Peter F.M. van der Ven

A Mutation in the Dimerization Domain of Filamin C Causes a Novel Type of Autosomal Dominant Myofibrillar Myopathy

Myofibrillar myopathy (MFM) is a human disease that is characterized by focal myofibrillar destruction and pathological cytoplasmic protein aggregations. In an extended German pedigree with a novel form of MFM characterized by clinical features of a limb-girdle myopathy and morphological features of MFM, we identified a co-segregating, heterozygous nonsense mutation (8130G-->A; W2710X) in the filamin c gene (FLNC) on chromosome 7q32.1. The mutation is the first found in FLNC and is localized in the dimerization domain of filamin c. Functional studies showed that, in the truncated mutant protein, this domain has a disturbed secondary structure that leads to the inability to dimerize properly. As a consequence of this malfunction, the muscle fibers of our patients display massive cytoplasmic aggregates containing filamin c and several Z-disk-associated and sarcolemmal proteins.

Mutations in the Human Muscle LIM Protein Gene in Families With Hypertrophic Cardiomyopathy

Background—Muscle LIM protein (MLP) is an essential nuclear regulator of myogenic differentiation. Additionally, it may act as an integrator of protein assembly of the actin-based cytoskeleton. MLP-knockout mice develop a marked cardiac hypertrophy reaction and dilated cardiomyopathy (DCM). MLP is therefore a candidate gene for heritable forms of hypertrophic cardiomyopathy (HCM) and DCM in humans. Methods and Results—We analyzed 1100 unrelated individuals (400 patients with DCM, 200 patients with HCM, and 500 controls) for mutations in the human CRP3 gene that encodes MLP. We found 3 different missense mutations in 3 unrelated patients with familial HCM but detected no mutation in the DCM group or the controls. All mutations predicted an amino acid exchange at highly conserved residues in the functionally important LIM1 domain, which is responsible for interaction with &agr;-actinin and with certain muscle-specific transcription factors. Protein-binding studies indicate that mutations in the CRP3 gene lead to a decreased binding activity of MLP to &agr;-actinin. All 3 index patients were characterized by typical asymmetrical septal hypertrophy. Family studies revealed cosegregation of clinically affected individuals with the respective mutations in MLP. Conclusion—Here, we present evidence that mutations in the CRP3/MLP gene can cause HCM.

Two immunoglobulin-like domains of the Z-disc portion of titin interact in a conformation-dependent way with telethonin.

The giant muscle protein titin/connectin plays a crucial role in myofibrillogenesis as a molecular ruler for sarcomeric protein sorting. We describe here that the N‐terminal titin immunoglobulin domains Z1 and Z2 interact specifically with telethonin in yeast two‐hybrid analysis and protein binding assays. Immunofluorescence with antibodies against the N‐terminal region of titin and telethonin detects both proteins at the Z‐disc of human myotubes. Longer titin fragments, comprising a serine‐proline‐rich phosphorylation site and the next domain, do not interact. The interaction of telethonin with titin is therefore conformation‐dependent, reflecting a possible phosphorylation regulation during myofibrillogenesis.

Transient association of titin and myosin with microtubules in nascent myofibrils directed by the MURF2 RING-finger protein

Assembly of muscle sarcomeres is a complex dynamic process and involves a large number of proteins. A growing number of these have regulatory functions and are transiently present in the myofibril. We show here that the novel tubulin-associated RING/B-box protein MURF2 associates transiently with microtubules, myosin and titin during sarcomere assembly. During sarcomere assembly, MURF2 first associates with microtubules at the exclusion of tyrosinated tubulin. Then, MURF2-labelled microtubules associate transiently with sarcomeric myosin and later with A-band titin when non-striated myofibrils differentiate into mature sarcomeres. Finally, MURF2 labelled microtubules disappear from the sarcomere after the incorporation of myosin filaments and the elongation of titin. This suggests that the incorporation of myosin into nascent sarcomeres and the elongation of titin require an active, microtubule-dependent transport process and that MURF2-associated microtubules play a role in the alignment and extension of nascent sarcomeres. MURF2 is expressed in at least four isoforms, of which a 27 kDa isoform is cardiac specific. A C-terminal isoform is generated by alternative reading frame use, a novelty in muscle proteins. In mature cardiac sarcomeres, endogenous MURF2 can associate with the M-band, and is translocated to the nucleus. MURF2 can therefore act as a transient adaptor between microtubules, titin and nascent myosin filaments, as well as being involved in signalling from the sarcomere to the nucleus.

