Using Drosophila to study the structural and signaling functions of filamin in muscles.

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The overall objective of this NSERC grant is to understand the mechanisms that mediate muscle contraction. Muscles have an impressive actin cytoskeleton, composed of thousands of repeated contractile units called sarcomeres that allow them to produce large contractile forces. Sarcomeres are composed of antiparallel actin and myosin filaments that slide along each other. We want to know the mechanisms that prevent sarcomeres from breaking apart during muscle contraction. We will focus on the actin-binding protein filamin because of three important observations. It has an elastic region that coils around itself and adopts either a coiled or a linear conformation upon pulling forces. It localizes between two opposing sarcomeres at the perfect spot to resist contractile forces. Most importantly, reducing filamin levels in Drosophila muscles cause the sarcomeres to break. We will use Drosophila to analyze in detail the function of filamin in maintaining sarcomere stability. First, we will use a recently generated scarless genetic engineering method to make mutant flies with specific mutations in the filamin gene. Then, we will analyze the muscles of these mutant flies using confocal, super-resolution and electron microscopy techniques. This combination will allow us to separate the elastic, the structural, and the signalling functions of filamin in muscles. Our preliminary results include the generation of a few mutant fly lines, which demonstrate the feasibility of the genetic manipulation method, and preliminary microscopy images of two filamin mutants in which the elastic property is compromised.