Nilsson H, Steffen W, Palazzo R E
Dept. Zoology, Univ. Göteborg, Göteborg, Sweden.
Cell Motil Cytoskeleton. 2001 Jan;48(1):1-10. doi: 10.1002/1097-0169(200101)48:1<1::AID-CM1>3.0.CO;2-D.
Movement and positioning of melanophore pigment organelles depend on microtubule- and actin-dependent motors, but the regulation of these forces are poorly understood. Here, we describe a cell free and fixed time motility assay for the study of the regulation of microtubule-dependent pigment organelle positioning in vitro. The assay involves introduction of microtubule-asters assembled in clam oocyte lysates into lysates prepared from Fundulus heteroclitus melanophores with either aggregated or dispersed pigment. When asters were introduced in lysates prepared from melanophores with dispersed pigment, pigment organelles bound astral microtubules and were evenly distributed throughout the aster. In contrast, when asters were introduced into lysates prepared from melanophores with aggregated pigment, pigment organelles accumulated around the centrosome, mimicking a pigment aggregate. The addition of anti-dynein intermediate chain antibody (m74-1), previously shown to interfere with binding of dynactin to dynein and thereby causing detachment of dynein from organelles, blocked the ATP-dependent aggregation of pigment in vitro and induced a depletion of pigment from the centrosomal area. The results show that dynein is essential for pigment aggregation and involved in maintenance of evenly dispersed pigment in vitro, analogous to cellular evidence, and suggest a possible role for dynactin in these processes as well.
黑素细胞色素细胞器的移动和定位依赖于微管和肌动蛋白依赖的马达蛋白，但对这些力的调节却知之甚少。在这里，我们描述了一种无细胞和固定时间的运动分析方法，用于研究体外微管依赖的色素细胞器定位的调节。该分析方法包括将在蛤蜊卵母细胞裂解物中组装的微管星状体引入从具有聚集或分散色素的底鳉黑素细胞制备的裂解物中。当星状体被引入从具有分散色素的黑素细胞制备的裂解物中时，色素细胞器结合星状微管并均匀分布在整个星状体中。相反，当星状体被引入从具有聚集色素的黑素细胞制备的裂解物中时，色素细胞器聚集在中心体周围，模拟色素聚集。添加抗动力蛋白中间链抗体（m74-1），先前已证明其会干扰动力蛋白激活蛋白与动力蛋白的结合，从而导致动力蛋白从细胞器上脱离，在体外阻断了ATP依赖的色素聚集，并导致中心体区域色素的耗尽。结果表明，动力蛋白对于色素聚集至关重要，并且在体外参与维持色素均匀分散，这与细胞证据类似，也表明动力蛋白激活蛋白在这些过程中可能也发挥作用。
