Human and animal muscles produce large mechanical forces that pump the blood across the body and enable voluntary body movements. These large forces are produced by mini-machines in the muscles called sarcomeres. Each sarcomere consists of actin-, myosin- and titin-filaments assembled in a pseudo-crystalline order to efficiently produce force. How such an extremely high order of proteins can be built-up during muscle development is an important question in biology. An interdisciplinary team consisting of three groups based in Marseille, Sophie Brasselet’s group at the Fresnel Institute as well as Pierre François Lenne’s and Frank Schnorrer’s groups at the Developmental Biology Institute of Marseille (IBDM) applied a sophisticated method called polarization resolved fluorescent microscopy to measure the molecular order of actin during muscle development. Molecular order reports the way molecules organize in more or less parallel orientations, here applied to actin filaments via their fluorescence label. The scientists used the insect flight muscle as a model system, which is a very amenable model to combine microscopy with defined genetic alterations (see image). Interestingly this combination discovered that molecular order of actin is built-up simultaneous across all developing sarcomeres of each muscle fiber at the same time. Surprisingly,  the motor activity of the muscle myosin motor protein, which produces force on actin, only plays a minor role in order generation during sarcomere development. These results have been recently published in PLoS Biology.

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The insect flight muscle is a particularly well suited system for studying sarcomere morphogenesis as flight muscle cells are large, about 1 mm in length, and contain many hundred myofibrils. Each myofibril is a long array of about 300 sarcomeres with a length of 3.2 µm that spans across the entire flight muscle cell. Thus, the formation of many thousands of sarcomeres can be investigated during development of each muscle cell.

Polarization-resolved microscopy is a method that can determine the molecular order of fluorescent dyes in a small volume within a cell. The scientists applied this technology to a dye coupled to an actin binding compound. Thus, they could determine the molecular order of actin filaments in flight muscles of Drosophila during the formation and maturation of sarcomeres. If all actin filaments point in the same direction, for example along the future contraction axis of the muscle, all actin binding dyes point in this direction and a high actin order is resolved by the microscope.

As each flight muscle cell contains thousands of sarcomeres the scientists could observe the formation and maturation of a large number of sarcomeres at the same time (see image). Interestingly, they discovered that the molecular order of actin is the same in all sarcomeres of a muscle cell at a given time of development. This suggests that the build-up of high molecular order is coordinated across the entire muscle cell.

How is this coordination achieved amongst the numerous sarcomeres? The scientists tested for a function of myosin motor proteins that pull on actin and become themselves ordered during sarcomere development. Indeed they found a role for muscle myosin in coordinating actin filament assembly into long parallel oriented filaments present in normal sarcomeres (see image). However, despite the absence of clearly identifiable sarcomeres in the myosin mutants actin is still highly ordered in these mutant muscle fibers, suggesting that myosin cannot be solely responsible for generating high molecular actin order during sarcomere formation and maturation. The scientists speculate that other actin cross-linking proteins such as the gigantic sarcomeric protein titin which links actin to myosin might be involved.

Schnorrer

Developing sarcomeres in a flight muscle fiber of Drosophila pupa.
Linear arrays of sarcomeres (myofibrils) in a flight muscle fiber of a 48 hour old pupa are visualized repetitive green and red stripes. Two red stripes labeling the Z-discs border a sarcomere. Flight muscle nuclei are labeled in grey. Zoom in shows the actin filament orientation in a mature sarcomere of a 90 hour old pupae. The red bar indicates the average direction of order. To visualize the small differences in orientation to deviation angle from the main axis has been amplified 50 times.