Immunogold labeling of endogenous and 3xFlag-tagged ectopic LRRK2 chromatographic fractions from cell lysates.

<p>(A) Relative chromatographic profiles built by measuring the dot blot immunoreactive signal intensity of each fraction and dividing it by the total signal of the protein in all fractions to determine the percentages shown in the graph. Endogenous LRRK2 from NIH-3T3 cells was detected using a rat monoclonal antibody <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043472#pone.0043472-Piccoli2" target="_blank">[68]</a>, which recognizes endogenous LRRK2 as shown by western blot of a total lysate (inset image). (B) Representative TEM images of immunogold labeled samples from chromatographic fractions corresponding to 12.5 mL peaks. 3xFlag-LRRK2 was labeled using monoclonal M2 anti-flag antibodies and anti-mouse secondary antibodies conjugated with 5 nm gold particles; endogenous LRRK2 from NIH-3T3 cells was labeled using a anti-LRRK2 rabbit monoclonal antibody (aa 100–500) and anti-rabbit secondary antibodies conjugated with 10 nm gold particles. (C) Frequency distribution plots of gold-particle distances for 3xFlag-LRRK2 (black bins) and endogenous (red bins) chromatographic fractions analyzed by immunogold EM.</p

Comparative kinase activities of LRRK1 and LRRK2.

<p>(A) Autophosphorylation assays of 3xFlag-LRRK1 wild-type, LRRK1 kinase dead, LRRK2 wild-type and LRRK2 kinase dead. End-point reactions (60 minutes) were resolved on a 4–20% SDS-PAGE and transferred onto PVDF membranes. Upper panel is autoradiography and lower panel western blot to correct activity for total loading (with anti-Flag antibody). The experiment is representative of n = 3 replicates. Markers to the right of the blots are in kilodaltons. (B) Quantitation of ³³P signal by densitometry normalized to total loading. (C–D) LRRKtide (C) and (D) Nictide phosphorylation assessed by P81 filter binding assay reveals that both peptides are specific substrates for LRRK2. (E) Rate of P³³ incorporation as a function of LRRK2 protein content (from 10 to 550 ng) measured by LRRKtide phosphorylation assays. (F) Kinetic constants of wild-type and G2019S LRRK2 for LRRKtide were determined by incubating 25 nM LRRK2 with varying concentrations of LRRKtide in the presence of 100 µM ATP and by fitting the data to a hyperbolic function. K_m was 171±20 µM for wild-type and 257±63 µM for G2019S. V_max were 1.92±0.06 pmol/min/µg for wild-type and 7.71±0.95 pmol/min/µg for G2019S. (G) ATP binding was tested for both LRRK1 and LRRK2 by affinity binding of the proteins to 4 different forms of ATP-bound agarose beads (ie ATP is coupled to the beads in different conformations) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043472#s4" target="_blank">materials and methods</a>. Both LRRK1 and LRRK2 bound to the beads when ATP is coupled via the adenine moiety (6-AH-ATP-A or 8-AH-ATP-A). Binding was negligible for ATP coupled via the gamma-phosphate (AP-ATP-A) or ribose group (EDA-ATP-A). The position of the 250 kilodalton M_w marker is shown.</p

LRRK1 and LRRK2 bind guanine nucleotides.

<p>(A) Nucleotide competition assays with purified 3xFLAG-LRRK1/2 bound to M2-Flag affinity resin and incubated with a fixed concentration of GTP-α-P³³ (10 nM) in the presence of 100 µM of cold nucleotides. Graph shows that loaded GTP- α-P³³ is outcompeted by guanine nucleotides but not by ATP or CTP. (B) Nucleotide competition assays with proteins incubated with a fixed concentration of GTP- α-P³³ (10 nM) and varying concentrations of cold GTP. Competition curves with GTP were used to generate IC50 values, which are apparent dissociation constants.</p

Analysis of LRRK1 and LRRK2 structure by transmission electron microscopy (TEM).

