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      Sarcomere Formation Occurs by the Assembly of Multiple Latent Protein Complexes

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      1 , * , 1 , 1 , 2 , *
      PLoS Genetics
      Public Library of Science

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          Abstract

          The stereotyped striation of myofibrils is a conserved feature of muscle organization that is critical to its function. Although most components that constitute the basic myofibrils are well-characterized biochemically and are conserved across the animal kingdom, the mechanisms leading to the precise assembly of sarcomeres, the basic units of myofibrils, are poorly understood. To gain insights into this process, we investigated the functional relationships of sarcomeric protein complexes. Specifically, we systematically analyzed, using either RNAi in primary muscle cells or available genetic mutations, the organization of myofibrils in Drosophila muscles that lack one or more sarcomeric proteins. Our study reveals that the thin and thick filaments are mutually dependent on each other for striation. Further, the tension sensor complex comprised of zipper/Zasp/α-actinin is involved in stabilizing the sarcomere but not in its initial formation. Finally, integrins appear essential for the interdigitation of thin and thick filaments that occurs prior to striation. Thus, sarcomere formation occurs by the coordinated assembly of multiple latent protein complexes, as opposed to sequential assembly.

          Author Summary

          Muscle functionality relies on the correct assembly of myofibrils, which are composed of tandem arrays of basic functional contractile units called the sarcomeres. Many mutations in genes encoding sarcomeric proteins cause muscle diseases such as congenital myopathy and dilated cardiac hypertrophy. Understanding the process of sarcomere assembly is not only relevant to the understanding of how protein complexes interact to form complex supra-molecular structures, but also of great significance to medicine for muscle diseases. Here, by taking advantage of our newly developed primary muscle cell culture method, we reevaluate sarcomere assembly by systematically analyzing the functional relationship of sarcomeric proteins using RNA interference or genetic ablation techniques. Our analysis leads us to propose a “two-state” model whereby sarcomeric proteins exist either in the “chaotic” state with independently assembled differential functional complexes or the “highly ordered suprastructure” state made from these complexes. Because we fail to detect any previously hypothesized sarcomere assembly intermediates in our system, our data support the model that sarcomere assembly is a highly coordinated process mediated by multiple latent protein complexes and does not occur in a step-wise fashion.

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          Most cited references40

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          Centriole assembly in Caenorhabditis elegans.

          Centrioles are necessary for flagella and cilia formation, cytokinesis, cell-cycle control and centrosome organization/spindle assembly. They duplicate once per cell cycle, but the mechanisms underlying their duplication remain unclear. Here we show using electron tomography of staged C. elegans one-cell embryos that daughter centriole assembly begins with the formation and elongation of a central tube followed by the peripheral assembly of nine singlet microtubules. Tube formation and elongation is dependent on the SAS-5 and SAS-6 proteins, whereas the assembly of singlet microtubules onto the central tube depends on SAS-4. We further show that centriole assembly is triggered by an upstream signal mediated by SPD-2 and ZYG-1. These results define a structural pathway for the assembly of a daughter centriole and should have general relevance for future studies on centriole assembly in other organisms.
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            Vector and parameters for targeted transgenic RNA interference in Drosophila melanogaster.

            The conditional expression of hairpin constructs in Drosophila melanogaster has emerged in recent years as a method of choice in functional genomic studies. To date, upstream activating site-driven RNA interference constructs have been inserted into the genome randomly using P-element-mediated transformation, which can result in false negatives due to variable expression. To avoid this problem, we have developed a transgenic RNA interference vector based on the phiC31 site-specific integration method.
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              Genetic dissection of structural and functional components of synaptic plasticity. I. Fasciclin II controls synaptic stabilization and growth.

              The glutamatergic neuromuscular synapse in Drosophila forms and differentiates into distinct boutons in the embryo and grows by sprouting new boutons throughout larval life. We demonstrate that two axons form approximately 18 boutons on muscles 7 and 6 by hatching and grow to approximately 180 boutons by third instar. We further show that, after synapse formation, the homophilic cell adhesion molecule Fasciclin II (Fas II) is localized both pre- and postsynaptically where it controls synapse stabilization. In FasII null mutants, synapse formation is normal, but boutons then retract during larval development. Synapse elimination and resulting lethality are rescued by transgenes that drive Fas II expression both pre- and postsynaptically; driving Fas II expression on either side alone is insufficient. Fas II can also control synaptic growth; various FasII alleles lead to either an increase or decrease in sprouting, depending upon the level of Fas II.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                1553-7390
                1553-7404
                November 2010
                November 2010
                18 November 2010
                : 6
                : 11
                : e1001208
                Affiliations
                [1 ]Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America
                [2 ]Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, United States of America
                Stanford University, United States of America
                Author notes

                ¤: Current address: Merck Research Laboratories, Boston, Massachusetts, United States of America

                Conceived and designed the experiments: YR NP. Performed the experiments: YR JB. Analyzed the data: YR JB. Contributed reagents/materials/analysis tools: YR. Wrote the paper: YR.

                Article
                10-PLGE-RA-2367R3
                10.1371/journal.pgen.1001208
                2987826
                21124995
                f756b244-6a77-4bb7-ac8f-f856f9be985b
                Rui et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 12 January 2010
                : 15 October 2010
                Page count
                Pages: 13
                Categories
                Research Article
                Cell Biology
                Developmental Biology
                Genetics and Genomics
                Genetics and Genomics/Gene Function

                Genetics
                Genetics

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