Myofibril Assembly Research Articles

Control of thin filament lengths by sarcomeric tropomodulin isoforms: insights from mouse models (1102.2)

The uniform lengths of skeletal muscle thin filaments are controlled by actin subunit exchange at pointed ends (P‐ends). Each P‐end is capped by two sarcomeric tropomodulin (Tmod) molecules, with Tmod1 and Tmod4 isoforms present in a 1:9 ratio. In Tmod1‐null sarcomeres, P‐ends are capped by Tmod3 (normally a sarcoplasmic reticulum‐associated Tmod isoform) and Tmod4, preserving normal thin filament lengths. In Tmod4‐null sarcomeres, P‐ends are capped solely by Tmod1, also preserving normal lengths. The mild phenotypes of Tmod1‐null and Tmod4‐null muscle highlight skeletal muscle’s ability to deploy compensatory mechanisms to specify correct thin filament lengths in the absence of one sarcomeric Tmod isoform during myofibril assembly and muscle development. To discern the effect of sarcomeric Tmod depletion from mature muscle, we measured thin filament lengths in the mdx and mdx/mTR mouse models of Duchenne muscular dystrophy, in which Tmods are calpain‐proteolyzed in a muscle use‐dependent manner. Proteolysis of Tmod1 and/or Tmod4 in mdx and mdx/mTR muscles results in 10‐12% increases in thin filament lengths, due to addition of actin subunits onto P‐ends. Collectively, these results show that Tmod1 and Tmod4 are individually dispensable for initial specification of muscle‐specific thin filament lengths, but important for maintenance of correct thin filament lengths in mature contracting myofibrils.Grant Funding Source: Supported by NIH and MDA.

Analysis of disease progression-associated gene expression profile in fibrillin-1 mutant mice: new insight into molecular pathogenesis of marfan syndrome.

Marfan syndrome (MFS) is a dominantly inherited connective tissue disorder caused by mutations in the gene encoding fibrillin-1 (FBN1) and is characterized by aortic dilatation and dissection, which is the primary cause of death in untreated MFS patients. However, disease progression-associated changes in gene expression in the aortic lesions of MFS patients remained unknown. Using a mouse model of MFS, FBN1 hypomorphic mouse (mgR/mgR), we characterized the aortic gene expression profiles during the progression of the MFS. Homozygous mgR mice exhibited MFS-like phenotypic features, such as fragmentation of elastic fibers throughout the vessel wall and were graded into mgR1–4 based on the pathological severity in aortic walls. Comparative gene expression profiling of WT and four mgR mice using microarrays revealed that the changes in the transcriptome were a direct reflection of the severity of aortic pathological features. Gene ontology analysis showed that genes related to oxidation/reduction, myofibril assembly, cytoskeleton organization, and cell adhesion were differentially expressed in the mgR mice. Further analysis of differentially expressed genes identified several candidate genes whose known roles were suggestive of their involvement in the progressive destruction of aorta during MFS. This study is the first genome-wide analysis of the aortic gene expression profiles associated with the progression of MFS. Our findings provide valuable information regarding the molecular pathogenesis during MFS progression and contribute to the development of new biomarkers as well as improved therapeutic strategies.

Tension and Force-Resistant Attachment Are Essential for Myofibrillogenesis in Drosophila Flight Muscle

Higher animals generate an elaborate muscle-tendon network to perform their movements. To build a functional network, developing muscles must establish stable connections with tendons and assemble their contractile apparatuses. Current myofibril assembly models do not consider the impact of muscle-tendon attachment on myofibrillogenesis. However, if attachment and myofibrillogenesis are not properly coordinated, premature muscle contractions can destroy an unstable myotendinous system, leading to severe myopathies. Here, we use Drosophila indirect flight muscles to investigate how muscle-tendon attachment and myofibrillogenesis are coordinated. We find that flight muscles first stably attach to tendons and then assemble their myofibrils. Interestingly, this myofibril assembly is triggered simultaneously throughout the entire muscle, suggesting a self-assembly mechanism. By applying laser-cutting experiments, we show that muscle attachment coincides with an increase in mechanical tension before periodic myofibrils can be detected. We manipulated tension buildup within the myotendinous system either by genetically compromising attachment initiation and integrin recruitment to the myotendinous junction or by optically severing tendons from muscle. Both treatments cause strong myofibrillogenesis defects. We find that myosin motor activity is required for both tension formation and myofibril assembly, suggesting that myofibril assembly itself contributes to tension buildup. Our results demonstrate that force-resistant attachment enables a stark tension increase in the myotendinous system. Subsequently, this tension increase triggers simultaneous myofibril self-assembly throughout the entire muscle fiber. As myofibril and sarcomeric architecture as well as their molecular components are evolutionarily conserved, we propose a similar tension-based mechanism to regulate myofibrillogenesis in vertebrates.

