Abstract
Sarcomeres, the smallest contractile unit of muscles, are arguably the most impressive actomyosin structure. Yet a complete understanding of sarcomere formation and maintenance is missing. The Drosophila indirect flight muscle (IFM) has proven to be a very valuable model to study sarcomeres. Here, we present a protocol for the rapid dissection of IFM and analysis of sarcomeres using fluorescently tagged proteins.
Keywords: Dissection, Drosophila, GFP, Indirect flight muscle, Sarcomere, Z-disc
Background
The cytoskeletal structures that enable contractility of striated muscle fibers are hundreds of cables called myofibrils. Myofibrils in turn are an array of serially arranged sarcomeres, all contracting simultaneously. The sarcomere is a perfectly symmetrical structure that contains all the elements required for contraction. At the center of the sarcomere lies the M-line, where myosin thick filaments are anchored. Flanking the sarcomere are the Z-discs, where actin thin filaments are anchored. Muscular dystrophies are inherited disorders that cause progressive skeletal muscle weakness (Schröder and Schoser, 2009). There is no cure for muscular dystrophy, likely due to an incomplete understanding of the molecular mechanisms that underlie muscular dystrophies (Olive et al., 2013). Drosophila melanogaster is an effective genetic model organism to study muscle biology owing to its short life span, economical maintenance, and abundant available resources (Hales et al., 2015; Wangler et al., 2015). Flight in Drosophila is powered by the synchronized action of the indirect flight muscles (IFM), the biggest muscles in flies, which are further subdivided into dorsal longitudinal muscles (DLM) and dorsal ventral muscles (DVM). The IFM share many fundamental similarities with human skeletal muscle: contraction mechanism, developmental steps, overall ultrastructure, and protein components (Vigoreaux, 2001). For example, the myopathy-related proteins ZASP and Filamin-C have fly homologs that when mutated develop muscle phenotypes (Liao et al., 2016; Gonzalez-Morales et al., 2017). Despite the advantages of using the IFM for muscle research, IFM dissection can be challenging and time-consuming. Here we present a protocol that combines fast and easy IFM dissection with high-quality imaging of the IFM using fluorescent proteins. We also provide a strategy for analyzing mutant phenotypes and quantifying sarcomeres by semi-automatic detection of sarcomere components.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Notes
Recipes
Acknowledgments
This protocol is adapted from the work of others. The original IFM dissection technique was described and distributed by Belinda Bullard and John Sparrow. We thank Anja Katzemich for technical advice, and the CIAN imaging facility for help with confocal microscopy. This work was supported by operating grant MOP-142475 from the Canadian Institutes of Health Research. We have no conflict of interest to declare.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.