanotransduction. RECENT FINDINGS: Multiple studies have highlighted the role of the nuclear lamina in maintaining the integrity of the nucleus. The lamina also has a critical role in 3D genome organization. Mutations in lamina proteins associated with various laminopathies result in the loss of organization of DNA at the nuclear periphery. However, it remains unclear if or how these two aspects of lamin function are connected. Recent data suggests that unlinking the cytoskeleton from the nuclear lamina may be beneficial to slow progress of deleterious phenotypes observed in laminopathies. In this review, we highlight emerging data that suggest interlinked chromatin- and mechanical biology-related pathways are interconnected in the pathogenesis of laminopathies.

COMPREHENSIVE CARDIOVASCULAR THERAPY IN EMERY-DREIFUSS MUSCULAR DYSTROPHY: A CASE REPORT.

A 25-year-old male with known EDMD was referred for the cardiology consultation due to symptoms of heart failure. Echocardiography showed decrease left ventricular ejection fraction (LVEF) and therapy with ramipril, torsemide and rivaroxaban was initiated. Despite initial improvement, the patient later developed presyncope, bradycardia, irregular heartbeat and worsening of dyspnea. Therefore, implantation of resynchronization pacemaker with the function of implantable cardioverter-defibrillator (CRT-D/P) was performed. Ramipril was substituted by sacubitril/valsartan, and mineralocorticoid receptor antagonist and beta-blocker were initiated. Genetic testing found AD mutation in lamin A/C gene LMNA c.746G>A, p.(Arg249Gln). Upon follow-up, the patient demonstrated resolution of dyspnea and reverse remodeling of the left ventricle with complete restoration of the LVEF.

Lamin A-mediated nuclear lamina integrity is required for proper ciliogenesis.

The primary cilium is a sensory organelle that receives specific signals from the extracellular environment important for vertebrate development and tissue homeostasis. Lamins, the major components of the nuclear lamina, are required to maintain the nuclear structure and are involved in most nuclear activities. In this study, we show that deficiency in lamin A/C causes defective ciliogenesis, accompanied by increased cytoplasmic accumulation of actin monomers and increased formation of actin filaments. Disruption of actin filaments by cytochalasin D rescues the defective ciliogenesis in lamin A/C-depleted cells. Moreover, lamin A/C-deficient cells display lower levels of nesprin 2 and defects in recruiting Arp2, myosin Va, and tau tubulin kinase 2 to the basal body during ciliogenesis. Collectively, our results uncover a functional link between nuclear lamina integrity and ciliogenesis and implicate the malfunction of primary cilia in the pathogenesis of laminopathy.

Lethal Restrictive Dermopathy with ZMPSTE24 Mutation.

Lethal restrictive dermopathy is genodermatoses associated with lamin protein defects resulting in connective tissue abnormalities of skin, musculoskeletal, and adipose tissue. We report one such case with a mutation in the ZMPSTE24 gene which is involved in lamin protein synthesis, resulting in fetal akinesia or hypokinesia deformation sequence. Early recognition in the perinatal period of distinctive clinical and skin histological features followed by molecular diagnosis enabled genetic counseling for the affected family.

Genotype Complements the Phenotype: Identification of the Pathogenicity of an LMNA Splice Variant by Nanopore Long-Read Sequencing in a Large DCM Family.