Tropomyosin is required for cardiac morphogenesis, myofibril assembly, and formation of adherens junctions in the developing mouse embryo

We explored a function for tropomyosin (TM) in mammalian myofibril assembly and cardiac development by analyzing a deletion in the mouse TPM1 gene targeting αTM1, the major striated muscle TM isoform. Mice lacking αTM1 are embryonic lethal at E9.5 with enlarged, misshapen, and non-beating hearts characterized by an abnormally thin myocardium and reduced trabeculae. αTM1-deficient cardiomyocytes do not assemble striated myofibrils, instead displaying aberrant non-striated F-actin fibrils with α-actinin puncta dispersed irregularly along their lengths. αTM1's binding partner, tropomodulin1 (Tmod1), is also disorganized, and both myomesin-containing thick filaments as well as titin Z1Z2 fail to assemble in a striated pattern. Adherens junctions are reduced in size in αTM1-deficient cardiomyocytes, α-actinin/F-actin adherens belts fail to assemble at apical cell-cell contacts, and cell contours are highly irregular, resulting in abnormal cell shapes and a highly folded cardiac surface. In addition, Tmod1-deficient cardiomyocytes exhibit failure of α-actinin/F-actin adherens belt assembly. Absence of αTM1 resulting in unstable F-actin may preclude sarcomere formation and/or lead to degeneration of partially assembled sarcomeres due to unregulated actomyosin interactions. Our data also identify a novel αTM1/Tmod1-based pathway stabilizing F-actin at cell-cell junctions, which may be required for maintenance of cell shapes during embryonic cardiac morphogenesis.

Severe protein aggregate myopathy in a knockout mouse model points to an essential role of cofilin2 in sarcomeric actin exchange and muscle maintenance

Mutations in the human actin depolymerizing factor cofilin2 result in an autosomal dominant form of nemaline myopathy. Here, we report on the targeted ablation of murine cofilin2, which leads to a severe skeletal muscle specific phenotype within the first two weeks after birth. Apart from skeletal muscle, cofilin2 is also expressed in heart and CNS, however the pathology was restricted to skeletal muscle. The two close family members of cofilin2 – ADF and cofilin1 – were co-expressed in muscle, but unable to compensate for the loss of cofilin2. While primary myofibril assembly and muscle development were unaffected in cofilin2 mutant mice, progressive muscle degeneration was observed between postnatal days 3 and 7. Muscle pathology was characterized by sarcoplasmic protein aggregates, fiber size disproportion, mitochondrial abnormalities and internal nuclei. The observed muscle pathology differed from nemaline myopathy, but showed combined features of actin-associated myopathy and myofibrillar myopathy. In cofilin2 mutant mice, the postnatal expression pattern and turnover of sarcomeric α-actin isoforms were altered. Levels of smooth muscle α-actin were increased and remained high in developing muscles, suggesting that cofilin2 plays a crucial role during the exchange of α-actin isoforms during the early postnatal remodeling of the sarcomere.

