Extended Data Fig. 5. Basic residues within the LRRK2 ROC domain are not conserved in LRRK1 and are involved in LRRK2’s binding to microtubules.

a, Cryo-EM structure determination of LRRK1^RCKW. Local resolution map, Fourier Shell Correlation, directional FSC plot, and the distribution of voxel resolutions are shown. b, Representative gel of supernatant from microtubule pelleting assay for 200 nM LRRK2^RCKW or LRRK1^RCKW with increasing tubulin concentrations. We performed 4 technical replicates. c, Sequence alignment of the ROC domains of LRRK2 and LRRK1 across several species made using Clustal Omega. Putative microtubule-contacting residues conserved in LRRK2 but not in LRRK1 are boxed. d, Close ups of the basic patches tested in this study, shown in the context of the cryo-EM maps for the ‘+’ and ‘−’ LRRK2^RCKW monomers in our reconstruction of the microtubule-associated filaments (Fig. 1e, f). The models shown here correspond to those in Fig. 5c. e, Example kymographs of kinesin motility in the presence of 100 nM LRRK2^RCKW (wild-type or K1358A/K1359A mutant). f, Representative images of 293 T cells expressing GFP-LRRK2 (wild type or ROC mutants) and treated with DMSO or 500 nM MLi-2 for 2 hours, corresponding to data plotted in Fig. 5g. Scale bar is 10 µm. g, Representative images of 293 T cells expressing GFP-LRRK2 (I2020T, I2020T/ROC mutant, or I2020T/R1501W), corresponding to data plotted in Fig. 5h, j. Scale bar is 10 µm. h, Representative images of 293 T cells expressing GFP-LRRK2 (wild type or R1501W mutant) and treated with DMSO or 500 nM MLi-2 for 2 hours, corresponding to data plotted in Fig. 5i. Scale bar is 10 µm. I, j, Rab10 phosphorylation in 293 T cells overexpressing WT LRRK2 or LRRK2 carrying the indicated mutations in the ROC domain. LRRK2[I2020T], which is known to increase Rab10 phosphorylation in cells, was tested as well. 293 T cells were transiently transfected with the indicated plasmids encoding for GFP-LRRK2 (wild-type or mutant) and GFP-Rab10. Thirty-six hours post-transfection the cells were lysed, immunoblotted for phospho-Rab10 (pT73), total GFP-Rab10, and total GFP-LRRK2. Quantification of immunoblotting data (i) is shown in (j) as ratios of pRab10/total GFP-Rab10 normalized to the average of all wildtype values. Individual data points represent separate populations of cells obtained across at least three independent experiments. Data are mean +/− s.e.m. ****p < 0.0001, ***p = 0.0004, **p = 0.0052, ***p = 0.0344, one-way ANOVA followed by a Fisher’s LSD test.

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