Sarcomeric pattern formation by actin cluster coalescence.
ABSTRACT: Contractile function of striated muscle cells depends crucially on the almost crystalline order of actin and myosin filaments in myofibrils, but the physical mechanisms that lead to myofibril assembly remains ill-defined. Passive diffusive sorting of actin filaments into sarcomeric order is kinetically impossible, suggesting a pivotal role of active processes in sarcomeric pattern formation. Using a one-dimensional computational model of an initially unstriated actin bundle, we show that actin filament treadmilling in the presence of processive plus-end crosslinking provides a simple and robust mechanism for the polarity sorting of actin filaments as well as for the correct localization of myosin filaments. We propose that the coalescence of crosslinked actin clusters could be key for sarcomeric pattern formation. In our simulations, sarcomere spacing is set by filament length prompting tight length control already at early stages of pattern formation. The proposed mechanism could be generic and apply both to premyofibrils and nascent myofibrils in developing muscle cells as well as possibly to striated stress-fibers in non-muscle cells.
Project description:The premyofibril model proposes a three-stage process for the de novo assembly of myofibrils in cardiac and skeletal muscles: premyofibrils to nascent myofibrils to mature myofibrils. FRAP experiments and jasplakinolide, a drug that stabilizes F-actin, permitted us to determine how decreasing the dynamics of actin filaments affected the dynamics of tropomyosin, troponin-T, troponin-C, and two Z-Band proteins (alpha-actinin, FATZ) in premyofibrils versus mature myofibrils. Jasplakinolide reduced markedly the dynamics of actin in premyofibrils and in mature myofibrils in skeletal muscles. Two isoforms of tropomyosin-1 (TPM1?, TPM1?) are more dynamic in premyofibrils than in mature myofibrils in control skeletal muscles. Jasplakinolide reduced the exchange rates of tropomyosins in premyofibrils but not in mature myofibrils. The reduced tropomyosin recoveries did not match the YFP-actin recoveries in premyofibrils in jasplakinolide. There were no significant differences in the effects of jasplakinolide on the dynamics of troponins in the thin filaments or of two Z-band proteins in premyofibrils or skeletal mature myofibrils. Cardiac control mature myofibrils lack nebulin, and small decreases in actin (?5%) and two tropomyosin isoforms (?10-15%) dynamics are detected in premyofibril to mature myofibril transformations compared with skeletal muscle. In contrast to skeletal muscle, jasplakinolide lowered the dynamics of actin and tropomyosin isoforms in the cardiac mature myofibrils. These results suggest that the dynamics of tropomyosins in control muscle cells are related to actin exchange. These results also suggest a stabilizing role for nebulin, an actin and tropomyosin-binding protein, present in mature myofibrils but not in premyofibrils of skeletal muscles.
Project description:Sarcomeres are stereotyped force-producing mini-machines of striated muscles. Each sarcomere contains a pseudocrystalline order of bipolar actin and myosin filaments, which are linked by titin filaments. During muscle development, these three filament types need to assemble into long periodic chains of sarcomeres called myofibrils. Initially, myofibrils contain immature sarcomeres, which gradually mature into their pseudocrystalline order. Despite the general importance, our understanding of myofibril assembly and sarcomere maturation in vivo is limited, in large part because determining the molecular order of protein components during muscle development remains challenging. Here, we applied polarization-resolved microscopy to determine the molecular order of actin during myofibrillogenesis in vivo. This method revealed that, concomitantly with mechanical tension buildup in the myotube, molecular actin order increases, preceding the formation of immature sarcomeres. Mechanistically, both muscle and nonmuscle myosin contribute to this actin order gain during early stages of myofibril assembly. Actin order continues to increase while myofibrils and sarcomeres mature. Muscle myosin motor activity is required for the regular and coordinated assembly of long myofibrils but not for the high actin order buildup during sarcomere maturation. This suggests that, in muscle, other actin-binding proteins are sufficient to locally bundle or cross-link actin into highly regular arrays.