Expression and Functional Characterization of Smyd1a in Myofibril Organization of Skeletal Muscles

BackgroundSmyd1, the founding member of the Smyd family including Smyd-1, 2, 3, 4 and 5, is a SET and MYND domain containing protein that plays a key role in myofibril assembly in skeletal and cardiac muscles. Bioinformatic analysis revealed that zebrafish genome contains two highly related smyd1 genes, smyd1a and smyd1b. Although Smyd1b function is well characterized in skeletal and cardiac muscles, the function of Smyd1a is, however, unknown.Methodology/Principal FindingsTo investigate the function of Smyd1a in muscle development, we isolated smyd1a from zebrafish, and characterized its expression and function during muscle development via gene knockdown and transgenic expression approaches. The results showed that smyd1a was strongly expressed in skeletal muscles of zebrafish embryos. Functional analysis revealed that knockdown of smyd1a alone had no significant effect on myofibril assembly in zebrafish skeletal muscles. However, knockdown of smyd1a and smyd1b together resulted in a complete disruption of myofibril organization in skeletal muscles, a phenotype stronger than knockdown of smyd1a or smyd1b alone. Moreover, ectopic expression of zebrafish smyd1a or mouse Smyd1 transgene could rescue the myofibril defects from the smyd1b knockdown in zebrafish embryos.Conclusion/SignificanceCollectively, these data indicate that Smyd1a and Smyd1b share similar biological activity in myofibril assembly in zebrafish embryos. However, Smyd1b appears to play a major role in this process.

The conserved transmembrane proteoglycan Perdido/Kon-tiki is essential for myofibrillogenesis and sarcomeric structure in Drosophila.

ABSTRACTMuscle differentiation requires the assembly of high-order structures called myofibrils, composed of sarcomeres. Even though the molecular organization of sarcomeres is well known, the mechanisms underlying myofibrillogenesis are poorly understood. It has been proposed that integrin-dependent adhesion nucleates myofibrils at the periphery of the muscle cell to sustain sarcomere assembly. Here, we report a role for the gene perdido (perd, also known as kon-tiki, a transmembrane chondroitin proteoglycan) in myofibrillogenesis. Expression of perd RNAi in muscles, prior to adult myogenesis, can induce misorientation and detachment of Drosophila adult abdominal muscles. In comparison to controls, perd-depleted muscles contain fewer myofibrils, which are localized at the cell periphery. These myofibrils are detached from each other and display a defective sarcomeric structure. Our results demonstrate that the extracellular matrix receptor Perd has a specific role in the assembly of myofibrils and in sarcomeric organization. We suggest that Perd acts downstream or in parallel to integrins to enable the connection of nascent myofibrils to the Z-bands. Our work identifies the Drosophila adult abdominal muscles as a model to investigate in vivo the mechanisms behind myofibrillogenesis.

Aciculin interacts with filamin C and Xin and is essential for myofibril assembly, remodeling and maintenance.

Filamin C (FLNc) and Xin actin-binding repeat-containing proteins (XIRPs) are multi-adaptor proteins that are mainly expressed in cardiac and skeletal muscles and which play important roles in the assembly and repair of myofibrils and their attachment to the membrane. We identified the dystrophin-binding protein aciculin (also known as phosphoglucomutase-like protein 5, PGM5) as a new interaction partner of FLNc and Xin. All three proteins colocalized at intercalated discs of cardiac muscle and myotendinous junctions of skeletal muscle, whereas FLNc and aciculin also colocalized in mature Z-discs. Bimolecular fluorescence complementation experiments in developing cultured mammalian skeletal muscle cells demonstrated that Xin and aciculin also interact in FLNc-containing immature myofibrils and areas of myofibrillar remodeling and repair induced by electrical pulse stimulation (EPS). Fluorescence recovery after photobleaching (FRAP) experiments showed that aciculin is a highly dynamic and mobile protein. Aciculin knockdown in myotubes led to failure in myofibril assembly, alignment and membrane attachment, and a massive reduction in myofibril number. A highly similar phenotype was found upon depletion of aciculin in zebrafish embryos. Our results point to a thus far unappreciated, but essential, function of aciculin in myofibril formation, maintenance and remodeling.

