JNK regulates ciliogenesis through the interflagellar transport complex and actin networks.

The c-Jun N-terminal kinase (JNK) regulates various important physiological processes. Although the JNK pathway has been under intense investigation for over 20 yr, its complexity is still perplexing, with multiple protein partners underlying the diversity of its activity. We show that JNK is associated with the basal bodies in both primary and motile cilia. Loss of JNK disrupts basal body migration and docking and leads to severe ciliogenesis defects. JNK's involvement in ciliogenesis stems from a dual role in the regulation of the actin networks of multiciliated cells (MCCs) and the establishment of the intraflagellar transport-B core complex. JNK signaling is also critical for the maintenance of the actin networks and ciliary function in mature MCCs. JNK is implicated in the development of diabetes, neurodegeneration, and liver disease, all of which have been linked to ciliary dysfunction. Our work uncovers a novel role of JNK in ciliogenesis and ciliary function that could have important implications for JNK's role in the disease.

Modulation of cytoskeleton in cardiomyopathy caused by mutations in LMNA gene.

Dilated cardiomyopathy caused by mutations in LMNA, encoding A-type lamins (i.e., LMNA cardiomyopathy), is characterized by a left ventricle enlargement and ultimately results in poor cardiac contractility associated with conduction defects. Despite current strategies to aggressively manage the symptoms, the disorder remains a common cause of sudden death and heart failure with decreased ejection fraction. Patient care includes cardioverter defibrillator implantation but the last therapeutic option remains cardiac transplantation. A-type lamins are intermediate filaments and are the main components of the nuclear lamina, a meshwork underlying the inner nuclear membrane, which plays an essential role in both maintaining the nuclear structure and organizing the cytoskeletal structures within the cell. Cytoskeletal proteins function as scaffold to resist external mechanical stress. An increasing amount of evidence demonstrates that LMNA mutations can lead to disturbances in several structural and cytoskeletal components of the cell such as microtubules, actin cytoskeleton, and intermediate filaments. Collectively, this review focuses on the significance of these cytoskeletal modulators and emphasizes their potential therapeutic role in LMNA cardiomyopathy. Indeed, molecular tuning of cytoskeletal dynamics has been successfully used in preclinical models and provides adequate grounds for a therapeutic approach for patients with LMNA cardiomyopathy.

Actin-microtubule cytoskeletal interplay mediated by MRTF-A/SRF signaling promotes dilated cardiomyopathy caused by LMNA mutations.

Mutations in the lamin A/C gene (LMNA) cause dilated cardiomyopathy associated with increased activity of ERK1/2 in the heart. We recently showed that ERK1/2 phosphorylates cofilin-1 on threonine 25 (phospho(T25)-cofilin-1) that in turn disassembles the actin cytoskeleton. Here, we show that in muscle cells carrying a cardiomyopathy-causing LMNA mutation, phospho(T25)-cofilin-1 binds to myocardin-related transcription factor A (MRTF-A) in the cytoplasm, thus preventing the stimulation of serum response factor (SRF) in the nucleus. Inhibiting the MRTF-A/SRF axis leads to decreased α-tubulin acetylation by reducing the expression of ATAT1 gene encoding α-tubulin acetyltransferase 1. Hence, tubulin acetylation is decreased in cardiomyocytes derived from male patients with LMNA mutations and in heart and isolated cardiomyocytes from Lmnap.H222P/H222P male mice. In Atat1 knockout mice, deficient for acetylated α-tubulin, we observe left ventricular dilation and mislocalization of Connexin 43 (Cx43) in heart. Increasing α-tubulin acetylation levels in Lmnap.H222P/H222P mice with tubastatin A treatment restores the proper localization of Cx43 and improves cardiac function. In summary, we show for the first time an actin-microtubule cytoskeletal interplay mediated by cofilin-1 and MRTF-A/SRF, promoting the dilated cardiomyopathy caused by LMNA mutations. Our findings suggest that modulating α-tubulin acetylation levels is a feasible strategy for improving cardiac function.