Project description:Assembly of contractile apparatuses in striated muscle requires precisely regulated reorganization of the actin cytoskeletal proteins into sarcomeric organization. Regulation of actin filament dynamics is one of the essential processes of myofibril assembly, but the mechanism of actin regulation in striated muscle is not clearly understood. Actin depolymerizing factor (ADF)/cofilin is a key enhancer of actin filament dynamics in striated muscle in both vertebrates and nematodes. Here, we report that CAS-1, a cyclase-associated protein in Caenorhabditis elegans, promotes ADF/cofilin-dependent actin filament turnover in vitro and is required for sarcomeric actin organization in striated muscle. CAS-1 is predominantly expressed in striated muscle from embryos to adults. In vitro, CAS-1 binds to actin monomers and enhances exchange of actin-bound ATP/ADP even in the presence of UNC-60B, a muscle-specific ADF/cofilin that inhibits the nucleotide exchange. As a result, CAS-1 and UNC-60B cooperatively enhance actin filament turnover. The two proteins also cooperate to shorten actin filaments. A cas-1 mutation is homozygous lethal with defects in sarcomeric actin organization. cas-1-mutant embryos and worms have aggregates of actin in muscle cells, and UNC-60B is mislocalized to the aggregates. These results provide genetic and biochemical evidence that cyclase-associated protein is a critical regulator of sarcomeric actin organization in striated muscle.
Project description:In striated muscle, the basic contractile unit is the sarcomere, which comprises myosin-rich thick filaments intercalated with thin filaments made of actin, tropomyosin and troponin. Troponin is required to regulate Ca(2+)-dependent contraction, and mutant forms of troponins are associated with muscle diseases. We have disrupted several genes simultaneously in zebrafish embryos and have followed the progression of muscle degeneration in the absence of troponin. Complete loss of troponin T activity leads to loss of sarcomere structure, in part owing to the destructive nature of deregulated actin-myosin activity. When troponin T and myosin activity are simultaneously disrupted, immature sarcomeres are rescued. However, tropomyosin fails to localise to sarcomeres, and intercalating thin filaments are missing from electron microscopic cross-sections, indicating that loss of troponin T affects thin filament composition. If troponin activity is only partially disrupted, myofibrils are formed but eventually disintegrate owing to deregulated actin-myosin activity. We conclude that the troponin complex has at least two distinct activities: regulation of actin-myosin activity and, independently, a role in the proper assembly of thin filaments. Our results also indicate that sarcomere assembly can occur in the absence of normal thin filaments.
Project description:The remarkable striation of muscle has fascinated many for centuries. In developing muscle cells, as well as in many adherent, nonmuscle cell types, striated, stress fiberlike structures with sarcomere-periodicity tend to register: Based on several studies, neighboring, parallel fibers at the basal membrane of cultured cells establish registry of their respective periodic sarcomeric architecture, but, to our knowledge, the mechanism has not yet been identified. Here, we propose for cells plated on an elastic substrate or adhered to a neighboring cell, that acto-myosin contractility in striated fibers close to the basal membrane induces substrate strain that gives rise to an elastic interaction between neighboring striated fibers, which in turn favors interfiber registry. Our physical theory predicts a dependence of interfiber registry on externally controllable elastic properties of the substrate. In developing muscle cells, registry of striated fibers (premyofibrils and nascent myofibrils) has been suggested as one major pathway of myofibrillogenesis, where it precedes the fusion of neighboring fibers. This suggests a mechanical basis for the optimal myofibrillogenesis on muscle-mimetic elastic substrates that was recently observed by several groups in cultures of mouse-, human-, and chick-derived muscle cells.
Project description:Actin filament assembly in nonmuscle cells is regulated by the actin polymerization machinery, including the Arp2/3 complex and formins. However, little is known about the regulation of actin assembly in muscle cells, where straight actin filaments are organized into the contractile unit sarcomere. Here, we show that Fhod3, a myocardial formin that localizes to thin actin filaments in a striated pattern, regulates sarcomere organization in cardiomyocytes. RNA interference-mediated depletion of Fhod3 results in a marked reduction in filamentous actin and disruption of the sarcomeric structure. These defects are rescued by expression of wild-type Fhod3 but not by that of mutant proteins carrying amino acid substitution for conserved residues for actin assembly. These findings suggest that actin dynamics regulated by Fhod3 are critical for sarcomere organization in striated muscle cells.