Cellular Mechanotransduction Relies on Tension-Induced and Chaperone-Assisted Autophagy

Mechanical tension is an ever-present physiological stimulus essential for the development and homeostasis of locomotory, cardiovascular, respiratory, and urogenital systems. Tension sensing contributes to stem cell differentiation, immune cell recruitment, and tumorigenesis. Yet, how mechanical signals are transduced inside cells remains poorly understood. Here, we identify chaperone-assisted selective autophagy (CASA) as a tension-induced autophagy pathway essential for mechanotransduction in muscle and immune cells. The CASA complex, comprised of the molecular chaperones Hsc70 and HspB8 and the cochaperone BAG3, senses the mechanical unfolding of the actin-crosslinking protein filamin. Together with the chaperone-associated ubiquitin ligase CHIP, the complex initiates the ubiquitin-dependent autophagic sorting of damaged filamin to lysosomes for degradation. Autophagosome formation during CASA depends on an interaction of BAG3 with synaptopodin-2 (SYNPO2). This interaction is mediated by the BAG3 WW domain and facilitates cooperation with an autophagosome membrane fusion complex. BAG3 also utilizes its WW domain to engage in YAP/TAZ signaling. Via this pathway, BAG3 stimulates filamin transcription to maintain actin anchoring and crosslinking under mechanical tension. By integrating tension sensing, autophagosome formation, and transcription regulation during mechanotransduction, the CASA machinery ensures tissue homeostasis and regulates fundamental cellular processes such as adhesion, migration, and proliferation.

Mutations in the N-terminal Actin-Binding Domain of Filamin C Cause a Distal Myopathy

Linkage analysis of the dominant distal myopathy we previously identified in a large Australian family demonstrated one significant linkage region located on chromosome 7 and encompassing 18.6 Mbp and 151 genes. The strongest candidate gene was FLNC because filamin C, the encoded protein, is muscle-specific and associated with myofibrillar myopathy. Sequencing of FLNC cDNA identified a c.752T>C (p.Met251Thr) mutation in the N-terminal actin-binding domain (ABD); this mutation segregated with the disease and was absent in 200 controls. We identified an Italian family with the same phenotype and found a c.577G>A (p.Ala193Thr) filamin C ABD mutation that segregated with the disease. Filamin C ABD mutations have not been described, although filamin A and filamin B ABD mutations cause multiple musculoskeletal disorders. The distal myopathy phenotype and muscle pathology in the two families differ from myofibrillar myopathies caused by filamin C rod and dimerization domain mutations because of the distinct involvement of hand muscles and lack of pathological protein aggregation. Thus, like the position of FLNA and B mutations, the position of the FLNC mutation determines disease phenotype. The two filamin C ABD mutations increase actin-binding affinity in a manner similar to filamin A and filamin B ABD mutations. Cell-culture expression of the c.752T>C (p.Met251)Thr mutant filamin C ABD demonstrated reduced nuclear localization as did mutant filamin A and filamin B ABDs. Expression of both filamin C ABD mutants as full-length proteins induced increased aggregation of filamin. We conclude filamin C ABD mutations cause a recognizable distal myopathy, most likely through increased actin affinity, similar to the pathological mechanism of filamin A and filamin B ABD mutations.

Immunogold EM reveals a close association of plectin and the desmin cytoskeleton in human skeletal muscle.

Plectin is a multifunctional cytoskeletal linker protein with an intermediate filament-binding site and sequence elements with high homology to actin-binding domains. Mutations of the human plectin gene as well as the targeted inactivation of its murine analog cause a generalized blistering skin disorder and muscular dystrophy, thus implying its essential role in cells that are exposed to mechanical stress. In the present study we report the characterization of two new domain-specific plectin antibodies as well as ultrastructural localization of plectin in normal human skeletal muscle. Using immunogold electron microscopy, we localized plectin at three prominent sites: 1) Plectin is found at regularly spaced intervals along the cytoplasmic face of the plasma membrane. 2) It is distinctly localized at filamentous bridges between Z-lines of peripheral myofibrils and the sarcolemma and 3) at structures forming the intermyofibrillar scaffold. At the latter two locations, plectin and desmin were found to colocalize. Our ultrastructural analysis suggests that plectin may have a central role in the structural and functional organization of the intermediate filament cytoskeleton in mature human skeletal muscle.