The apical ciliary adhesion complex is established at the basal foot of motile cilia and depends on the microtubule network.

The Ciliary Adhesion (CA) complex forms in close association with the basal bodies of cilia during the early stages of ciliogenesis and is responsible for mediating complex interactions with the actin networks of multiciliated cells (MCCs). However, its precise localization with respect to basal body accessory structures and the interactions that lead to its establishment in MCCs are not well understood. Here, we studied the distribution of the CA proteins using super-resolution imaging and possible interactions with the microtubule network. The results of this study reveal that the apical CA complex forms at the distal end of the basal foot and depends on microtubules. Our data also raise the possibility that CAs may have additional roles in the regulation of the organization of the microtubule network of MCCs.

53BP1-mediated recruitment of RASSF1A to ribosomal DNA breaks promotes local ATM signaling.

DNA lesions occur across the genome and constitute a threat to cell viability; however, damage at specific genomic loci has a relatively greater impact on overall genome stability. The ribosomal RNA gene repeats (rDNA) are emerging fragile sites. Recent progress in understanding how the rDNA damage response is organized has highlighted a key role of adaptor proteins. Here, we show that the scaffold tumor suppressor RASSF1A is recruited to rDNA breaks. RASSF1A recruitment to double-strand breaks is mediated by 53BP1 and depends on RASSF1A phosphorylation at Serine 131 by ATM kinase. Employing targeted rDNA damage, we uncover that RASSF1A recruitment promotes local ATM signaling. RASSF1A silencing, a common epigenetic event during malignant transformation, results in persistent breaks, rDNA copy number alterations and decreased cell viability. Overall, we identify a novel role for RASSF1A at rDNA break sites, provide mechanistic insight into how the DNA damage response is organized in a chromatin context, and provide further evidence for how silencing of the RASSF1A tumor suppressor contributes to genome instability.

The non-muscle ADF/cofilin-1 controls sarcomeric actin filament integrity and force production in striated muscle laminopathies.

Cofilins are important for the regulation of the actin cytoskeleton, sarcomere organization, and force production. The role of cofilin-1, the non-muscle-specific isoform, in muscle function remains unclear. Mutations in LMNA encoding A-type lamins, intermediate filament proteins of the nuclear envelope, cause autosomal Emery-Dreifuss muscular dystrophy (EDMD). Here, we report increased cofilin-1 expression in LMNA mutant muscle cells caused by the inability of proteasome degradation, suggesting a protective role by ERK1/2. It is known that phosphorylated ERK1/2 directly binds to and catalyzes phosphorylation of the actin-depolymerizing factor cofilin-1 on Thr25. In vivo ectopic expression of cofilin-1, as well as its phosphorylated form on Thr25, impairs sarcomere structure and force generation. These findings present a mechanism that provides insight into the molecular pathogenesis of muscular dystrophies caused by LMNA mutations.

Mutated lamin A modulates stiffness in muscle cells.

The cytoskeleton is a complex network interlinking filaments that extend throughout the cytoplasm from the nucleus to the plasma membrane. Three major types of filaments are found in the cytoskeleton: actin filaments, microtubules, and intermediate filaments. They play a key role in the ability of cells to both resist mechanical stress and generate force. However, the precise involvement of intermediate filament proteins in these processes remains unclear. Here, we focused on nuclear A-type lamins, which are connected to the cytoskeleton via the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Using micro-constriction rheology, we investigated the impact of A-type lamins (p.H222P) mutation on the mechanical properties of muscle cells. We demonstrate that the expression of point mutation of lamin A in muscle cells increases cellular stiffness compared with cells expressing wild type lamin A and that the chemical agent selumetinib, an inhibitor of the ERK1/2 signaling, reversed the mechanical alterations in mutated cells. These results highlight the interplay between A-type lamins and mechano-signaling, which are supported by cell biology measurements.

