Dictyostelium mutants lacking multiple classic myosin I isoforms reveal combinations of shared and distinct functions.
Dictyostelium mutants lacking multiple classic myosin I isoforms reveal combinations of shared and distinct functions.
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DOI：
10.1083/jcb.133.2.305
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发表时间：
1996-04
期刊：
影响因子：
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通讯作者：
Hammer JA 3rd
中科院分区：
文献类型：
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作者：
Jung G;Wu X;Hammer JA 3rd
缺乏myoB同种型的网骨藻细胞先前显示出吞噬作用和趋化性聚集（“流”）的效率降低，并且以野生型细胞的约一半速度爬行.在迄今为止鉴定的四种其他网囊藻肌球蛋白I同种型中，myoC和myoD在尾部结构域序列方面与myoB最相似。此外，我们在这里表明，myoC，像myoB和myoD，是集中在肌动蛋白丰富的皮层区域，如迁移细胞的前缘。为了寻找这些亚型之间功能重叠的证据，我们分析了myoB、myoC和myoD单突变体、myoB/myoD双突变体和myoB/myoC/myoD三突变体，这些突变体是使用基因靶向技术和反义RNA组成型表达的组合产生的。在三种单突变体中，关于增殖、聚集阶段细胞的速度，只有myoB突变体明显较慢。此外，双突变体和三突变体仅比myoB单突变体稍慢。与此相一致的是，在早期发育过程中，myoB的蛋白质水平急剧上升，这表明当细胞变得高度活跃时，对这种亚型有特殊的需求。然而，我们还发现，聚集期细胞中myoB蛋白的绝对量远高于myoC和myoD，这表明似乎是非重叠功能的情况可能是功能重叠同种型的量的巨大差异的结果。流式分析还表明，myoC在运动性的某些方面而不是细胞速度中起着重要作用。关于吞噬作用，myoB和myoC单突变体表现出显着降低初始速率，这表明这两个亚型执行非冗余的作用，支持吞噬过程。然而，在三重突变体中，这些缺陷不是累加的。最后，因为双和三突变体表现出显着的和渐进的倍增时间减少，我们还测量了流体相内吞通量（摄取，通过时间，流出）的动力学。不仅所有三种亚型都有助于这一过程，而且它们的贡献是协同的。虽然这些结果，当放在一起，驳斥了简单的概念，这三个“经典”肌球蛋白I亚型执行完全相同的功能，他们确实揭示了所有三个共享支持至少一个细胞过程（内吞作用），他们确定了其他几个过程（运动，流，吞噬作用），支持在很大程度上无论是个别亚型或它们的各种组合。
Dictyostelium cells that lack the myoB isoform were previously shown to exhibit reduced efficiencies of phagocytosis and chemotactic aggregation ("streaming") and to crawl at about half the speed of wild- type cells. Of the four other Dictyostelium myosin I isoforms identified to date, myoC and myoD are the most similar to myoB in terms of tail domain sequence. Furthermore, we show here that myoC, like myoB and myoD, is concentrated in actin-rich cortical regions like the leading edge of migrating cells. To look for evidence of functional overlap between these isoforms, we analyzed myoB, myoC, and myoD single mutants, myoB/myoD double mutants, and myoB/myoC/myoD triple mutants, which were created using a combination of gene targeting techniques and constitutive expression of antisense RNA. With regard to the speed of locomoting, aggregation-stage cells, of the three single mutants, only the myoB mutant was significantly slower. Moreover, double and triple mutants were only slightly slower than the myoB single mutant. Consistent with this, the protein level of myoB alone rises dramatically during early development, suggesting that a special demand is placed on this one isoform when cells become highly motile. We also found, however, that the absolute amount of myoB protein in aggregation- stage cells is much higher than that for myoC and myoD, suggesting that what appears to be a case of nonoverlapping function could be the result of large differences in the amounts of functionally overlapping isoforms. Streaming assays also suggest that myoC plays a significant role in some aspect of motility other than cell speed. With regard to phagocytosis, both myoB and myoC single mutants exhibited significant reductions in initial rate, suggesting that these two isoforms perform nonredundant roles in supporting the phagocytic process. In triple mutants these defects were not additive, however. Finally, because double and triple mutants exhibited significant and progressive decreases in doubling times, we also measured the kinetics of fluid phase endocytic flux (uptake, transit time, efflux). Not only do all three isoforms contribute to this process, but their contributions are synergistic. While these results, when taken together, refute the simple notion that these three "classic" myosin I isoforms perform exclusively identical functions, they do reveal that all three share in supporting at least one cellular process (endocytosis), and they identify several other processes (motility, streaming, and phagocytosis) that are supported to a significant extent by either individual isoforms or various combinations of them.