Membrane-myofibril cross-talk in myofibrillogenesis and in muscular dystrophy pathogenesis: lessons from the zebrafish

Striated muscle has a highly ordered structure in which specialized domains of the cell membrane involved in force transmission (costameres) and excitation-contraction coupling (T tubules) as well as the internal membranes of the sarcoplasmic reticulum are organized over specific regions of the sarcomere. Optimal muscle function is dependent on this high level of organization but how it established and maintained is not well understood. Due to its ex utero development and transparency, the zebrafish embryo is an excellent vertebrate model for the study of dynamic relationships both within and between cells during development. Transgenic models have allowed the delineation of cellular migration and complex morphogenic rearrangements during the differentiation of skeletal myocytes and the assembly and organization of new myofibrils. Molecular targeting of genes and transcripts has allowed the identification of key requirements for myofibril assembly and organization. With the recent advances in gene editing approaches, the zebrafish will become an increasingly important model for the study of human myopathies and muscular dystrophies. Its high fecundity and small size make it well suited to high-throughput screenings to identify novel pharmacologic and molecular therapies for the treatment of a broad range of neuromuscular conditions. In this review, we examine the lessons learned from the zebrafish model regarding the complex interactions between the sarcomere and the sarcolemma that pattern the developing myocyte and discuss the potential for zebrafish as a model system to examine the pathophysiology of, and identify new treatments for, human myopathies and muscular dystrophies.

Leiomodin 3 and tropomodulin 4 have overlapping functions during skeletal myofibrillogenesis.

Precise regulation of thin filament length is essential for optimal force generation during muscle contraction. The thin filament capping protein tropomodulin (Tmod) contributes to thin filament length uniformity by regulating elongation and depolymerization at thin filament ends. The leiomodins (Lmod1-3) are structurally related to Tmod1-4 and also localize to actin filament pointed ends, but in vitro biochemical studies indicate that Lmods act instead as robust nucleators. Here, we examined the roles of Tmod4 and Lmod3 during Xenopus skeletal myofibrillogenesis. Loss of Tmod4 or Lmod3 resulted in severe disruption of sarcomere assembly and impaired embryonic movement. Remarkably, when Tmod4-deficient embryos were supplemented with additional Lmod3, and Lmod3-deficient embryos were supplemented with additional Tmod4, sarcomere assembly was rescued and embryonic locomotion improved. These results demonstrate for the first time that appropriate levels of both Tmod4 and Lmod3 are required for embryonic myofibrillogenesis and, unexpectedly, both proteins can function redundantly during in vivo skeletal muscle thin filament assembly. Furthermore, these studies demonstrate the value of Xenopus for the analysis of contractile protein function during de novo myofibril assembly.

The switch role of the Tmod4 in the regulation of balanced development between myogenesis and adipogenesis

Tmod4 (Tropomodulin 4) is a member of Tmod family that plays important role in thin filament length regulation and myofibril assembly. We found that the expression levels of Tmod4 were higher in skeletal muscle and adipose tissues. However, the function and regulation of the Tmod4 gene in the myogenesis and adipogenesis remains unclear. In this study, we found that the expression of Tmod4 was decreased in myogenesis while increased in adipogenesis. Then, the transcriptional regulation analysis of Tmod4 promoter showed that Tmod4 could be regulated directly by myogenic factors and adipogenic factors. Furthermore, the roles of Tmod4 in the myogenesis and adipogenesis were confirmed by its over-expression in C2C12 cells and 3T3 cells, which suggested that Tmod4 could promote adipogenesis by up-regulating the adipogenic factors but moderately delay the myogenesis. These results indicated that the Tmod4 gene may play as a switch between myogenesis and adipogenesis, which resulted in the balanced development between skeletal muscle and adipose tissue. Therefore, the model for switch role of the Tmod4 in the balanced regulation between myogenesis and adipogenesis was proposed. It is showed that the expression of Tmod4 was activated in adipogenesis by adipogenic factors while inhibited in myogenesis by myogenic factors. Moreover, Tmod4 could promote adipogenesis by up-regulating the expression of adipogenic factors while moderately delaying the myogenesis. Our study provides an important basis for further understanding the regulation and function of porcine Tmod4 in muscle and fat development.

