Mapping of Rab32 binding motif within LRRK2 aminoterminus.

<p>After co-transformation of the yeast strain Y190 with the indicated plasmids with and without the hypothetical coiled-coil motif, cells were grown on synthetic media lacking histidine, supplemented with 30 mM 3 AT. Cell growth on these media and blue staining in subsequent β-gal-filter assays indicated direct interaction of the proteins.</p><p>no growth on selection media or staining in ß-galactosidase filter assay, + growth on selection media and blue staining in in ß-galactosidase filter assay. n = 3 independent experiments.</p><p>Mapping of Rab32 binding motif within LRRK2 aminoterminus.</p

Co-localization analysis of LRRK2-GFP and DsRed-Monomer-Rab32.

<p>(A) NIH3T3 cells were co-transfected with plasmids encoding DsRed-Monomer-Rab32 wt and LRRK2-GFP. Living cells were imaged using a Zeiss LSM5 live microscope. The image shows a still frame from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111632#pone.0111632.s008" target="_blank">Movie S1</a>. Scale bar = 10 µm. (B) NIH3T3 cells were co-transfected with plasmids encoding DsRed-Monomer-Rab32 Q85L and LRRK2-GFP. After 48 hours the cells were fixed in 4% PFA and subsequently analyzed with a laser scanning microscope. Scale bar = 10 µm. (C) Co-transport and sorting of LRRK2-GFP by DsRed-Monomer-Rab32 wt. Image series are a detail view from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111632#pone.0111632.s009" target="_blank">Movie S2</a>. Both channels were recorded simultaneously every second. Scale bar = 2 µm. (D and E) NIH3T3 cells expressing either DsRed-Monomer-Rab32 wt or LRRK2-GFP were fixed and subsequently subjected to immunofluorescence labeling of β-tubulin (D) or the Golgi marker Rab6A (E). Scale bar = 10 µm.</p

Co-localization analysis of Rab32 wt and the constitutively active mutant Rab32 Q85L with different endosomal markers.

<p>(A) NIH3T3 cells were co-transfected with plasmids encoding the recycling endosome marker GFP-Rab11B and DsRed-Monomer-Rab32 wt or DsRed-Monomer-Rab32 Q85L, fixed and analyzed by fluorescence microscopy. Scale bar = 10 µm. (B) Cells were transfected with plasmids encoding for GFP-Rab32 wt or GFP-Rab32 Q85L followed by fixation and subsequent immunofluorescence staining of Rab7. Scale bar = 10 µm. (C–E) NIH3T3 cells expressing either GFP-Rab32 wt or GFP-Rab32 Q85L were fixed and stained for Rab7. (C) Microscopic analysis of GFP-Rab32 Q85L that co-localized with endogenous Rab7 (arrows) in the perinuclear area. Non co-localizing Rab7 was indicated by arrowheads. The perinuclear area was defined by the red (outlines nucleus) and the yellow line (outer border for perinuclear area). The image illustrates the cellular area used for the following analysis. (D) Rab7 perinuclear aggregates co-localizing with GFP-Rab32 wt or GFP-Rab32 Q85L and non co-localizing ones. ctrl. = control (untransfected IHKE-1 cells) (E) Quantification of perinuclear Rab7-positive structures. ctrl. (control): perinuclear Rab7 in untransfected cells; wt/Q85L: perinuclear Rab7 co-localizing with GFP-Rab32 constructs. control/Rab32 wt/Rab32 Q85L: n = 1660/18/167 structures in 67/18/60 cells, 1/1/3 independent experiments. Statistical significance was tested by a Student's T-test: p_control-wt = 0.02; p_control-Q85L<0.005; p_wt-Q85L = 0.02. Scale bar = 10 µm.</p

Binding of endogenous LRRK2 by the small GTPase Rab32.

