Acute Inhibition of Heterotrimeric Kinesin-2 Function Reveals Mechanisms of Intraflagellar Transport in Mammalian Cilia
Acute Inhibition of Heterotrimeric Kinesin-2 Function Reveals Mechanisms of Intraflagellar Transport in Mammalian Cilia
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DOI：
10.1016/j.cub.2019.02.043
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发表时间：
2019-04-01
期刊：
影响因子：
9.2
通讯作者：
Verhey, Kristen J.
中科院分区：
文献类型：
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作者：
Engelke, Martin F.;Waas, Bridget;Verhey, Kristen J.
纤毛内组分的运输，称为鞭毛内运输（IFT），由驱动蛋白-2和动力蛋白-2马达提供动力。异源三聚体KIF 3A/KIF 3B/KAP驱动蛋白-2马达的任何亚基中的功能丧失阻止哺乳动物细胞中的纤毛发生，并且阻碍了对驱动蛋白-2马达如何在纤毛组装和IFT中起作用的理解。我们使用化学遗传学方法来产生可复制的KIF 3A/KIF 3B/KAP驱动蛋白-2马达（i3 A/i3 B），其能够在Kif 3a/Kif 3b双敲除细胞中拯救用于纤毛组装和Hedgehog信号传导的野生型（WT）马达功能。我们证明，KIF 3A/KIF 3B功能不仅是纤毛组装所需的，也是纤毛维持所需的，因为抑制i3 A/i3 B在2分钟内阻断IFT，并在8小时内导致初级纤毛完全丧失。相反，在相同的时间范围内，抑制动力蛋白-2对纤毛维持没有影响。纤毛损失的动力学表明，两个过程有助于纤毛拆卸停止顺行IFT：一个缓慢的缩短，是稳定的，随着时间的推移和快速decilization发生随机发病。我们还证明，驱动蛋白-2家族成员KIF 3A/KIF 3C和KIF 17不能拯救Kif 3a/Kif 3b双敲除细胞中的纤毛发生或延迟i3 A/i3 B抑制后组装纤毛的损失。这些结果表明KIF 3A/KIF 3B/KAP是哺乳动物细胞中纤毛组装和维持的唯一和必需的马达。这些发现突出了驱动蛋白-2马达如何适应纤毛组装和IFT功能的差异。
The trafficking of components within cilia, called intraflagellar transport (IFT), is powered by kinesin-2 and dynein-2 motors. Loss of function in any subunit of the heterotrimeric KIF3A/KIF3B/KAP kinesin-2 motor prevents ciliogenesis in mammalian cells and has hindered an understanding of how kinesin-2 motors function in cilium assembly and IFT. We used a chemical-genetic approach to generate an inhibitable KIF3A/KIF3B/KAP kinesin-2 motor (i3A/i3B) that is capable of rescuing wild-type (WT) motor function for cilium assembly and Hedgehog signaling in Kif3a/Kif3b double-knockout cells. We demonstrate that KIF3A/KIF3B function is required not just for cilium assembly but also for cilium maintenance, as inhibition of i3A/i3B blocks IFT within 2 min and leads to a complete loss of primary cilia within 8 h. In contrast, inhibition of dynein-2 has no effect on cilium maintenance within the same time frame. The kinetics of cilia loss indicate that two processes contribute to ciliary disassembly in response to cessation of anterograde IFT: a slow shortening that is steady over time and a rapid deciliation that occurs with stochastic onset. We also demonstrate that the kinesin-2 family members KIF3A/KIF3C and KIF17 cannot rescue ciliogenesis in Kif3a/Kif3b double-knockout cells or delay the loss of assembled cilia upon i3A/i3B inhibition. These results demonstrate that KIF3A/KIF3B/KAP is the sole and essential motor for cilium assembly and maintenance in mammalian cells. These findings highlight differences in how kinesin-2 motors were adapted for cilium assembly and IFT function across species.