Localization of the Elastic Proteins in the Flight Muscle of Manduca sexta

Abstract

The flight muscle of Manduca sexta (DLM₁) is an emerging model system for biophysical studies of muscle contraction. Unlike the well-studied indirect flight muscle of Lethocerus and Drosophila, the DLM₁ of Manduca is a synchronous muscle, as are the vertebrate cardiac and skeletal muscles. Very little has been published regarding the ultrastructure and protein composition of this muscle. Previous studies have demonstrated that DLM₁ express two projectin isoform, two kettin isoforms, and two large Salimus (Sls) isoforms. Such large Sls isoforms have not been observed in the asynchronous flight muscles of Lethocerus and Drosophila. The spatial localization of these proteins was unknown. Here, immuno-localization was used to show that the N-termini of projectin and Salimus are inserted into the Z-band. Projectin spans across the I-band, and the C-terminus is attached to the thick filament in the A-band. The C-terminus of Sls was also located in the A-band. Using confocal microscopy and experimental force-length curves, thin filament lengths were estimated as ~1.5 µm and thick filament lengths were measured as ~2.5 µm. This structural information may help provide an interpretive framework for future studies using this muscle system.

Keywords: Manduca sexta; elastic proteins; myofilament lengths; sarcomere structures; synchronous insect flight muscle.

Figure 1

Estimating filament Length. (A) Top, Z-band labelled by α-actinin. Middle, actin labelled by phalloidin. Bottom, phase-contrast image of the fiber. (B) Phalloidin labelled thin filament length (double headed arrows) was defined by the distance between two adjacent H-zone (dark bands between two arrows). (C) Thick filament length (double headed arrows) was estimated after the fiber was mildly stretched.

Figure 2

Filament integrity upon stretching. (A) Fiber was stretched manually to 5.4 μm. Top, actin labeled by Phalloidin. Bottom, phase-contrast image of the fiber. Phalloidin labeled actin was found concentrating in the A-band, leaving the I-band dark, indicating thin filaments have been detached from the Z-band. (B) Slow stretched fiber. Top, actin labeled by Phalloidin. Bottom, phase-contrast image. Slow stretch has left some phalloidin signal to be dispersed near Z-band, indicating some thin filaments have remained attached to the Z-bands.

Figure 3

Force-length tension curve of outermost ventral muscle. (A) Length-force curve before correct for rundown. (B) Length-force curve after correction. Force is normalized to its value at 3.2 μm.

Figure 4

Location of projectin in the sarcomere. (A) From top to bottom. α-Actinin labeled Z-band, NT2 labeled N-terminal of projectin, MAC150 labeled middle portion of projectin, 3B11 labeled C-terminal of projectin, and phase-contrast image of the fiber. (B) From top to bottom. Phalloidin labeled actin, MAC150 labeled middle portion of projectin, phase-contrast image of the fiber, and overlaid image.

Figure 5

Position of Sallimus in the Sarcomere. (A) Top, N-Sls labelled N-terminal of Sallimus (blue) with phallodin labelled actin (red). Bottom, phase-contrast image of the fiber. The N-terminus of Sls is found in the Z-band. (B) Top, M-Sls labelled Sallimus (green) overlap with phalloidin labelled actin (red). Bottom, phase-contrast image of the fiber. M-Sls is found across the A and I-band.

Figure 6

A schematic representation of a Manduca sexta moth and the location of the DLM₁ flight muscle. The five layers of muscle are labeled from A to E with A being outermost ventral muscle and E being the outermost dorsal muscle.