PDF(Pubmed)
Abstract：
Neurexins are key adhesion proteins that coordinate extracellular and intracellular synaptic components. Nonetheless, the low abundance of these multidomain proteins has complicated any localization and structure-function studies. Here we combine an ALFA tag (AT)/nanobody (NbALFA) tool with classic genetics, cell biology and electrophysiology to examine the distribution and function of the Drosophila Nrx-1 in vivo. We generate full-length and ΔPDZ ALFA-tagged Nrx-1 variants and find that the PDZ binding motif is key to Nrx-1 surface expression. A PDZ binding motif provided in trans, via genetically encoded cytosolic NbALFA-PDZ chimera, fully restores the synaptic localization and function of NrxΔPDZ-AT. Using cytosolic NbALFA-mScarlet intrabody, we achieve compartment-specific detection of endogenous Nrx-1, track live Nrx-1 transport along the motor neuron axons, and demonstrate that Nrx-1 co-migrates with Rab2-positive vesicles. Our findings illustrate the versatility of the ALFA system and pave the way towards dissecting functional domains of complex proteins in vivo.
摘要：
Neurexins是协调细胞外和细胞内突触组分的关键粘附蛋白。尽管如此，这些多域蛋白的低丰度使任何定位和结构功能研究变得复杂。在这里，我们将ALFA标签（AT）/纳米抗体（NbALFA）工具与经典遗传学相结合，细胞生物学和电生理学检查果蝇Nrx-1在体内的分布和功能。我们产生全长和ΔPDZALFA标记的Nrx-1变体，发现PDZ结合基序是Nrx-1表面表达的关键。反式提供的PDZ结合基序，通过基因编码的胞质NbALFA-PDZ嵌合体，完全恢复NrxΔPDZ-AT的突触定位和功能。使用胞质NbALFA-mScarletintrabody，我们实现了内源性Nrx-1的区室特异性检测，跟踪沿着运动神经元轴突的活Nrx-1运输，并证明Nrx-1与Rab2阳性囊泡共同迁移。我们的发现说明了ALFA系统的多功能性，并为在体内解剖复杂蛋白质的功能域铺平了道路。
