Chen T L, Kowalczyk P A, Ho G, Chisholm R L
Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611, USA.
J Cell Sci. 1995 Oct;108 ( Pt 10):3207-18. doi: 10.1242/jcs.108.10.3207.
We have previously demonstrated that the myosin essential light chain (ELC) is required for myosin function in a Dictyostelium cell line, 7-11, in which the expression of ELC was inhibited by antisense RNA overexpression. We have now disrupted the gene encoding the ELC (mlcE) in Dictyostelium by gene targeting. The mlcE- mutants provide a clean genetic background for phenotypic analysis and biochemical characterization by removing complications arising from the residual ELC present in 7-11 cells, as well as the possibility of mutations due to insertion of the antisense construct at multiple sites in the genome. The mlcE- mutants, when grown in suspension, exhibited the typical multinucleate phenotype observed in both myosin heavy chain mutants and 7-11 cells. This phenotype was rescued by introducing a construct that expressed the wild-type Dictyostelium ELC cDNA. Myosin purified from the mlcE- cells exhibited significant calcium ATPase activity, but the actin-activated ATPase activity was greatly reduced. The results obtained from the mlcE- mutants strengthen our previous conclusion based on the antisense cell line 7-11 that ELC is critical for myosin function. The proper localization of myosin in mlcE- cells suggests that its phenotypic defects primarily arise from defective contractile function of myosin rather than its mislocalization. The enzymatic defect of myosin in mlcE- cells also suggests a possible mechanism for the observed chemotactic defect of mlcE- cells. We have shown that while mlcE- cells were able to respond to chemoattractant with proper directionality, their rate of movement was reduced. During chemotaxis, proper directionality toward chemoattractant may depend primarily on proper localization of myosin, while efficient motility requires contractile function. In addition, we have analyzed the morphogenetic events during the development of mlcE- cells using lacZ reporter constructs expressed from cell type specific promoters. By analyzing the morphogenetic patterns of the two major cell types arising during Dictyostelium development, prespore and prestalk cells, we have shown that the localization of prespore cells is more susceptible to the loss of ELC than prestalk cells, although localization of both cell types is abnormal when developed in chimeras formed by mixing equal numbers of wild-type and mutant cells. These results suggest that the morphogenetic events during Dictyostelium development have different requirements for myosin.
我们之前已经证明，在盘基网柄菌细胞系7-11中，肌球蛋白必需轻链（ELC）对于肌球蛋白的功能是必需的，在该细胞系中，ELC的表达受到反义RNA过表达的抑制。我们现在通过基因靶向破坏了盘基网柄菌中编码ELC的基因（mlcE）。mlcE突变体通过消除7-11细胞中残留的ELC所带来的复杂性，以及由于反义构建体在基因组多个位点插入而导致突变的可能性，为表型分析和生化特性鉴定提供了一个纯净的遗传背景。mlcE突变体在悬浮培养时，表现出在肌球蛋白重链突变体和7-11细胞中都观察到的典型多核表型。通过引入表达野生型盘基网柄菌ELC cDNA的构建体，这种表型得以挽救。从mlcE细胞中纯化的肌球蛋白表现出显著的钙ATP酶活性，但肌动蛋白激活的ATP酶活性大大降低。从mlcE突变体获得的结果强化了我们之前基于反义细胞系7-11得出的结论，即ELC对肌球蛋白功能至关重要。肌球蛋白在mlcE细胞中的正确定位表明，其表型缺陷主要源于肌球蛋白收缩功能的缺陷而非其定位错误。mlcE细胞中肌球蛋白的酶缺陷也提示了mlcE细胞观察到的趋化缺陷的一种可能机制。我们已经表明，虽然mlcE细胞能够以正确的方向性对趋化剂做出反应，但其运动速率降低。在趋化过程中，对趋化剂的正确方向性可能主要取决于肌球蛋白的正确定位，而高效的运动性则需要收缩功能。此外，我们使用从细胞类型特异性启动子表达的lacZ报告构建体分析了mlcE细胞发育过程中的形态发生事件。通过分析盘基网柄菌发育过程中出现的两种主要细胞类型，即前孢子细胞和前柄细胞的形态发生模式，我们已经表明，前孢子细胞的定位比前柄细胞更容易受到ELC缺失的影响，尽管当由等量的野生型和突变细胞混合形成嵌合体时，两种细胞类型的定位都是异常的。这些结果表明，盘基网柄菌发育过程中的形态发生事件对肌球蛋白有不同的要求。
