Dipartimento di Farmacia e Biotecnologie, Alma Mater Studiorum Università di Bologna, Via Selmi, 3, Bologna 40126, Italy.
BMC Biol. 2014 Feb 14;12:12. doi: 10.1186/1741-7007-12-12.
The Drosophila abnormal wing discs (awd) belongs to a highly conserved family of genes implicated in metastasis suppression, metabolic homeostasis and epithelial morphogenesis. The cellular function of the mammalian members of this family, the Nm23 proteins, has not yet been clearly defined. Previous awd genetic analyses unraveled its endocytic role that is required for proper internalization of receptors controlling different signaling pathways. In this study, we analyzed the role of Awd in controlling Notch signaling during development.
To study the awd gene function we used genetic mosaic approaches to obtain cells homozygous for a loss of function allele. In awd mutant follicle cells and wing disc cells, Notch accumulates in enlarged early endosomes, resulting in defective Notch signaling. Our results demonstrate that awd function is required before γ-secretase mediated cleavage since over-expression of the constitutively active form of the Notch receptor in awd mutant follicle cells allows rescue of the signaling. By using markers of different endosomal compartments we show that Notch receptor accumulates in early endosomes in awd mutant follicle cells. A trafficking assay in living wing discs also shows that Notch accumulates in early endosomes. Importantly, constitutively active Rab5 cannot rescue the awd phenotype, suggesting that awd is required for Rab5 function in early endosome maturation.
In this report we demonstrate that awd is essential for Notch signaling via its endocytic role. In addition, we identify the endocytic step at which Awd function is required for Notch signaling and we obtain evidence indicating that Awd is necessary for Rab5 function. These findings provide new insights into the developmental and pathophysiological function of this important gene family.
果蝇异常翅盘（awd）属于一个高度保守的基因家族，该家族与转移抑制、代谢稳态和上皮形态发生有关。该家族的哺乳动物成员，Nm23 蛋白的细胞功能尚未明确界定。先前的 awd 遗传分析揭示了其内吞作用，这对于控制不同信号通路的受体的正确内化是必需的。在这项研究中，我们分析了 Awd 在发育过程中控制 Notch 信号的作用。
为了研究 awd 基因的功能，我们使用遗传嵌合体方法获得了纯合缺失功能等位基因的细胞。在 awd 突变滤泡细胞和翅盘细胞中，Notch 在增大的早期内体中积累，导致 Notch 信号传导缺陷。我们的结果表明，awd 功能在 γ-分泌酶介导的切割之前是必需的，因为在 awd 突变滤泡细胞中过表达组成型激活形式的 Notch 受体允许信号的挽救。通过使用不同内体区室的标记物，我们表明 Notch 受体在 awd 突变滤泡细胞中积累在早期内体中。在活体翅盘中进行的运输测定也表明 Notch 在早期内体中积累。重要的是，组成型激活 Rab5 不能挽救 awd 表型，这表明 awd 对于 Rab5 在早期内体成熟中的功能是必需的。
在本报告中，我们证明 awd 通过其内吞作用对 Notch 信号传导是必需的。此外，我们确定了 awd 对 Notch 信号传导所必需的内吞步骤，并且我们获得了表明 awd 对于 Rab5 功能是必需的证据。这些发现为这个重要的基因家族的发育和病理生理学功能提供了新的见解。
