The Jungers Center for Neurosciences Research, Oregon Health and Science University, Portland, OR 97239, USA.
J Cell Biol. 2012 Aug 20;198(4):749-61. doi: 10.1083/jcb.201205070.
Identifying the kinesin motors that interact with different vesicle populations is a longstanding and challenging problem with implications for many aspects of cell biology. Here we introduce a new live-cell assay to assess kinesin-vesicle interactions and use it to identify kinesins that bind to vesicles undergoing dendrite-selective transport in cultured hippocampal neurons. We prepared a library of "split kinesins," comprising an axon-selective kinesin motor domain and a series of kinesin tail domains that can attach to their native vesicles; when the split kinesins were assembled by chemical dimerization, bound vesicles were misdirected into the axon. This method provided highly specific results, showing that three Kinesin-3 family members-KIF1A, KIF13A, and KIF13B-interacted with dendritic vesicle populations. This experimental paradigm allows a systematic approach to evaluate motor-vesicle interactions in living cells.
鉴定与不同囊泡群体相互作用的驱动蛋白一直是一个具有挑战性的问题，它对细胞生物学的许多方面都有影响。在这里，我们引入了一种新的活细胞测定法来评估驱动蛋白-囊泡相互作用，并利用它来鉴定与培养的海马神经元中树突选择性运输的囊泡结合的驱动蛋白。我们制备了一个“分裂驱动蛋白”文库，该文库包含一个轴突选择性驱动蛋白马达结构域和一系列可以连接到其天然囊泡的驱动蛋白尾部结构域；当通过化学二聚化组装分裂驱动蛋白时，结合的囊泡被错误引导到轴突中。这种方法提供了高度特异性的结果，表明三种驱动蛋白-3 家族成员-KIF1A、KIF13A 和 KIF13B-与树突状囊泡群体相互作用。这种实验范式允许系统地评估活细胞中的马达-囊泡相互作用。
