Department of Biochemistry and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, USA.
Methods Mol Biol. 2022;2478:585-608. doi: 10.1007/978-1-0716-2229-2_21.
KIF1A is a neuron-specific member of the kinesin-3 family of microtubule (MT) plus-end-directed motor proteins. It powers the migration of nuclei in differentiating brain stem cells and the transport of synaptic precursors and dense core vesicles in axons. Its dysfunction causes severe neurodevelopmental and neurodegenerative diseases termed KIF1A-associated neurological disorders (KAND). KAND mutations span the entirety of the KIF1A protein sequence, of which the majority are located within the motor domain and are thus predicted to affect the motor's motility and force-generating properties. Unfortunately, the molecular etiologies of KAND remain poorly understood, in part because KIF1A's molecular mechanism remains unclear. Here, we describe detailed methods for how to express a tail-truncated dimeric KIF1A in E. coli cells and provide step-by-step protocols for performing single-molecule studies with total internal reflection fluorescence microscopy and optical tweezers assays, which, when combined with structure-function studies, help to decipher KIF1A's molecular mechanism.
KIF1A 是驱动蛋白-3 家族的一个神经元特异性成员，属于微管（MT）正端导向的运动蛋白。它为分化中的脑干细胞中的核迁移以及轴突中突触前体和致密核心囊泡的运输提供动力。其功能障碍会导致严重的神经发育和神经退行性疾病，称为 KIF1A 相关神经障碍（KAND）。KAND 突变跨越 KIF1A 蛋白序列的全部，其中大多数位于马达结构域内，因此预计会影响马达的运动性和产生力的特性。不幸的是，KAND 的分子病因仍知之甚少，部分原因是 KIF1A 的分子机制尚不清楚。在这里，我们描述了在大肠杆菌细胞中表达截短二聚体 KIF1A 的详细方法，并提供了使用全内反射荧光显微镜和光学镊子测定进行单分子研究的分步方案，这些方法与结构功能研究相结合，有助于破译 KIF1A 的分子机制。