Dilated cardiomyopathy (DCM) is a common cause of heart failure (HF) and is of familial origin in 20−40% of cases. Genetic testing by next-generation sequencing (NGS) has yielded a definite diagnosis in many cases; however, some remain elusive. In this study, we used a combination of NGS, human-induced pluripotent-stem-cell-derived cardiomyocytes (iPSC-CMs) and nanopore long-read sequencing to identify the causal variant in a multi-generational pedigree of DCM. A four-generation family with familial DCM was investigated. Next-generation sequencing (NGS) was performed on 22 family members. Skin biopsies from two affected family members were used to generate iPSCs, which were then differentiated into iPSC-CMs. Short-read RNA sequencing was used for the evaluation of the target gene expression, and long-read RNA nanopore sequencing was used to evaluate the relevance of the splice variants. The pedigree suggested a highly penetrant, autosomal dominant mode of inheritance. The phenotype of the family was suggestive of laminopathy, but previous genetic testing using both Sanger and panel sequencing only yielded conflicting evidence for LMNA p.R644C (rs142000963), which was not fully segregated. By re-sequencing four additional affected family members, further non-coding LMNA variants could be detected: rs149339264, rs199686967, rs201379016, and rs794728589. To explore the roles of these variants, iPSC-CMs were generated. RNA sequencing showed the LMNA expression levels to be significantly lower in the iPSC-CMs of the LMNA variant carriers. We demonstrated a dysregulated sarcomeric structure and altered calcium homeostasis in the iPSC-CMs of the LMNA variant carriers. Using targeted nanopore long-read sequencing, we revealed the biological significance of the variant c.356+1G>A, which generates a novel 5' splice site in exon 1 of the cardiac isomer of LMNA, causing a nonsense mRNA product with almost complete RNA decay and haploinsufficiency. Using novel molecular analysis and nanopore technology, we demonstrated the pathogenesis of the rs794728589 (c.356+1G>A) splice variant in LMNA. This study highlights the importance of precise diagnostics in the clinical management and workup of cardiomyopathies.

LMNA Co-Regulated Gene Expression as a Suitable Readout after Precise Gene Correction.

LMNA-related muscular dystrophy is an autosomal-dominant progressive disorder caused by mutations in LMNA. LMNA missense mutations are becoming correctable with CRISPR/Cas9-derived tools. Evaluating the functional recovery of LMNA after gene editing bears challenges as there is no reported direct loss of function of lamin A/C proteins in patient-derived cells. The proteins encoded by LMNA are lamins A/C, important ubiquitous nuclear envelope proteins but absent in pluripotent stem cells. We induced lamin A/C expression in induced pluripotent stem cells (iPSCs) of two patients with LMNA-related muscular dystrophy, NM_170707.4 (LMNA): c.1366A > G, p.(Asn456Asp) and c.1494G > T, p.(Trp498Cys), using a short three-day, serum-induced differentiation protocol and analyzed expression profiles of co-regulated genes, examples being COL1A2 and S100A6. We then performed precise gene editing of LMNA c.1366A > G using the near-PAMless (PAM: protospacer-adjacent motif) cytosine base editor. We show that the mutation can be repaired to 100% efficiency in individual iPSC clones. The fast differentiation protocol provided a functional readout and demonstrated increased lamin A/C expression as well as normalized expression of co-regulated genes. Collectively, our findings demonstrate the power of CRISPR/Cas9-mediated gene correction and effective outcome measures in a disease with, so far, little perspective on therapies.

Heterozygous lamin B1 and lamin B2 variants cause primary microcephaly and define a novel laminopathy.

PURPOSE: Lamins are the major component of nuclear lamina, maintaining structural integrity of the nucleus. Lamin A/C variants are well established to cause a spectrum of disorders ranging from myopathies to progeria, termed laminopathies. Phenotypes resulting from variants in LMNB1 and LMNB2 have been much less clearly defined. METHODS: We investigated exome and genome sequencing from the Deciphering Developmental Disorders Study and the 100,000 Genomes Project to identify novel microcephaly genes. RESULTS: Starting from a cohort of patients with extreme microcephaly, 13 individuals with heterozygous variants in the two human B-type lamins were identified. Recurrent variants were established to be de novo in nine cases and shown to affect highly conserved residues within the lamin ɑ-helical rod domain, likely disrupting interactions required for higher-order assembly of lamin filaments. CONCLUSION: We identify dominant pathogenic variants in LMNB1 and LMNB2 as a genetic cause of primary microcephaly, implicating a major structural component of the nuclear envelope in its etiology and defining a new form of laminopathy. The distinct nature of this lamin B-associated phenotype highlights the strikingly different developmental requirements for lamin paralogs and suggests a novel mechanism for primary microcephaly warranting future investigation.