Cardioprotective effects of α-cardiac actin on oxidative stress in a dilated cardiomyopathy mouse model.

The expression of α-cardiac actin, a major constituent of the cytoskeleton of cardiomyocytes, is dramatically decreased in a mouse model of dilated cardiomyopathy triggered by inducible cardiac-specific serum response factor (Srf) gene disruption that could mimic some forms of human dilated cardiomyopathy. To investigate the consequences of the maintenance of α-cardiac actin expression in this model, we developed a new transgenic mouse based on Cre/LoxP strategy, allowing together the induction of SRF loss and a compensatory expression of α-cardiac actin. Here, we report that maintenance of α-cardiac actin within cardiomyocytes temporally preserved cytoarchitecture from adverse cardiac remodeling through a positive impact on both structural and transcriptional levels. These protective effects were accompanied in vivo by the decrease of ROS generation and protein carbonylation and the downregulation of NADPH oxidases NOX2 and NOX4. We also show that ectopic expression of α-cardiac actin protects HEK293 cells against oxidative stress induced by H2 O2 . Oxidative stress plays an important role in the development of cardiac remodeling and contributes also to the pathogenesis of heart failure. Taken together, these findings indicate that α-cardiac actin could be involved in the regulation of oxidative stress that is a leading cause of adverse remodeling during dilated cardiomyopathy development.

When Hippo meets actin in the nucleus.

Nuclear actin is exported from the nucleus via the Exportin-6 (XPO6)/RAN GTPase complex. We recently identified that RASSF1A and the hippo pathway kinase Mammalian STE20-like protein kinase 2 (MST2) play a pivotal role in nucleocytoplasmic shuttling of actin by regulating the association of XPO6 with RAN GTPase. Importantly, loss of Ras association domain family 1A (RASSF1A) and hippo signaling in cancer cells highlights a key mechanism by which nuclear actin promotes tumorigenesis.

RASSF1A is required for the maintenance of nuclear actin levels.

Nuclear actin participates in many essential cellular processes including gene transcription, chromatin remodelling and mRNA processing. Actin shuttles into and out the nucleus through the action of dedicated transport receptors importin-9 and exportin-6, but how this transport is regulated remains unclear. Here, we show that RASSF1A is a novel regulator of actin nucleocytoplasmic trafficking and is required for the active maintenance of nuclear actin levels through supporting binding of exportin-6 (XPO6) to RAN GTPase. RASSF1A (Ras association domain family 1 isoform A) is a tumour suppressor gene frequently silenced by promoter hypermethylation in all major solid cancers. Specifically, we demonstrate that endogenous RASSF1A localises to the nuclear envelope (NE) and is required for nucleocytoplasmic actin transport and the concomitant regulation of myocardin-related transcription factor A (MRTF-A), a co-activator of the transcription factor serum response factor (SRF). The RASSF1A/RAN/XPO6/nuclear actin pathway is aberrant in cancer cells where RASSF1A expression is lost and correlates with reduced MRTF-A/SRF activity leading to cell adhesion defects. Taken together, we have identified a previously unknown mechanism by which the nuclear actin pool is regulated and uncovered a previously unknown link of RASSF1A and MRTF-A/SRF in tumour suppression.

RASSF1A controls tissue stiffness and cancer stem-like cells in lung adenocarcinoma.

Lung cancer remains the leading cause of cancer-related death due to poor treatment responses and resistance arising from tumour heterogeneity. Here, we show that adverse prognosis associated with epigenetic silencing of the tumour suppressor RASSF1A is due to increased deposition of extracellular matrix (ECM), tumour stiffness and metastatic dissemination in vitro and in vivo. We find that lung cancer cells with RASSF1A promoter methylation display constitutive nuclear YAP1 accumulation and expression of prolyl 4-hydroxylase alpha-2 (P4HA2) which increases collagen deposition. Furthermore, we identify that elevated collagen creates a stiff ECM which in turn triggers cancer stem-like programming and metastatic dissemination in vivo. Re-expression of RASSF1A or inhibition of P4HA2 activity reverses these effects and increases markers of lung differentiation (TTF-1 and Mucin 5B). Our study identifies RASSF1A as a clinical biomarker associated with mechanical properties of ECM which increases the levels of cancer stemness and risk of metastatic progression in lung adenocarcinoma. Moreover, we highlight P4HA2 as a potential target for uncoupling ECM signals that support cancer stemness.

