Imaging Approaches to Investigate Myonuclear Positioning in Drosophila

Abstract

In the skeletal muscle, nuclei are positioned at the periphery of each myofiber and are evenly distributed along its length. Improper positioning of myonuclei has been correlated with muscle disease and decreased muscle function. Several mechanisms required for regulating nuclear position have been identified using the fruit fly, Drosophila melanogaster. The conservation of the myofiber between the fly and vertebrates, the availability of advanced genetic tools, and the ability to visualize dynamic processes using fluorescent proteins in vivo makes the fly an excellent system to study myonuclear positioning. This chapter describes time-lapse and fixed imaging methodologies using both the Drosophila embryo and the larva to investigate mechanisms of myonuclear positioning.

Muscle cofilin alters neuromuscular junction postsynaptic development to strengthen functional neurotransmission

“…Third-instar larvae at the wandering stage were dissected as previously described in (Azevedo et al, 2016; Brent et al, 2009) to expose the body wall muscles. Fixation was done using 4% paraformaldehyde in HL3.1 buffer for 15 minutes at room temperature for all antibodies except GluRIIA for which Bouin’s fixative was used for 5 min at room temperature.…”

Analyzing muscle structure and function throughout the larval instars in live Drosophila

“…It is recommended to track the larvae at the same time every day. Once late 3 rd instar larvae have been tracked, dissect, fix, stain, and mount each larva individually as described in Azevedo et al. (2016) .…”

“…Transfer cleaned larvae into a fresh apple juice plate at room temperature. Note: For further information and images on laying pot setup and larval collection please refer to Azevedo et al. (2016) .…”

“…Note: For further information and images on laying pot setup and larval collection please refer to Azevedo et al. (2016) .…”

“…Examination of muscle structure through heat fixation is limited by the availability of fluorescent transgenes. To analyze muscle structure in detail, larval dissection, followed by fixation and staining of musculature in larval fillet should be performed [as described in ( Azevedo et al., 2016 , Brent et al., 2009 )]. Dissection is technically challenging in the 1 st and 2 nd instar larvae.…”