Genotype-phenotype analysis of LMNA-related diseases predicts phenotype-selective alterations in lamin phosphorylation.

Laminopathies are rare diseases associated with mutations in LMNA, which encodes nuclear lamin A/C. LMNA variants lead to diverse tissue-specific phenotypes including cardiomyopathy, lipodystrophy, myopathy, neuropathy, progeria, bone/skin disorders, and overlap syndromes. The mechanisms underlying these heterogeneous phenotypes remain poorly understood, although post-translational modifications, including phosphorylation, are postulated as regulators of lamin function. We catalogued all known lamin A/C human mutations and their associated phenotypes, and systematically examined the putative role of phosphorylation in laminopathies. In silico prediction of specific LMNA mutant-driven changes to lamin A phosphorylation and protein structure was performed using machine learning methods. Some of the predictions we generated were validated via assessment of ectopically expressed wild-type and mutant LMNA. Our findings indicate phenotype- and mutant-specific alterations in lamin phosphorylation, and that some changes in phosphorylation may occur independently of predicted changes in lamin protein structure. Therefore, therapeutic targeting of phosphorylation in the context of laminopathies will likely require mutant- and kinase-specific approaches.

Management of heart failure with concomitant complete atrioventricular block caused by a novel missense LMNA mutation.

A 30-year-old lady was admitted to the hospital with progressive exertional dyspnoea and bradycardia. A complete atrioventricular block was diagnosed using 12­lead electrocardiography and a transthoracic echocardiography revealed a severely impaired left ventricular systolic dysfunction with an ejection fraction of 20%. Following hospitalization, her coronary angiography was normal, so a whole exome sequencing was conducted. The novel Lamin A/C Gene missense mutation c.263C > A,p.Ala88Asp in exon 3 was identified. A CRT-D was implanted due to the high risk of life-threatening ventricular arrhythmias and low potential for left ventricular reverse remodelling. The patient is undergoing follow-ups at the outpatient clinic, showing a 25% improvement in left ventricular ejection fraction during the last visit.

Super-Resolution Imaging of the A- and B-Type Lamin Networks: A Comparative Study of Different Fluorescence Labeling Procedures.

A- and B-type lamins are type V intermediate filament proteins. Mutations in the genes encoding these lamins cause rare diseases, collectively called laminopathies. A fraction of the cells obtained from laminopathy patients show aberrations in the localization of each lamin subtype, which may represent only the minority of the lamina disorganization. To get a better insight into more delicate and more abundant lamina abnormalities, the lamin network can be studied using super-resolution microscopy. We compared confocal scanning laser microscopy and stimulated emission depletion (STED) microscopy in combination with different fluorescence labeling approaches for the study of the lamin network. We demonstrate the suitability of an immunofluorescence staining approach when using STED microscopy, by determining the lamin layer thickness and the degree of lamin A and B1 colocalization as detected in fixed fibroblasts (co-)stained with lamin antibodies or (co-)transfected with EGFP/YFP lamin constructs. This revealed that immunofluorescence staining of cells does not lead to consequent changes in the detected lamin layer thickness, nor does it influence the degree of colocalization of lamin A and B1, when compared to the transfection approach. Studying laminopathy patient dermal fibroblasts (LMNA c.1130G>T (p.(Arg377Leu)) variant) confirmed the suitability of immunofluorescence protocols in STED microscopy, which circumvents the need for less convenient transfection steps. Furthermore, we found a significant decrease in lamin A/C and B1 colocalization in these patient fibroblasts, compared to normal human dermal fibroblasts. We conclude that super-resolution light microscopy combined with immunofluorescence protocols provides a potential tool to detect structural lamina differences between normal and laminopathy patient fibroblasts.