Extended Data Fig. 4. Mechanism of LRRK2^RCKW binding to microtubules.

a, Representative images of 293 T cells expressing GFP-LRRK2 (wild-type or the WD40 mutants L23443D and S2345D) and treated with DMSO or 500 nM MLi-2 for 2 hours. Scale bar is 10 µm. b, Representative images of 293 T cells expressing GFP-LRRK2 (wild-type or the COR-B mutants R1731L and R1731D) and treated with DMSO or 500 nM MLi-2 for 2 hours. Scale bar is 10 µm. c,d, Rab10 phosphorylation in 293 T cells overexpressing WT LRRK2 or LRRK2 carrying mutations in the WD40 (c) or COR-B (d) domains. LRRK2[I2020T], which is known to increase Rab10 phosphorylation in cells, was tested as well. 293 T cells were transiently co-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 data in (c) and (d) is shown in Fig. 2d, f, respectively. e, A representative gel of the supernatants from the microtubule pelleting assays used to generate the data shown in Fig. 2g. f, Cryo-EM analysis of filament formation in vitro by LRRK2^RCKW mutants. Top row, cryo-EM images of an individual microtubule (left) or combinations of microtubules and LRRK2^RCKW mutants. Middle, Diffraction patterns calculated from the images above. Arrowheads point to layer lines arising from the microtubule (white) or from the LRRK2^RCKW filaments (open), and their frequencies are indicated below the images. Bottom, 2D class averages from multiple images equivalent to those shown at the top. g, Example kymographs of single-molecule kinesin motility assays in the presence or absence of 50 nM LRRK2^RCKW wild-type or indicated mutant. h, Comparison of 2D class averages from cryo-EM images of different LRRK2^RCKW mutants with the corresponding 2D projection from a LRRK2^RCKW molecular model (PDB: 6VNO). Two different views are shown for each mutant. i, Representative in vitro kinase reaction. Rab8a phosphorylation was measured via western blotting with a phospho-T72-specific Rab8a antibody, and total LRRK2^RCKW concentration was measured by Sypro Red staining. Phosphorylation reactions were terminated after 30 minutes. j, Quantification of data shown in (i). For each reaction, phospho-Rab8a band intensity (chemiluminescence) was divided by LRRK2^RCKW band intensity (Sypro red); for each western blot, an average normalized value was calculated for all replicates of unlabeled LRRK2^RCKW, and all data was then normalized to this value. Data are mean +/− s.d., n = 5 replicates, p=ns, two-tailed unpaired t-test. k, Cumulative distribution of run lengths for kinesin in the absence or presence of 25 nM TMR-LRRK2^RCKW. The run lengths were significantly different between 0 nM and 25 nM TMR-LRRK2^RCKW (Mann-Whitney test). Mean decay constants (tau) are shown. The effect on kinesin motility is similar to that previously shown using unlabeled LRRK2^RCKW. l, m, Representative microtubule pelleting assay gel for LRRK2^RCKW in the presence of 100 mM and 150 mM sodium chloride. Co-sedimentation was measured as depletion from the supernatant. For each reaction, 200 nM LRRK2^RCKW was mixed with a given concentration of microtubules, microtubules were pelleted by a high-speed spin, and a gel sample was taken of the supernatant. Quantification of data represented in (l) is shown in (m). Data are mean ± s.d., n = 4. The solid line represents a hyperbolic curve fit to the data. n, Representative images of coverslip-tethered Alexa Fluor 488-labeled MTs (cyan) bound to 100 nM TMR-LRRK2^RCKW (magenta) in the presence of increasing concentrations of sodium chloride, used to generate the data in Fig. 3d. o, Subtilisin treatment of taxol-stabilized microtubules. Serial dilutions of taxol-stabilized microtubules were treated with subtilisin (left) or left untreated as a control (right). The experiment was performed twice with similar results. p, Representative images of untreated (top) and subtilisin-treated (bottom) Alexa Fluor 488-labeled MTs (cyan) bound to 50 nM TMR-LRRK2^RCKW (magenta), used to generate the data shown in Fig. 3e.

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