Fig. 3.
Conditions that impact LRRK2-dependent liposome tubulation. (A) Software was generated to enhance visualization of tubules in order to allow their quantification. Representative fluorescence micrographs showing LRRK2-induced liposome tubulation in the presence of GMPPNP before (Top) and after image processing (Bottom). Liposome tubules are labeled in green; nontubular liposomes are labeled in red. (B and C) Quantification of the tubulation efficiency of full length LRRK2 (B) or the RCKW fragment (C) in the presence of different nucleotides. (D) Quantification of the tubulation efficiency of autophosphorylated LRRK2, showing that autophosphorylation does not affect tubulation efficiency. (E) Representative fields, after image processing to highlight membrane tubules, of liposome incubated with wild-type (Upper) and kinase domain mutant (I2020T) LRRK2, at the concentration indicated. (F) Quantification of the tubulation efficiency of WT and mutant LRRK2I2020T at the indicated protein concentration. Bars represent mean ± SD n ≥ 10 images from at least 3 independent experiments. Data from (B) and (F) were analyzed using ordinary one-way ANOVA by Prism 9; Data from (C) and (D) were analyzed using t tests by Prism 9. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, P > 0.05.