Project description:Heart development requires organized integration of actin filaments into the sarcomere, the contractile unit of myofibrils, although it remains largely unknown how actin filaments are assembled during myofibrillogenesis. Here we show that Fhod3, a member of the formin family of proteins that play pivotal roles in actin filament assembly, is essential for myofibrillogenesis at an early stage of heart development. Fhod3(-/-) mice appear normal up to embryonic day (E) 8.5, when the developing heart, composed of premyofibrils, initiates spontaneous contraction. However, these premyofibrils fail to mature and myocardial development does not continue, leading to embryonic lethality by E11.5. Transgenic expression of wild-type Fhod3 in the heart restores myofibril maturation and cardiomyogenesis, which allow Fhod3(-/-) embryos to develop further. Moreover, cardiomyopathic changes with immature myofibrils are caused in mice overexpressing a mutant Fhod3, defective in binding to actin. These findings indicate that actin dynamics, regulated by Fhod3, participate in sarcomere organization during myofibrillogenesis and thus play a crucial role in heart development.
Project description:Among many essential genes in the nematode Caenorhabditis elegans, let-330 is located on the left arm of chromosome V and was identified as the largest target of a mutagen in this region. However, let-330 gene has not been characterized at the molecular level. Here, we report that two sequenced let-330 alleles are nonsense mutations of ketn-1, a previously characterized gene encoding kettin. Kettin is a large actin-binding protein of 472 kDa with 31 immunoglobulin domains and is expressed in muscle cells in C. elegans. let-330/ketn-1 mutants are homozygous lethal at the first larval stage with mild defects in body elongation. These mutants have severe defects in sarcomeric actin and myosin assembly in striated muscle. However, ?-actinin and vinculin, which are components of the dense bodies anchoring actin to the membranes, were not significantly disorganized by let-330/ketn-1 mutation. Kettin localizes to embryonic myofibrils before ?-actinin is expressed, and ?-actinin deficiency does not affect kettin localization in larval muscle. Depletion of vinculin minimally affects kettin localization but significantly reduces colocalization of actin with kettin in embryonic muscle cells. These results indicate that kettin is an essential protein for sarcomeric assembly of actin filaments in muscle cells.
Project description:A transient increase in Ca<sup>2+</sup> concentration in sarcomeres is essential for their proper function. Ca<sup>2+</sup> drives striated muscle contraction via binding to the troponin complex of the thin filament to activate its interaction with the myosin thick filament. In addition to the troponin complex, the myosin essential light chain and myosin-binding protein C were also found to be Ca<sup>2+</sup> sensitive. However, the effects of Ca<sup>2+</sup> on the function of the tropomodulin family proteins involved in regulating thin filament formation have not yet been studied. Leiomodin, a member of the tropomodulin family, is an actin nucleator and thin filament elongator. Using pyrene-actin polymerization assay and transmission electron microscopy, we show that the actin nucleation activity of leiomodin is attenuated by Ca<sup>2+</sup> . Using circular dichroism and nuclear magnetic resonance spectroscopy, we demonstrate that the mostly disordered, negatively charged region of leiomodin located between its first two actin-binding sites binds Ca<sup>2+</sup> . We propose that Ca<sup>2+</sup> binding to leiomodin results in the attenuation of its nucleation activity. Our data provide further evidence regarding the role of Ca<sup>2+</sup> as an ultimate regulator of the ensemble of sarcomeric proteins essential for muscle function. SUMMARY STATEMENT: Ca<sup>2+</sup> fluctuations in striated muscle sarcomeres modulate contractile activity via binding to several distinct families of sarcomeric proteins. The effects of Ca<sup>2+</sup> on the activity of leiomodin-an actin nucleator and thin filament length regulator-have remained unknown. In this study, we demonstrate that Ca<sup>2+</sup> binds directly to leiomodin and attenuates its actin nucleating activity. Our data emphasizes the ultimate role of Ca<sup>2+</sup> in the regulation of the sarcomeric protein interactions.
Project description:The precise assembly of actin-based thin filaments is crucial for muscle contraction. Dysregulation of actin dynamics at thin filament pointed ends results in skeletal and cardiac myopathies. Here, we discovered adenylyl cyclase-associated protein 2 (CAP2) as a unique component of thin filament pointed ends in cardiac muscle. CAP2 has critical functions in cardiomyocytes as it depolymerizes and inhibits actin incorporation into thin filaments. Strikingly distinct from other pointed-end proteins, CAP2's function is not enhanced but inhibited by tropomyosin and it does not directly control thin filament lengths. Furthermore, CAP2 plays an essential role in cardiomyocyte maturation by modulating pre-sarcomeric actin assembly and regulating α-actin composition in mature thin filaments. Identification of CAP2's multifunctional roles provides missing links in our understanding of how thin filament architecture is regulated in striated muscle and it reveals there are additional factors, beyond Tmod1 and Lmod2, that modulate actin dynamics at thin filament pointed ends.