Identification of Xin-repeat proteins as novel ligands of the SH3 domains of nebulin and nebulette and analysis of their interaction during myofibril formation and remodeling

The Xin actin-binding repeat-containing proteins Xin and XIRP2 are exclusively expressed in striated muscle cells, where they are believed to play an important role in development. In adult muscle, both proteins are concentrated at attachment sites of myofibrils to the membrane. In contrast, during development they are localized to immature myofibrils together with their binding partner, filamin C, indicating an involvement of both proteins in myofibril assembly. We identify the SH3 domains of nebulin and nebulette as novel ligands of proline-rich regions of Xin and XIRP2. Precise binding motifs are mapped and shown to bind both SH3 domains with micromolar affinity. Cocrystallization of the nebulette SH3 domain with the interacting XIRP2 peptide PPPTLPKPKLPKH reveals selective interactions that conform to class II SH3 domain-binding peptides. Bimolecular fluorescence complementation experiments in cultured muscle cells indicate a temporally restricted interaction of Xin-repeat proteins with nebulin/nebulette during early stages of myofibril development that is lost upon further maturation. In mature myofibrils, this interaction is limited to longitudinally oriented structures associated with myofibril development and remodeling. These data provide new insights into the role of Xin actin-binding repeat-containing proteins (together with their interaction partners) in myofibril assembly and after muscle damage.

Abstract 346: Genome-wide Bisulfite Sequencing After Isoproterenol Identifies DNA Methylation Modules Influencing Heart Failure Susceptibility

DNA methylation is an epigenetic mechanism that controls gene expression. Recent studies of human samples linked dysregulation of DNA methylation to cardiovascular diseases. However, whether DNA methylation is mechanistically involved in disease pathogenesis is unknown. To determine how DNA methylation participates in the development of heart failure, we measured the DNA methylome in healthy and hypertrophic mouse hearts (and in isolated cardiac myocytes in parallel experiments) using reduced representational bisulfite sequencing. Isoproterenol (ISO) minipumps were implanted in two mouse strains with opposite phenotypes: BUB/BnJ, which is resistant to ISO-induced hypertrophy/failure, and BALB/cJ, which is susceptible. DNA and mRNA were isolated from hearts and myocytes and analyzed by bisulfite sequencing and microarrays, respectively. The results reveal three levels of information about the methylome: basal differences between the resistant and susceptible strains, loci affected in a disease-associated manner, and strain-specific changes in methylation following ISO treatment_which serve as novel targets to understand the genetic basis of differential incidence of heart failure. We observed 1,122 loci undergoing a >20% change in methylation after ISO in either strain (p<0.05). Our data also showed significant ISO-induced methylation differences in genes related to myofibril assembly (GO:0031032, p=6e-7) and increased heart weight (MP:0002833, p=2e-6). We selected the top 15,000 promoter-associated CpGs with high variation among samples and carried out an unbiased weighted correlation network analysis (WGCNA) of these methylation data. We identified a module of promoter-associated CpGs whose demethylation is highly correlated with ISO treatment in both strains (75 CpGs, Pearson correlation=0.898, p=0.001). We also identified modules with strain-specific responses to ISO treatment. Interestingly, we found a module of 109 CpGs whose methylation is increased in BALB/cJ strain after ISO treatment, but decreased in BUB/BnJ strain. Our data demonstrate global changes in DNA methylation in the adult heart during the development of disease and reveal networks of modified loci that influence heart failure susceptibility.

Alp/Enigma Family Proteins Cooperate in Z-Disc Formation and Myofibril Assembly

The Drosophila Alp/Enigma family protein Zasp52 localizes to myotendinous junctions and Z-discs. It is required for terminal muscle differentiation and muscle attachment. Its vertebrate ortholog ZASP/Cypher also localizes to Z-discs, interacts with α-actinin through its PDZ domain, and is involved in Z-disc maintenance. Human mutations in ZASP cause myopathies and cardiomyopathies. Here we show that Drosophila Zasp52 is one of the earliest markers of Z-disc assembly, and we use a Zasp52-GFP fusion to document myofibril assembly by live imaging. We demonstrate that Zasp52 is required for adult Z-disc stability and pupal myofibril assembly. In addition, we show that two closely related proteins, Zasp66 and the newly identified Zasp67, are also required for adult Z-disc stability and are participating with Zasp52 in Z-disc assembly resulting in more severe, synergistic myofibril defects in double mutants. Zasp52 and Zasp66 directly bind to α-actinin, and they can also form a ternary complex. Our results indicate that Alp/Enigma family members cooperate in Z-disc assembly and myofibril formation; and we propose, based on sequence analysis, a novel class of PDZ domain likely involved in α-actinin binding.

