以果蝇为模式系统解析微管剪切蛋白fidgetin的功能

Fidgetin is a member of the AAA (ATPases associated with different cellular activities) domain protein superfamily, which uses energy from ATP hydrolysis to disrupt tubulin contacts within the microtubule (MT) lattice and is highly conserved from bacteria to vertebrates. .Drosophila fidgetin could disrupt MT arrays when overexpressed in S2 cells. Up .to now, the in vivo functions of fidgetin have not well characterized in model animals including mice and nematodes. Our preliminary study showed that when fidgetin was overexpressed in Drosophila muscle cells, the density of the microtubule network decreased in areas away from nucleus compared to wild type. Meanwhile, overexpression of fidgetin in motor neurons increased the number of satellite boutons in neuromuscular junction synaptic terminals, demonstrating that it plays a critical role at synapses. The goal of this project is to further define the neuronal and developmental functions of fidgetin with an interdisciplinary approach of biochemistry, cell biology, genetics, electrophysiology, electromicroscopy, live imagingand so on.

fidgetin是含有AAA功能域的ATP酶超家族的一员。它具有催化ATP水解活性及微管剪切功能。fidgetin在进化上高度保守,从细菌到脊椎动物中均有它的同源基因存在。体外培养细胞研究显示高表达fidgetin可以完全破坏微管网络。目前，除小鼠及线虫尚外未见到其它模式动物用于研究figetin的功能。但在小鼠及线虫中也极少有对其功能的解析。我们初步研究结果显示果蝇体内高表达fidgetin可造成远离细胞核区域微管网络的破坏。神经系统特异性高表达fidgetin可导致神经肌肉突触发育异常，表明其在神经肌肉突触发育中起重要作用。本计划拟通过生物化学，细胞生物学，电生理学，遗传学，活体成像和电子显微镜术等多学科研究方法，解析fidgetin在微管网络调控和在神经系统特别是神经突触发育中的功能。该研究不但能促进我们对fidgetin体内功能的认识，而且还能加深我们对微管调控突触发育机制的理解。

微管在多种的生物过程包括细胞分裂，细胞的生长和活力，细胞内运输，及维持细胞的形状等起重要功能。微管异常与神经发育和神经退行性疾病如遗传性痉挛性截瘫密切相关。在微管调控蛋白中，fidgetin是一个微管剪切蛋白，但其在神经系统的功能尚未在多细胞生物的体内进行研究。为了解析其在体内的功能，我们得到了fidgetin突变体果蝇和抗体以及可以组织特异性高表达fidgetin及敲减的转基因果蝇。免疫染色结果显示fidgetin在果蝇大脑及神经突触有明显分布。fidgetin在神经系统高表达导致神经肌肉接头（neuromuscular junction，NMJ）的卫星扣结（bouton）增多，而突变体及神经系统敲减fidgetin导致则导致NMJ的分枝减少，神经扣结数目减少。综上所述，我们的结果阐明了fidgetin是神经肌肉突触正常发育所必需的。该项目资助的研究已完成文章一篇，投往Genetica。