Regulation of Commissureless by the ubiquitin ligase DNedd4 is required for neuromuscular synaptogenesis in Drosophila melanogaster
Regulation of Commissureless by the ubiquitin ligase DNedd4 is required for neuromuscular synaptogenesis in Drosophila melanogaster
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DOI：
10.1128/mcb.00463-06
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发表时间：
2007-01-01
影响因子：
5.3
通讯作者：
Rotin, Daniela
中科院分区：
文献类型：
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作者：
Ing, Bryant;Shteiman-Kotler, Alina;Rotin, Daniela
黑腹果蝇的肌肉突触发生需要commissuless （Comm）的内吞作用，commissuless （Comm）是泛素连接酶dNedd4的结合伙伴。我们研究了dNedd4和泛素化是否介导了这一过程。在这里，我们发现Comm在肌肉的细胞内囊泡中表达，而Comm在负责dNedd4结合的两个PY基（L/PPXY）中携带突变[Comm (2PY -> AY)]，或在作为泛素受体位点的所有Lys残基中携带Lys -> Arg突变[Comm (10K -> R)]，定位于肌肉表面，表明它们不能内噬。因此，在肌肉发育早期表达的Comm（2PY -> AY）和Comm（10K -> R）突变体中观察到异常的肌肉神经支配。当dNedd4在肌肉、dNedd4杂合子幼虫或过度表达无催化活性的dNedd4的肌肉中被双链RNA干扰敲除时，观察到类似的Comm和神经支配缺陷的肌肉表面积累。Comm突变体融合成一种不能多泛素化并模仿单泛素化的单一泛素表达[Comm (2PY -> AY)- monoub或Comm (10K -> R) - monoub]可防止Comm内吞和突触发生缺陷，表明单泛素化足以在肌肉中实现Comm内吞。在突触神经支配后，Comm突变体在肌肉发育后期的表达没有影响。这些结果表明，dNedd4和泛素化是无联胞吞作用和正常神经肌肉突触发生所必需的。
Muscle synaptogenesis in Drosophila melanogaster requires endocytosis of Commissureless (Comm), a binding partner for the ubiquitin ligase dNedd4. We investigated whether dNedd4 and ubiquitination mediate this process. Here we show that Comm is expressed in intracellular vesicles in the muscle, whereas Comm bearing mutations in the two PY motifs (L/PPXY) responsible for dNedd4 binding [Comm (2PY -> AY)], or bearing Lys -> Arg mutations in all Lys residues that serve as ubiquitin acceptor sites [Comm (10K -> R)], localize to the muscle surface, suggesting they cannot endocytose. Accordingly, aberrant muscle innervation is observed in the Comm(2PY -> AY) and Comm(10K -> R) mutants expressed early in muscle development. Similar muscle surface accumulation of Comm and innervation defects are observed when dNedd4 is knocked down by double-stranded RNA interference in the muscle, in dNedd4 heterozygote larvae, or in muscles overexpressing catalytically inactive dNedd4. Expression of the Comm mutants fused to a single ubiquitin that cannot be polyubiquitinated and mimics monoubiquitination [Comm (2PY -> AY)-monoUb or Comm (10K -> R) -monoUb] prevents the defects in both Comm endocytosis and synaptogenesis, suggesting that monoubiquitination is sufficient for Comm endocytosis in muscles. Expression of the Comm mutants later in muscle development, after synaptic innervation, has no effect. These results demonstrate that dNedd4 and ubiquitination are required for Commissureless endocytosis and proper neuromuscular synaptogenesis.