<p>Distributions of gold particle distances of double-gold labeled LRRK1 (A) and LRRK2 (B) particles and representative images. Particles were stained with uranyl acetate and subsequently labeled with primary anti-Flag (M2) antibody and secondary 5 nm gold-labeled secondary anti-mouse antibody. (C and D) Distributions of particles diameters of purified LRRK1 and LRRK2 negative stained with uranyl acetate and representative images of protein shapes. (E) Scatter plots of gold distances measured for double-gold labeled LRRK1 and LRRK2. Distributions are significantly different as assessed by t-test (**, <i>P</i> = 0.001). (F) Scatter plots of particle size for LRRK1 and LRRK2. Distribution of LRRK1 and LRRK2 are significantly different by t-test (***, <i>P</i><0.0001).</p

Characterization of HEK293T cell line stably expressing 3xFlag-LRRK1 and LRRK2.

<p>(A) Schematic alignment of LRRK1 and LRRK2. Predicted functional domains are drawn to scale at the relative location within the full protein sequence. For domains containing repeat sequences, predicted individual repeat units are depicted. The sequence identity and similarity for the LRR, ROC, COR and Kinase domains are given below the schematic. Also given are detailed alignments of LRRK1 and LRRK2 at the level of common LRRK2 clinical mutations. Abbreviations for the domains: ARM, armadillo repeat domain; ANK, ankyrin repeat domain; LRR, leucine rich repeat domain; ROC, Ras of comple proteins domain; COR, C-terminal of ROC domain; Kin, kinase domain; WD40, WD40 repeat domain. (B) Representative western blot analysis of HEK293T cells stably expressing (from lane 1 to 7) 3xFlag-tagged LRRK2 wild-type, T1348N GTP deficient binding mutant, K1906M kinase dead, G2019S pathogenic mutant and LRRK1 wild-type K650A GTP deficient binding mutant, K1269M kinase dead. Upper panel shows membranes probed with Flag (M2) antibody (note that LRRK2 and LRRK1 have different exposure time due to the very low expression of T1348N mutant). Lower panel shows β-tubulin loading control. (C) Representative confocal images of stable HEK293T cells expressing LRRK1 and LRRK2 wild-type and mutants. Scale bar 10 µm.</p

Purification of soluble full-length 3xFlag-LRRK1 and 3xFlag-LRRK2.

<p>(A) Representative silver staining of purified 3xFlag-LRRK1 and LRRK2 purification indicates highly pure protein fractions. Markers are in kilodaltons (B) Circular dichroism analysis of purified 3xFlag LRRK1 and LRRK2. Representative spectra are reported as mean residue molar ellipticity (deg cm² dmol⁻¹). (C) Representative fluorescence spectra of purified LRRK1 (right) and LRRK2 (left) before (solid line) and after (dashed line) addition of 6M GdHCl using an excitation wavelength of 280 nm. Fluorescence intensity was normalized to the highest peak.</p

Analysis of different LRRK2 variants by immunogold EM reveals existence of dimeric proteins.

<p>Distributions of gold particle distances of gold-labeled LRRK2 wild-type (A), G2019S pathological mutant (B), kinase dead K1906M (C) and GTP deficient binding mutant T1348N (D). Distances between particles were measured within 200 nm and weighted by the area of the annulus of thickness correspondent to the bin size (2.5 nm). See the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043472#s4" target="_blank">materials and methods</a> section for a more detailed explanation of the analysis.</p

Additional file 1: Figure S1. of Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation

Distinct pathogenic LRRK2 mutants cause deficits in centrosome cohesion in transfected HEK293T cells. (DOCX 1160 kb

Additional file 3: Figure S3. of Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation

LRRK2 phosphorylates Rab8a at T72, and phosphomimetic mutants do not display altered nucleotide binding or retention. (DOCX 1005 kb

Additional file 5: Figure S5. of Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation

Golgi dispersal/disruption has no effect on LRRK2-mediated pericentrosomal/centrosomal accumulation of Rab8a. (DOCX 1670 kb