Holweg Carola Luise
Institute of Biology II, Freiburg, Germany.
Methods Mol Biol. 2009;586:265-73. doi: 10.1007/978-1-60761-376-3_14.
A key to understanding cytoskeletal mechanisms of eukaryotic cells is found in their internal motility. In many plant cell types, these motile events, termed "cytoplasmic streaming", are very impressive with rapid movement of organelles over long distances. Like many other features of cytoplasmic streaming, organelle velocity is determined by acto-myosin-related mechanisms. Therefore, the quantification of streaming velocity aids the characterization of important factors contributing to cytoskeleton function. Usually, the movement velocity varies greatly between particles and exhibits rapid changes. This complexity makes measurements very cumbersome and requires large cell numbers and a lot of imaging data for statistical evaluation. Focusing on a triplet of rapidly moving organelles in a single cell proved to be an efficient method for determining organelle displacement in a direct, standardized manner. This approach requires only a few cells and allows the evaluation of potential factors involved in cytoplasmic streaming with a relatively low temporal and technical effort. This chapter evaluates two examples that show the high sensitivity of the method in the detection of differences in organelle streaming velocities. These include the retardation of streaming upon myosin inhibition and a similar, but much less expected, response following the overexpression of actin-binding proteins.
理解真核细胞细胞骨架机制的关键在于其内部运动。在许多植物细胞类型中，这些被称为“细胞质环流”的运动事件非常引人注目，细胞器能在长距离内快速移动。与细胞质环流的许多其他特征一样，细胞器速度由肌动蛋白-肌球蛋白相关机制决定。因此，对环流速度的量化有助于表征影响细胞骨架功能的重要因素。通常，粒子之间的移动速度差异很大，并且会迅速变化。这种复杂性使得测量非常繁琐，需要大量细胞和大量成像数据进行统计评估。事实证明，聚焦于单个细胞中快速移动的一组三个细胞器是一种直接、标准化地确定细胞器位移的有效方法。这种方法只需要少量细胞，并且能够以相对较少的时间和技术投入来评估参与细胞质环流的潜在因素。本章评估了两个例子，展示了该方法在检测细胞器环流速度差异方面的高灵敏度。这些例子包括肌球蛋白抑制后环流的减慢，以及肌动蛋白结合蛋白过表达后出现的类似但出人意料得多的反应。