Kif5b controls the localization of myofibril components for their assembly and linkage to the myotendinous junctions

Controlled delivery of myofibril components to the appropriate sites of assembly is crucial for myofibrillogenesis. Here, we show that kinesin-1 heavy chain Kif5b plays important roles in anterograde transport of α-sarcomeric actin, non-muscle myosin IIB, together with intermediate filament proteins desmin and nestin to the growing tips of the elongating myotubes. Mice with Kif5b conditionally knocked out in myogenic cells showed aggregation of actin filaments and intermediate filament proteins in the differentiating skeletal muscle cells, which further affected myofibril assembly and their linkage to the myotendinous junctions. The expression of Kif5b in mutant myotubes rescued the localization of the affected proteins. Functional mapping of Kif5b revealed a 64-amino acid α-helix domain in the tail region, which directly interacted with desmin and might be responsible for the transportation of these proteins in a complex.

Defining Myosin Relay Domain Interactions with the Converter Domain and with the SH1-SH2 Helix Region and their Significance in Muscle Contraction

Interaction of the myosin relay domain with the converter domain and with the SH1-SH2 helix region appears to be vital for the power stroke and muscle contraction. To define involvement of specific residues in these interactions we used mutation and compensatory mutation approach. The mutant myosins were expressed transgenically in Drosophila indirect flight muscles, which allow assessment of myosin function in vitro and in vivo. We mutated residue R714 near the SH1-SH2 helix in the main body of myosin. We found that R714E myosin ATPase is reduced by ∼80% and that no in vitro motility is detected. R714E organisms show myofibril assembly defects that are exacerbated with age and these organisms cannot fly. Putative compensatory mutation E499R in the relay domain improves ATPase activity so that there is only ∼35% reduction compared to wild type. Further, R714E/E499R myosin moves actin filaments at ∼30% of wild-type velocity. Normal myofibril assembly is restored and myofibrils are more stable. We next studied mutation R759E in the converter domain. It depresses myosin ATPase activity, in vitro motility and flight ability. Although myofibrils assemble normally, they degenerate as flies age. We inserted a lysine mutation at putative interacting relay domain loop residue N509 in an attempt to suppress the R759E mutant phenotypes. N509K dramatically rescues muscle structure and function, with significant increases in ATPase rates and in vitro motility. These interactions are critical for normal ATPase activity, in vitro motility, myofibril assembly and/or stability and flight muscle function. Thus the specific residue contacts identified permit the myosin relay domain to communicate information between the nucleotide binding pocket and the converter domain and are essential for progress through the mechanochemical cycle that powers muscle contraction.

Zasp PDZ Domain Proteins Cooperate in Z-Disc Formation and Myofibril Assembly

Zasp52 is a multi-domain protein, composed of an N-terminal PDZ domain, ZM motif, and four LIM domains, and is found predominantly in Drosophila muscle. It is part of the PDZ-LIM domain protein family, which comprises Enigma, ENH, ZASP, Alp, CLP36, RIL, and Mystique in vertebrates. In Drosophila embryos, Zasp52 was shown to co-localize with α-actinin at Z-discs and with integrins at myotendinous junctions. Mutations in Zasp52 cause first instar larval lethality with defects in Z-disc assembly and maintenance as well as muscle attachment. During Drosophila indirect flight muscle (IFM) development in the pupa, Zasp52 is present at very early stages of myofibril assembly. It localizes with α-actinin in rudimentary Z-bodies along the assembling myofibril, before other muscle proteins show any periodicity. RNA interference against the last exon encoding the most C-terminal LIM domain results in the depletion of all high molecular weight isoforms. This gives rise to viable but flightless adult animals. IFM sarcomeres show thin and interrupted Z-discs as well as distorted H-zones from early stages of myofibril assembly onwards. These findings suggest that Zasp52 is required for the establishment of normal Z-discs in the IFM and subsequent sarcomere stability after onset of contractility. A closely related PDZ domain containing protein, Zasp66, is also required for Z-disc formation and stability. Simultaneous knock down of Zasp52 and Zasp66 lead to more severe, synergistic myofibril defects, demonstrating that both proteins act together during Z-disc formation, likely because both biochemically bind α-actinin.

