6). (Fig. 2test; *= 0.029. (test; = 0.94. (= 0.80), all decapitations (= 0.45), and cilia loss (= 0.85) are shown. Values represent the mean SEM. Significance was determined by the unpaired test. (represents the lifetime of an individual cilium, with horizontal lines indicating the timing of decapitation events. The left-most bars in each set (red or blue) indicate cilia that were stable over the imaging time frame; the adjacent bars of decreasing height represent cilia with the indicated, shorter lifetimes ( 8 h). Note that in the absence of MLi-2 (blue bars), there were fewer stable cilia and proportionately more cilia loss; the percent of cilia displaying ARN 077 decapitation was similar between treated and untreated cells (Fig. 2and and and and = 0.047, control versus shRILPL1: = 0.159). (and = 0.037]; at 7 h, control +MLi-2 versus control ?MLi-2 [= 0.009], control +MLi+2 versus shRab10 [= 0.024]; at 10 h, control +MLi-2 versus control ?MLi-2 [= 0.037], control +MLi-2 versus shRab10 [= 0.022], and control +MLi+2 versus shRILPL1 [= 0.029]). Cilia dynamics are tightly linked to the cell cycle. Cilia are formed during G0/G1 phase and resorbed before mitosis begins (20). We next tested whether LRRK2 hyperactivity accelerated cell cycle reentry to enhance cilia loss after serum stimulation. After 24 h serum starvation to trigger cilia formation, cells were treated with serum to initiate ARN 077 cilia loss MLi-2 (for 8 h). Cell cycle status was then determined using propidium iodide staining flow cytometry (and and and and and Movies S4 and S5). This protocol examines the intrinsic stability of cilia after LRRK2 reactivation by MLi-2 washout. LRRK2 activity had no influence on ARN 077 the percent of cells with persistent cilia (open bars) or absence (filled bars) of pathogenic LRRK2 activity. Under these conditions, we did not detect differences in enhanced cilia loss probability ( 30 in each experiment). Significance was determined by the paired test, = 0.054. (Scale bars, 10 m.) To test if pathogenic LRRK2 influenced the recruitment of TTBK2 to the mother centriole, we stably introduced GFP tagged TTBK2 at barely detectable levels into R1441C LRRK2 MEF cells and monitored its recruitment to the mother centriole upon serum starvation. (Endogenous TTBK2 was not detectable by immunofluorescence staining or by Western blot using a commercial antibody in this cell type). In addition to R1441C LRRK2 expression blocking release of CP110 from mother centrioles in MEF cells (Fig. 4), hyperactive LRRK2 also blocked the recruitment of TTBK2 to the centriolar region marked by gamma-tubulin (Fig. 5) and, more specifically, to the mother centriole, marked by distal appendage protein CEP164 (Fig. 6). In contrast, in cells treated overnight with the LRRK2-specific MLi-2 inhibitor, 90% of cells showed a bright TTBK2 spot at the mother centriole, as expected for cells in which CP110 is released (Fig. 6 60 in each experiment). Significance was determined by the paired test; *= 0.004. (Scale bars, 10 m.) Open in a separate window Fig. 6. Pathogenic LRRK2 activity blocks TTBK2 recruitment without affecting CEP164. ( 30 in each experiment). Significance was determined by the paired test; *= 0.034. Colored shapes represent the mean of each separate experiment. ( 30). Colored shapes represent the mean of each separate experiment. Rabbit Polyclonal to ZC3H11A = 0.036. ( 40). Colored shapes represent the mean of each experiment. Significance was determined by the paired test, = 0.034. (Scale bars, 10 m.) In untreated R1441C LRRK2 MEF cells, we often saw a weak signal of TTBK2 adjacent to gamma-tubulin positive centrioles, which we ascribe to the established localization of TTBK2 to the plus ends of microtubules (22, 23). Lo et al. (14) also report.
