The population of rapidly transported outposts did not colocalize with EB1 comet origins; however, we did observe that some moving outposts became stationary Cyclopamine price followed by nucleation of EB1 comets (Figures 3A and 3D). Overall, we observed 45% of total EB1 comets originating from Golgi outposts and 44% of total Golgi outposts correlating with EB1 comet formation (Figures 3E and 3F). These results strongly
support a role for Golgi outposts as microtubule nucleation sites within da neurons. We next asked whether these Golgi outposts could support microtubule nucleation in vitro. We partially purified Golgi vesicles from ppk-Gal4 > UAS-ManII-eGFP fly embryos. We separated the vesicles from other membranous structures via vesicle flotation through a sucrose step gradient, and then tested the ability of these vesicles
to nucleate microtubules when incubated with purified tubulin and GTP ( Hendricks et al., 2010; Kollman et al., 2010; Macurek et al., 2008; Mitchison and Kirschner, 1984; Ori-McKenney et al., 2010). We observed numerous microtubules extending from GFP-labeled Golgi vesicles, indicating that these vesicles are competent to promote microtubule nucleation in vitro ( Figures 4A–4D). Forskolin price Quantification revealed that 54% of the GFP-labeled Golgi vesicles were associated with one or more microtubules, and 60% of the microtubules extended from Golgi vesicles, consistent with our in vivo results ( Figures 4A–4C). To investigate the differences between the Golgi vesicles that could support nucleation and those that could not, we immunostained the vesicles for γ-tubulin and CP309, the Drosophila homolog of AKAP450 ( Figures 4B and 4C; Kawaguchi and Zheng, 2004). Both proteins were present on the Golgi outpost vesicles associated with microtubules. Strikingly, 85% of the γ-tubulin positive vesicles and 68% of the CP309 positive
vesicles nucleated microtubules ( Figure 4D). In contrast, we did not observe microtubule nucleation from any of the γ-tubulin negative vesicles. We Ketanserin were also able to inhibit microtubule nucleation from Golgi vesicles with a γ-tubulin function blocking antibody ( Figure S4; Joshi et al., 1992). Together these results reveal that Golgi outposts are novel sites of microtubule nucleation both in vivo and in vitro, and that this activity requires γ-tubulin and CP309. Next, we asked whether γ-tubulin and CP309 were essential for microtubule nucleation by Golgi outposts in vivo. We combined the UAS-γ-tubulin-23C RNAi with the loss of function allele, γ-tubulin-23C(A15-2), to generate viable mutant larvae with a robust phenotype.