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

这项NSERC资助的总体目标是了解调节肌肉收缩的机制。肌肉有一个令人印象深刻的肌动蛋白细胞骨架，它由数千个被称为肌节的重复收缩单元组成，使它们能够产生强大的收缩力量。肌节由相互滑动的反平行肌动蛋白和肌球蛋白细丝组成。我们想知道在肌肉收缩过程中防止肌节断裂的机制。我们将重点关注肌动蛋白结合蛋白丝氨酸，因为有三个重要的观察结果。它有一个弹性区域，缠绕在自己周围，在拉力作用下采取卷曲或线性构象。它位于两个相对的肌节之间的完美位置，以抵抗收缩力量。最重要的是，降低果蝇肌肉中的细丝素水平会导致肌节断裂。我们将以果蝇为研究对象，详细分析丝素在维持肌节稳定中的作用。首先，我们将使用最近产生的一种无疤痕基因工程方法来制造具有丝蛋白基因特定突变的突变果蝇。然后，我们将使用共聚焦、超分辨率和电子显微镜技术分析这些突变果蝇的肌肉。这种结合将使我们能够分离肌肉中细丝的弹性、结构和信号功能。我们的初步结果包括产生了几个突变的苍蝇品系，这证明了遗传操作方法的可行性，以及两个丝胺突变体的初步显微镜图像，在这些突变体中，弹性特性受到了损害。

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.