Mutations that activate LRRK2 protein kinase cause Parkinson's disease. We showed previously that Rab10 phosphorylation by LRRK2 enhances its binding to RILPL1, and together, these proteins block cilia formation in a variety of cell types, including patient derived iPS cells. We have used live-cell fluorescence microscopy to identify, more precisely, the effect of LRRK2 kinase activity on both the formation of cilia triggered by serum starvation and the loss of cilia seen upon serum readdition. LRRK2 activity decreases the overall probability of ciliation without changing the rates of cilia formation in R1441C LRRK2 MEF cells. Cilia loss in these cells is accompanied by ciliary decapitation, and kinase activity does not change the timing or frequency of decapitation or the rate of cilia loss but increases the percent of cilia that are lost upon serum addition. LRRK2 activity, or overexpression of RILPL1 protein, blocks release of CP110 from the mother centriole, a step normally required for early ciliogenesis; LRRK2 blockade of CP110 uncapping requires Rab10 and RILPL1 proteins and is due to failure to recruit TTBK2, a kinase needed for CP110 release. In contrast, deciliation probability does not change in cells lacking Rab10 or RILPL1 and relies on a distinct LRRK2 pathway. These experiments provide critical detail to our understanding of the cellular consequences of pathogenic LRRK2 mutation and indicate that LRRK2 blocks ciliogenesis upstream of TTBK2 and enhances the deciliation process in response to serum addition.

激活 LRRK2 蛋白激酶的突变会导致帕金森病。我们之前表明，LRRK2 磷酸化 Rab10 会增强其与 RILPL1 的结合，并且这些蛋白一起阻断多种细胞类型（包括患者来源的 iPS 细胞）中纤毛的形成。我们使用活细胞荧光显微镜来更准确地识别 LRRK2 激酶活性对血清饥饿引发的纤毛形成和血清重新添加时观察到的纤毛损失的影响。 LRRK2 活性降低了纤毛形成的总体概率，但不改变 R1441C LRRK2 MEF 细胞中纤毛形成的速率。这些细胞中的纤毛损失伴随着纤毛断头，激酶活性不会改变断头的时间或频率或纤毛损失的速率，但会增加添加血清后纤毛损失的百分比。 LRRK2 活性或 RILPL1 蛋白的过度表达会阻止 CP110 从母体中心粒的释放，这是早期纤毛发生通常所需的步骤； LRRK2 阻断 CP110 脱帽需要 Rab10 和 RILPL1 蛋白，这是由于未能招募 TTBK2（CP110 释放所需的激酶）所致。相反，在缺乏 Rab10 或 RILPL1 的细胞中，脱纤概率不会改变，并且依赖于不同的 LRRK2 途径。这些实验为我们理解致病性 LRRK2 突变的细胞后果提供了关键细节，并表明 LRRK2 阻断 TTBK2 上游的纤毛发生，并增强响应血清添加的脱纤过程。