Sallimus and the Dynamics of Sarcomere Assembly in Drosophila Flight Muscles Zacharias Orfanos 1 , Kevin Leonard 2 , Chris Elliott 1 , Anja Katzemich 1,3 , Belinda Bullard 1 and John Sparrow 1 1 - Department of Biology, University of York, York YO10 5DD, UK 2 - European Molecular Biology Laboratory European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire CB10 1SD, UK 3 - Present address: Department of Biology, McGill University, Montreal, Quebec, Canada H3A 1B1 Correspondence to Zacharias Orfanos: Institute for Cell Biology, Department of Molecular Cell Biology, University of Bonn, Ulrich-Haberland-Strasse 61a, 53121 Bonn, Germany. Fax: + 49 228735302. orfanos@uni-bonn.de http://dx.doi.org/10.1016/j.jmb.2015.04.003 Edited by J. Sellers Abstract The Drosophila indirect flight muscles (IFM) can be used as a model for the study of sarcomere assembly. Here we use a transgenic line with a green fluorescent protein (GFP) exon inserted into the Z-disc-proximal portion of sallimus (Sls), also known as Drosophila titin, to observe sarcomere assembly during IFM development. Firstly, we confirm that Sls-GFP can be used in the heterozygote state without an obvious phenotype in IFM and other muscles. We then use Sls-GFP in the IFM to show that sarcomeres grow individually and uniformly throughout the fibre, growing linearly in length and in diameter. Finally, we show that limiting the amounts of Sls in the IFM using RNAi leads to sarcomeres with smaller Z-discs in their core, whilst the thick/thin filament lattice can form peripherally without a Z-disc. Thick filament preparations from those muscles show that although the Z-disc-containing core has thick filaments of a regular length, filaments from the peripheral lattice are longer and asymmetrical around the bare zone. Therefore, the Z-disc and Sls are required for thick filament length specification but not for the assembly of the thin/thick filament lattice. © 2015 Elsevier Ltd. All rights reserved. Introduction The sarcomere is a macromolecular assembly that is the fundamental contractile subunit of striated muscle. A central question in muscle biology is how the constituent proteins of the sarcomere come together during development to form the complex regular, almost crystalline, structure. From work on vertebrate muscles, various models have been proposed to describe the process. The most popular of these is the premyofibril model [1–3]. This describes the process of sarcomere maturation from the early precursors, called premyofibrils, by the progressive assembly of different proteins in a hierarchical fashion. The Drosophila indirect flight muscles (IFM) are an ideal model for the study of myofibril assembly, as they display some of the most regular sarcomeres seen in nature. Besides the known advantages of Drosophila, such as the ease of genetic manipula- tion, IFM sarcomeres grow individually (without lateral fusion of premyofibrils) and synchronously throughout the muscle fibre [4,5], in a relatively slow process that takes approximately 3 days, allowing for a precise study of the different stages of development. Sallimus, also known as D-titin, is a large modular protein similar to the I-band region of vertebrate titin. However, the largest known isoform is only large enough to span the I-band, and in Drosophila, there are no large modular proteins such as the vertebrate titin, extending from the Z-disc to the M-line, that can function as sarcomere “rulers”. The length of sallimus isoforms found in different muscles can be correlated with the length of the I-band and with the extensibility of the sarcomere [6]. IFM have two isoforms: kettin (500 kDa) and a minor isoform of 700 kDa. The shorter isoform, kettin, links the A-band to the Z-disc and is responsible for the passive stiffness 0022-2836/© 2015 Elsevier Ltd. All rights reserved. J Mol Biol (2015) 427, 2151–2158 Communication