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 Nrx^ΔPDZ-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.

神经毒素是协调细胞外和细胞内突触成分的关键粘附蛋白。尽管如此，这些多域蛋白的低丰度使得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、细胞生物学和电生理学相结合，检查果蝇Nrx-1 在体内的分布和功能。我们生成全长和 ΔPDZ ALFA 标记的 Nrx-1 变体，并发现 PDZ 结合基序是 Nrx-1 表面表达的关键。通过基因编码的胞质 NbALFA-PDZ 嵌合体，反式提供的 PDZ 结合基序完全恢复了 Nrx ^ΔPDZ-AT的突触定位和功能。使用胞质 NbALFA-mScarlet 体内，我们实现了内源性 Nrx-1 的区室特异性检测，跟踪沿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。我们的研究结果说明了 ALFA 系统的多功能性，并为剖析体内复杂蛋白质的功能域铺平了道路。