The limits of promiscuity: isoform-specific dimerization of filamins.

Filamins are a family of actin cross-linking proteins that are primarily localized in the cortical cytoplasm of all mammalian cells. Until now, three major isoforms (filamins a, b, and c) have been identified, that were shown to be differentially expressed and/or localized in different tissues. An amino-terminal double CH-domain actin binding domain, and a dimerization region in the carboxy-terminal portion of the protein are the molecular basis for its actin cross-linking activity. Chemical cross-linking of bacterially expressed recombinant proteins was used to demonstrate that in all three filamin isoforms the most carboxy-terminally situated immunoglobulinlike domain is required and sufficient for dimerization. The efficiency of the dimerization was increased upon inclusion of the preceding hinge 2 region, indicating a function for this region in the regulation of dimerization. By mixing recombinant proteins derived from different filamin isoforms, we found that heterodimer formation is possible between filamins b and c but not between filamin a and the other two filamins. This selectivity of dimerization might provide a further molecular explanation for the differential intracellular sorting of filamin isoforms and their distinct properties.

Differential involvement of sarcomeric proteins in myofibrillar myopathies: a morphological and immunohistochemical study.

Myofibrillar myopathies (MFMs) are rare inherited or sporadic progressive neuromuscular disorders with considerable clinical and genetic heterogeneity. In the current study, we have analyzed histopathological and immunohistochemical characteristics in genetically identified MFMs. We performed a morphological and morphometrical study in a cohort of 24 genetically identified MFM patients (12 desmin, 6 αB-crystallin, 4 ZASP, 2 myotilin), and an extensive immunohistochemical study in 15 of these patients, using both well-known and novel antibodies directed against distinct compartments of the muscle fibers, including Z-disc and M-band proteins. Our morphological data revealed some significant differences between the distinct MFM subgroups: the consistent presence of ‘rubbed-out’ fibers in desminopathies and αB-crystallinopathies, an elevated frequency of vacuoles in ZASPopathies and myotilinopathies, and the presence of a few necrotic fibers in the two myotilinopathy patients. Immunohistochemistry showed that in MFM only a subset of Z-disc proteins, such as filamin C and its ligands myotilin and Xin, exhibited significant alterations in their localization, whereas other Z-disc proteins like α-actinin, myopodin and tritopodin, did not. In contrast, M-band proteins revealed no abnormalities in MFM. We conclude that the presence of ‘rubbed-out’ fibers are a suggestive feature for desminopathy or αB-crystallinopathy, and that MFM is not a general disease of the myofibril, but primarily affects a subgroup of stress-responsive Z-disc proteins.

Decreased interactions of mutant muscle LIM protein (MLP) with N-RAP and α-actinin and their implication for hypertrophic cardiomyopathy

Abstract.Previous work has shown that mutations in muscle LIM protein (MLP) can cause hypertrophic cardiomyopathy (HCM). In order to gain an insight into the molecular basis of the disease phenotype, we analysed the binding characteristics of wild-type MLP and of the (C58G) mutant MLP that causes hypertrophic cardiomyopathy. We show that MLP can form a ternary complex with two of its previously documented myofibrillar ligand proteins, N-RAP and α-actinin, which indicates the presence of distinct, non-overlapping binding sites. Our data also show that, in comparison to wild-type MLP, the capacity of the mutated MLP protein to bind both N-RAP and α-actinin is significantly decreased. In addition, this single point mutation prevents zinc coordination and proper folding of the second zinc-finger in the first LIM domain, which consequently renders the protein less stable and more susceptible to proteolysis. The molecular basis for HCM-causing mutations in the MLP gene might therefore be an alteration in the equilibrium of interactions of the ternary complex MLP–N-RAP–α-actinin. This assumption is supported by the previous observation that in the pathological situation accompanied by MLP down regulation, cardiomyocytes try to compensate for the decreased stability of MLP protein by increasing the expression of its ligand N-RAP, which might finally result in the development of myocyte disarray that is characteristic of this disease.