<p>(A) GST-Rab32 wt, GST-Rab32 Q85L or GST as control was applied to glutathione agarose beads followed by incubation with NIH3T3 lysate overnight. Samples were analyzed by 6% SDS-PAGE and subsequent Western blot analysis to detect LRRK2. n≥3 independent experiments. (B) Lysates from IHKE-1 cells stably expressing GFP-Rab32 wt were incubated overnight with an anti-LRRK2 antibody (1E11). IP control = no antibody was added. Co-precipitated GFP-Rab32 wt was detected using an anti-Rab32 antibody. n = 3 independent experiments. (C) Lysates from IHKE-1 cells expressing GFP-Rab32 wt, GFP-Rab32 Q85L or GFP as control were subjected to immunoprecipitation by the GFP-Trap kit. Co-precipitated endogenous LRRK2 was detected using an anti LRRK2 antibody. n = 2 independent experiments.</p

LRRK2-GFP subcellular localization in DsRed-Monomer-Rab32 wt and DsRed-Monomer-Rab32 Q85L overexpressing NIH3T3 cells.

<p>(A) Graphical representation of the most common subcellular features of LRRK2-GFP in DsRed-Monomer-Rab32 wt or DsRed-Monomer-Rab32 Q85L expressing cells. 1: pericentriolar endosome; 2: transport vesicles; 3: perinuclear aggregates. (B–D) Microscopic analysis of NIH3T3 cells co-transfected with plasmids encoding DsRed-Monomer-Rab32 wt or DsRed-Monomer-Rab32 Q85L and LRRK2-GFP. In every cell the LRRK2-GFP channel was analyzed for the occurrence of the features transport vesicles, pericentriolar endosomes and perinuclear aggregates. (-) LRRK2-GFP alone: n = 71 cells from 5 independent experiments; LRRK2-GFP and DsRed-Monomer-Rab32 wildtype: n = 15 cells from 3 independent experiments, LRRK2-GFP and DsRed-Monomer-Rab32 Q85L: n = 21 cells from 5 independent experiments.</p

LRRK2 constructs used in this study.

<p>ARM: armadillo repeats; ANK: ankyrin repeats; LRR: leucine rich repeats; ROC: Ras of complex; COR: C-terminal of ROC; Kin: kinase domain; WD40: WD40 domain; c/c: coiled coil motif (aa 319–348).</p

A New Mint1 Isoform, but Not the Conventional Mint1, Interacts with the Small GTPase Rab6

<div><p>Small GTPases of the Rab family are important regulators of a large variety of different cellular functions such as membrane organization and vesicle trafficking. They have been shown to play a role in several human diseases. One prominent member, Rab6, is thought to be involved in the development of Alzheimer’s Disease, the most prevalent mental disorder worldwide. Previous studies have shown that Rab6 impairs the processing of the amyloid precursor protein (APP), which is cleaved to β-amyloid in brains of patients suffering from Alzheimer’s Disease. Additionally, all three members of the Mint adaptor family are implied to participate in the amyloidogenic pathway. Here, we report the identification of a new Mint1 isoform in a yeast two-hybrid screening, Mint1 826, which lacks an eleven amino acid (aa) sequence in the conserved C-terminal region. Mint1 826, but not the conventional Mint1, interacts with Rab6 via the PTB domain. This interaction is nucleotide-dependent, Rab6-specific and influences the subcellular localization of Mint1 826. We were able to detect and sequence a corresponding proteolytic peptide derived from cellular Mint1 826 by mass spectrometry proving the absence of aa 495–505 and could show that the deletion does not influence the ability of this adaptor protein to interact with APP. Taking into account that APP interacts and co-localizes with Mint1 826 and is transported in Rab6 positive vesicles, our data suggest that Mint1 826 bridges APP to the small GTPase at distinct cellular sorting points, establishing Mint1 826 as an important player in regulation of APP trafficking and processing.</p></div

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 6: Figure S6. of Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation

Rab8a protein levels and pericentrosomal/centrosomal accumulation of phosphorylated Rab8a in lymphoblasts from control and G2019S mutant LRRK2 PD patients. (DOCX 636 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