Gardini Lucia, Arbore Claudia, Capitanio Marco, Pavone Francesco Saverio
LENS - European Laboratory for Non-Linear Spectroscopy, Via Nello Carrara 1, 50019, Sesto Fiorentino, Italy.
National Institute of Optics-National Research Council, Largo Fermi 6, 50125, Florence, Italy.
MethodsX. 2019 Aug 23;6:1854-1862. doi: 10.1016/j.mex.2019.08.011. eCollection 2019.
Myosin is a large family of actin-based molecular motors, which includes efficient intracellular transporters that move cargoes and material essential for cell's life. Here, we describe protocols for labelling single myosin motors with quantum dots, tracking them in an in vitro reconstituted single-molecule motility assay, acquiring image stacks and analyzing them. We describe the required steps to obtain trajectories of single myosin motors from which fundamental biophysical parameters such as the motor velocity, run length and step size can be derived. We also describe protocols for an ensemble actin gliding assay, which is valuable to test the motor viability and its ensemble properties. The protocols allow probing the effect of changes in nucleotides, ions, and buffer composition on the motor properties and are easily generalizable to track the movements of different motor proteins.
肌球蛋白是一个基于肌动蛋白的大型分子马达家族，其中包括高效的细胞内转运蛋白，这些转运蛋白可移动对细胞生命至关重要的货物和物质。在这里，我们描述了用量子点标记单个肌球蛋白马达、在体外重组的单分子运动分析中跟踪它们、获取图像堆栈并进行分析的实验方案。我们描述了获得单个肌球蛋白马达轨迹所需的步骤，从中可以得出诸如马达速度、运行长度和步长等基本生物物理参数。我们还描述了整体肌动蛋白滑动分析的实验方案，这对于测试马达的活力及其整体特性很有价值。这些实验方案允许探究核苷酸、离子和缓冲液成分变化对马达特性的影响，并且很容易推广到跟踪不同运动蛋白的运动。
