2 DROSOPHILA BETA TUBULIN ISOFORMS ARE NOT FUNCTIONALLY EQUIVALENT
2 DROSOPHILA BETA TUBULIN ISOFORMS ARE NOT FUNCTIONALLY EQUIVALENT
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DOI：
10.1083/jcb.111.3.1009
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发表时间：
1990-09-01
影响因子：
7.8
通讯作者：
RAFF, EC
中科院分区：
文献类型：
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作者：
HOYLE, HD;RAFF, EC
我们通过在果蝇雄性生殖系中表达β3-微管蛋白来替代或补充β2-微管蛋白，在体内测试了不同β微管蛋白亚型的功能能力。睾丸特异的异构体β2相对于主要的后生动物β微管蛋白是保守的，而发育调节的异构体β3在序列上有相当大的差异。β3-微管蛋白通常在发育过程中的特定时间在不同的细胞亚群中表达，但在雄性生殖系中不表达。β2-微管蛋白通常只在睾丸的有丝分裂后生殖细胞中表达，是这些细胞中所有基于微管的功能所必需的。β2-微管蛋白的正常功能包括组装减数分裂纺锤体、轴丝和至少两类细胞质微管，包括与分化的线粒体衍生物相关的微管。构建了由β2基因的5‘’序列与β3基因的3‘’序列和蛋白质编码序列连接的杂交基因。转杂交基因的果蝇在有丝分裂后的雄性生殖细胞中表达β3-微管蛋白。当在没有正常睾丸异构体的情况下表达时，β3-微管蛋白支持一类功能细胞质微管的组装。在这种雄性中，与线粒体衍生物膜相关的微管被组装，正常的线粒体衍生物伸长，但轴丝组装和其他微管介导的过程，包括减数分裂和核成形，不发生。这些数据表明，β3微管蛋白只能支持β2正常执行的多种功能的一部分，也表明与线粒体衍生物相关的微管介导了它们的延长。当β3与β2在雄性胚系中共表达时，在任何水平上，纺锤体和所有类别的细胞质微管都被组装并正常发挥功能。然而，当β3-微管蛋白超过总睾丸β微管蛋白池的20%时，它会以主导方式扰乱正常的轴丝组装。在这种雄性的轴丝集合体中，双线小管具有中央对微管和辅助小管的单线微管的某些形态特征。因此，这些数据明确地表明，果蝇β微管蛋白亚型β2和β3在内在功能能力上并不等同，并进一步表明轴丝双线小管的组装对β微管功能的限制不同于单个微管的组装。
We have tested the functional capacity of different beta tubulin isoforms in vivo by expressing .beta.3-tubulin either in place of or in addition to .beta.2-tubulin in the male germ line of Drosophila melanogaster. The testes-specific isoform, .beta.2, is conserved relative to major metazoan beta tubulins, while the developmentally regulated isoform, .beta.3, is considerably divergent in sequence. .beta.3-tubulin is normally expressed in discrete subsets of cells at specific times during development, but is not expressed in the male germ line. .beta.2-Tubulin is normally expressed only in the postmitotic germ cells of the testis, and is required for all microtubule-based functions in these cells. The normal functions of .beta.2-tubulin include assembly of meiotic spindles, axonemes, and at least two classes of cytoplasmic microtubules, including those associated with the differentiating mitochondrial derivatives. A hybrid gene was constructed in which 5'' sequences from the .beta.2 gene were jointed to protein coding and 3'' sequences of the .beta.3 gene. Drosophila transfromed with the hybrid gene express .beta.3-tubulin in the postmitotic male germ cells. When expressed in the absence of the normal testis isoform, .beta.3-tubulin supports assembly of one class of functional cytoplasmic microtubules. In such males the microtubules associated with the membranes of the mitochondrial derivatives are assembled and normal mitochondrial derivative elongation occurs, but axoneme assembly and other microtubule-mediated processes, including meiosis and nuclear shaping, do not occur. These data show that .beta.3 tubulin can support only a subset of the multiple functions normally performed by .beta.2, and also suggest that the microtubules associated with the mitochondrial derivatives mediate their elongation. When .beta.3 is coexpressed in the male germ line with .beta.2, at any level, spindles and all classes of cytoplasmic microtubules are assembled and function normally. However, when .beta.3-tubulin exceeds 20% of the total testis beta tubulin pool, it acts in a dominant way to disrupt normal axoneme assembly. In the axonemes assembles in such males, the doublet tubules acquire some of the morphological characteristics of the singlet microtubules of the central pair and accessory tubules. These data therefore unambiguously demonstrate that the Drosophila beta tubulin isoforms .beta.2 and .beta.3 are not equivalent in intrinsic funcitonal capacity, and furthermore show that assembly of the doublet tubules of the axoneme imposes different constraints on beta tubulin function than does assembly of single microtubules.