Lasp regulates Actin Filament Dynamics in Drosophila Myofibrils Assembly

The actin cytoskeleton plays a key role in a number of motile and morphogenetic processes. The coordinated assembly and disassembly of actin filaments promotes cell shape changes, mediates motility, contractility, and many other processes. In each case, actin dynamics is finely regulated by a large number of actin-binding proteins (ABPs) that control actin filament polymerization, nucleation and crosslinking.One group of ABPs is the nebulin family. To date, this family comprises 5 members, each containing from 2 to 185 actin-binding nebulin repeats. In our previous work, we showed that Lasp is the single member of this family in Drosophila. Lasp has an N-terminal LIM domain, two nebulin repeats, and a C-terminal SH3 domain. Lasp null mutants are homozygous viable, but male sterile. The stem cell niche is not properly anchor and actin cone migration is impaired, resulting in failure of spermatid individualization. Recently, we discovered that Lasp also functions in myofibril assembly. In Lasp mutants, sarcomere length is reduced and muscle contractility is weaker than in wild type flies. These results are consistent with a function of nebulin family proteins as scaffolding and actin filament organizing proteins.Using a double-tagged Lasp, I performed a pull down assay to identify interacting partners. As expected Lasp binds muscle-specific actin isoforms. More surprisingly, most of the isolated proteins are components of thick filaments, suggesting new functions for Lasp. We will report which domain of Lasp regulates actin dynamics, protein interactions and proper localization in sarcomere assembly. By deciphering Lasp function in both Z-disc and A-band, we will obtain further insights into the mechanism of myogenesis.

Impaired embryonic motility indusp27mutants reveals a developmental defect in myofibril structure

ABSTRACTAn essential step in muscle fiber maturation is the assembly of highly ordered myofibrils that are required for contraction. Much remains unknown about the molecular mechanisms governing the formation of the contractile apparatus. We identified an early embryonic motility mutant in zebrafish caused by integration of a transgene into the pseudophosphatase dual specificity phosphatase 27 (dusp27) gene. dusp27 mutants exhibit near complete paralysis at embryonic and larval stages, producing extremely low levels of spontaneous coiling movements and a greatly diminished touch response. Loss of dusp27 does not prevent somitogenesis but results in severe disorganization of the contractile apparatus in muscle fibers. Sarcomeric structures in mutants are almost entirely absent and only rare triads are observed. These findings are the first to implicate a functional role of dusp27 as a gene required for myofiber maturation and provide an animal model for analyzing the mechanisms governing myofibril assembly.

Myopodin is an F-actin bundling protein with multiple independent actin-binding regions

The assembly of striated muscle myofibrils is a multistep process in which a variety of proteins is involved. One of the first and most important steps in myofibrillogenesis is the arrangement of thin myofilaments into ordered I-Z-I brushes, requiring the coordinated activity of numerous actin binding proteins. The early expression of myopodin prior to sarcomeric α-actinin, as well as its binding to actin, α-actinin and filamin indicate an important role for this protein in actin cytoskeleton remodelling with the precise function of myopodin in this process yet remaining to be resolved. While myopodin was previously described as a protein capable of cross-linking actin filaments into thick bundles upon transient transfections, it has remained unclear whether myopodin alone is capable of bundling actin, or if additional proteins are involved. We have therefore investigated the in vitro actin binding properties of myopodin. High speed cosedimentation assays with skeletal muscle actin confirmed direct binding of myopodin to F-actin and showed that this interaction is mediated by at least two independent actin binding sites, found in all myopodin isoforms identified to date. Furthermore, low-speed cosedimentation assays revealed that not only full length myopodin, but also the fragment containing only the second binding site, bundles microfilaments in the absence of accessory proteins. Ultrastructural analysis demonstrated that this bundling activity resembled that of α-actinin. Biochemical experiments revealed that bundling was not achieved by myopodin's ability to dimerize, indicating the presence of two individual F-actin binding sites within the second binding segment. Thus full length myopodin contains at least three F-actin binding sites. These data provide further understanding of the mechanisms by which myopodin contributes to actin reorganization during myofibril assembly.