Microtubule cytoskeleton regulates Connexin 43 localization and cardiac conduction in cardiomyopathy caused by mutation in A-type lamins gene.

Mutations in the lamin A/C gene (LMNA) cause an autosomal dominant inherited form of dilated cardiomyopathy associated with cardiac conduction disease (hereafter referred to as LMNA cardiomyopathy). Compared with other forms of dilated cardiomyopathy, mutations in LMNA are responsible for a more aggressive clinical course owing to a high rate of malignant ventricular arrhythmias. Gap junctions are intercellular channels that allow direct communication between neighboring cells, which are involved in electrical impulse propagation and coordinated contraction of the heart. For gap junctions to properly control electrical synchronization in the heart, connexin-based hemichannels must be correctly targeted to intercalated discs, Cx43 being the major connexin in the working myocytes. We here showed an altered distribution of Cx43 in a mouse model of LMNA cardiomyopathy. However, little is known on the molecular mechanisms of Cx43 remodeling in pathological context. We now show that microtubule cytoskeleton alteration and decreased acetylation of α-tubulin lead to remodeling of Cx43 in LMNA cardiomyopathy, which alters the correct communication between cardiomyocytes, ultimately leading to electrical conduction disturbances. Preventing or reversing this process could offer a strategy to repair damaged heart. Stabilization of microtubule cytoskeleton using Paclitaxel improved intraventricular conduction defects. These results indicate that microtubule cytoskeleton contributes to the pathogenesis of LMNA cardiomyopathy and that drugs stabilizing the microtubule may be beneficial for patients.

Nuclear/Cytoplasmic Fractionation to Study Hippo Effectors.

The translocation or shuttling of Hippo proteins between the nucleus and cytoplasm is a rapid event following cytoskeletal or mechanical cues as well as stimulation with extracellular growth factors. Here we describe an experimental procedure for a simple and fast separation of nuclear and cytoplasmic fractions which maintains protein integrity and integrity of protein-protein complexes, indicating that it should be applicable to many experimental questions.

Rescue of biosynthesis of nicotinamide adenine dinucleotide protects the heart in cardiomyopathy caused by lamin A/C gene mutation.

Cardiomyopathy caused by lamin A/C gene (LMNA) mutations (hereafter referred as LMNA cardiomyopathy) is an anatomic and pathologic condition associated with muscle and electrical dysfunction of the heart, often leading to heart failure-related disability. There is currently no specific therapy available for patients that target the molecular pathophysiology of LMNA cardiomyopathy. Recent studies suggested that nicotinamide adenine dinucleotide (NAD+) cellular content could be a critical determinant for heart function. Biosynthesis of NAD+ from vitamin B3 (known as salvage pathways) is the primary source of NAD+. We showed here that NAD+ salvage pathway was altered in the heart of mouse and human carrying LMNA mutation, leading to an alteration of one of NAD+ co-substrate enzymes, PARP-1. Oral administration of nicotinamide riboside, a natural NAD+ precursor and a pyridine-nucleoside form of vitamin B3, leads to a marked improvement of the NAD+ cellular content, an increase of PARylation of cardiac proteins and an improvement of left ventricular structure and function in a model of LMNA cardiomyopathy. Collectively, our results provide mechanistic and therapeutic insights into dilated cardiomyopathy caused by LMNA mutations.

