Kuczmarski E R, Palivos L, Aguado C, Yao Z L
Department of Physiology and Biophysics, Chicago Medical School, Illinois 60064.
J Cell Biol. 1991 Sep;114(6):1191-9. doi: 10.1083/jcb.114.6.1191.
Cytoskeletons provide valuable information on the composition and organization of the cell's contractile machinery, and in many cases these cell models retain the ability to contract. To quantitate contraction rates, we developed a novel stopped-flow assay permitting simultaneous analysis of thousands of Dictyostelium cytoskeletons within milliseconds of mixing with Mg-ATP. Cytoskeletons were placed in one syringe of the stopped flow apparatus and the appropriate buffer was placed in the second syringe. Mixing with Mg-ATP caused an immediate increase in the absorbance at 310 nm. Rapid fixation of the cytoskeletons during the reaction confirmed that this change in absorbance was highly correlated with contraction of the cytoskeletons. This spectroscopic change was used to measure the effects of temperature, pH, ionic strength, and nucleotides on contraction rate. Treatment with high salt and ATP removed most of the myosin, some actin, and small amounts of minor proteins. These extracted cytoskeletons lost the ability to contract, but after the addition of purified Dictyostelium myosin they regained full function. In contrast, rabbit skeletal muscle myosin was unable to restore contractility, even though it bound to the extracted cytoskeletons. Cytoskeletons prepared from a myosin-null mutant did not contract. Upon the addition of purified ameba myosin, however, they became contractile. These results suggest that filamentous Dictyostelium myosin II is essential for contraction, and that the actin cytoskeleton and associated proteins retain their functional organization in the absence of myosin.
细胞骨架提供了有关细胞收缩机制的组成和组织的有价值信息，并且在许多情况下，这些细胞模型保留了收缩能力。为了定量收缩率，我们开发了一种新型的停流测定法，能够在与Mg-ATP混合后的几毫秒内同时分析数千个盘基网柄菌细胞骨架。将细胞骨架置于停流装置的一个注射器中，将合适的缓冲液置于第二个注射器中。与Mg-ATP混合导致310nm处的吸光度立即增加。在反应过程中对细胞骨架进行快速固定，证实这种吸光度变化与细胞骨架的收缩高度相关。这种光谱变化用于测量温度、pH、离子强度和核苷酸对收缩率的影响。用高盐和ATP处理可去除大部分肌球蛋白、一些肌动蛋白和少量次要蛋白质。这些提取的细胞骨架失去了收缩能力，但在添加纯化的盘基网柄菌肌球蛋白后，它们恢复了全部功能。相比之下，兔骨骼肌肌球蛋白即使与提取的细胞骨架结合，也无法恢复收缩能力。从肌球蛋白缺失突变体制备的细胞骨架不收缩。然而，加入纯化的变形虫肌球蛋白后，它们变得具有收缩性。这些结果表明，丝状盘基网柄菌肌球蛋白II对收缩至关重要，并且在没有肌球蛋白的情况下，肌动蛋白细胞骨架和相关蛋白保留了它们的功能组织。
