Structure and function of the cytoskeleton of a Dictyostelium myosin-defective mutant.
Structure and function of the cytoskeleton of a Dictyostelium myosin-defective mutant.
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DOI：
10.1083/jcb.110.2.367
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发表时间：
1990-02
影响因子：
7.8
通讯作者：
Spudich, J A
中科院分区：
文献类型：
--
作者：
Fukui, Y;De Lozanne, A;Spudich, J A
为了研究传统肌球蛋白在非肌肉细胞中的作用，我们确定了盘基网柄菌肌球蛋白缺陷突变体的细胞骨架组织和生理反应。通过肌球蛋白重链基因的插入诱变产生盘基网柄菌hmm细胞(De Lozanne，A.和J.A.Spudich.1987.Science(Wash.DC).236：1086-1091)。使用不同 mAb 的蛋白质印迹分析证实，hmm 细胞表达 140 kD 的截短肌球蛋白片段（HMM-140 蛋白），而不是正常的 243 kD 肌球蛋白重链 (MHC)。自发回复体出现的频率小于 4 x 10(-5)，其合成正常肌球蛋白并能够形成粗丝。在hmm细胞中，HMM-140蛋白广泛分布在细胞质中，表明它不能组装成粗丝。这些突变细胞中的肌动蛋白分布与野生型细胞相似。然而，渗透到板状足区域的细胞质微管的组织存在显着异常。微管网络平均由约13个微管组成，其模式异常。尽管 hmm 细胞可以形成有丝分裂纺锤体，但有丝分裂与正常的皱纹形成并不协调。 hmm 细胞在导致正常胞质分裂的收缩事件中明显存在缺陷。细胞不同区域的收缩可能会导致细胞的一部分偶尔被夹断。该过程与有丝分裂纺锤体的形成无关。在这些收缩处没有 HMM-140 的特异性积累，而 73% 的此类细胞显示肌动蛋白集中在这些区域。突变的 hmm 细胞也缺乏 Con-A 结合表面受体的加帽，而是将这种复合物内化到细胞质中。 hmm 细胞表现出对细菌的活跃吞噬作用。虽然肌动蛋白集中在吞噬杯中，但 HMM-140 蛋白并不集中在这些区域。 cAMP 是一种化学引诱剂，可在野生型细胞中诱导剧烈的四舍五入并形成表面气泡，但不会在 hmm 细胞中诱导四舍五入。野生型阿米巴的 Triton 透化细胞模型在用 Mg-ATP 重新激活时收缩，而 hmm 细胞模型则没有显示出可检测到的收缩。我们的数据表明，传统肌球蛋白参与盘基网柄菌细胞的重要皮质运动活动，包括聚拢、收缩裂沟、加盖表面受体和建立细胞极性。
To study the role of conventional myosin in nonmuscle cells, we determined the cytoskeletal organization and physiological responses of a Dictyostelium myosin-defective mutant. Dictyostelium hmm cells were created by insertional mutagenesis of the myosin heavy chain gene (De Lozanne, A., and J. A. Spudich. 1987. Science (Wash. DC). 236: 1086- 1091). Western blot analysis using different mAbs confirms that hmm cells express a truncated myosin fragment of 140 kD (HMM-140 protein) instead of the normal 243-kD myosin heavy chain (MHC). Spontaneous revertants appear at a frequency less than 4 x 10(-5), which synthesize normal myosin and are capable of forming thick filaments. In hmm cells, the HMM-140 protein is diffusely distributed in the cytoplasm, indicating that it cannot assemble into thick filaments. The actin distribution in these mutant cells appears similar to that of wild-type cells. However, there is a significant abnormality in the organization of cytoplasmic microtubules, which penetrate into lamellipodial regions. The microtubule networks consist of approximately 13 microtubules on average and their pattern is abnormal. Although hmm cells can form mitotic spindles, mitosis is not coordinated with normal furrow formation. The hmm cells are clearly defective in the contractile events that lead to normal cytokinesis. The retraction of different regions of the cell can result in the occasional pinching off of part of the cell. This process is not coupled with formation of mitotic spindles. There is no specific accumulation of HMM-140 in such constrictions, whereas 73% of such cells show actin concentrated in these regions. The mutant hmm cells are also deficient in capping of Con-A-bound surface receptors, but instead internalize this complex into the cytoplasm. The hmm cells display active phagocytosis of bacteria. Whereas actin is concentrated in the phagocytic cups, HMM-140 protein is not localized in these regions. cAMP, a chemoattractant that induces drastic rounding up and formation of surface blebs in wild type cells, does not induce rounding up in the hmm cells. A Triton- permeabilized cell model of the wild-type amebae contracts on reactivation with Mg-ATP, whereas a model of the hmm cell shows no detectable contraction. Our data demonstrate that the conventional myosin participates in the significant cortical motile activities of Dictyostelium cells, which include rounding up, constriction of cleavage furrows, capping surface receptors, and establishing cell polarity.