Cofilin-1 phosphorylation catalyzed by ERK1/2 alters cardiac actin dynamics in dilated cardiomyopathy caused by lamin A/C gene mutation.

Hyper-activation of extracellular signal-regulated kinase (ERK) 1/2 contributes to heart dysfunction in cardiomyopathy caused by mutations in the lamin A/C gene (LMNA cardiomyopathy). The mechanism of how this affects cardiac function is unknown. We show that active phosphorylated ERK1/2 directly binds to and catalyzes the phosphorylation of the actin depolymerizing factor cofilin-1 on Thr25. Cofilin-1 becomes active and disassembles actin filaments in a large array of cellular and animal models of LMNA cardiomyopathy. In vivo expression of cofilin-1, phosphorylated on Thr25 by endogenous ERK1/2 signaling, leads to alterations in left ventricular function and cardiac actin. These results demonstrate a novel role for cofilin-1 on actin dynamics in cardiac muscle and provide a rationale on how increased ERK1/2 signaling leads to LMNA cardiomyopathy.

S6K2-mediated regulation of TRBP as a determinant of miRNA expression in human primary lymphatic endothelial cells.

MicroRNAs (miRNAs) are short non-coding RNAs that silence mRNAs. They are generated following transcription and cleavage by the DROSHA/DGCR8 and DICER/TRBP/PACT complexes. Although it is known that components of the miRNA biogenesis machinery can be phosphorylated, it remains poorly understood how these events become engaged during physiological cellular activation. We demonstrate that S6 kinases can phosphorylate the extended C-terminal domain of TRBP and interact with TRBP in situ in primary cells. TRBP serines 283/286 are essential for S6K-mediated TRBP phosphorylation, optimal expression of TRBP, and the S6K-TRBP interaction in human primary cells. We demonstrate the functional relevance of this interaction in primary human dermal lymphatic endothelial cells (HDLECs). Angiopoietin-1 (ANG1) can augment miRNA biogenesis in HDLECs through enhancing TRBP phosphorylation and expression in an S6K2-dependent manner. We propose that the S6K2/TRBP node controls miRNA biogenesis in HDLECs and provides a molecular link between the mTOR pathway and the miRNA biogenesis machinery.

ERK1/2 directly acts on CTGF/CCN2 expression to mediate myocardial fibrosis in cardiomyopathy caused by mutations in the lamin A/C gene.

Cardiomyopathy caused by lamin A/C gene mutations (LMNA cardiomyopathy) is characterized by increased myocardial fibrosis, which impairs left ventricular relaxation and predisposes to heart failure, and cardiac conduction abnormalities. While we previously discovered abnormally elevated extracellular signal-regulated kinase 1/2 (ERK1/2) activities in heart in LMNA cardiomyopathy, its role on the development of myocardial fibrosis remains unclear. We now showed that transforming growth factor (TGF)-ß/Smad signaling participates in the activation of ERK1/2 signaling in LMNA cardiomyopathy. ERK1/2 acts on connective tissue growth factor (CTGF/CCN2) expression to mediate the myocardial fibrosis and left ventricular dysfunction. Studies in vivo demonstrate that inhibiting CTGF/CCN2 using a specific antibody decreases myocardial fibrosis and improves the left ventricular dysfunction. Together, these findings show that cardiac ERK1/2 activity is modulated in part by TGF-ß/Smad signaling, leading to altered activation of CTGF/CCN2 to mediate fibrosis and alter cardiac function. This identifies a novel mechanism in the development of LMNA cardiomyopathy.

Nuclear envelope and striated muscle diseases.

The nuclear lamina is a mesh-like network of intermediate filaments localized mainly at the inner surface of the inner nuclear membrane and is composed of proteins called lamins. Many inherited diseases are linked with mutations in nuclear lamins and integral proteins of the inner nuclear membrane. In this article, we summarize basic aspects of the nuclear envelope architecture and provide some remarkable findings of the involvement of lamins in striated muscle disorders.