Abstract
Mutations in human leucine-rich repeat kinase 2 (Lrrk2), a protein of yet unknown function, are linked to Parkinson's disease caused by degeneration of midbrain dopaminergic neurons. The protein comprises several domains including a GTPase and a kinase domain both affected by several pathogenic mutations. To elucidate the molecular interaction network of endogenous Lrrk2 under stoichiometric constraints, we applied QUICK (quantitative immunoprecipitation combined with knockdown) in NIH3T3 cells. The identified interactome reveals actin isoforms as well as actin-associated proteins involved in actin filament assembly, organization, rearrangement, and maintenance, suggesting that the biological function of Lrrk2 is linked to cytoskeletal dynamics. In fact, we demonstrate Lrrk2 de novo binding to F-actin and its ability to modulate its assembly in vitro. When tested in intact cells, knockdown of Lrrk2 causes morphological alterations in NIH3T3 cells. In developing dopaminergic midbrain primary neurons, Lrrk2 knockdown results in shortened neurite processes, indicating a physiological role of Lrrk2 in cytoskeletal organization and dynamics of dopaminergic neurons. Hence, our results demonstrate that molecular interactions as well as the physiological function of Lrrk2 are closely related to the organization of the actin-based cytoskeleton, a crucial feature of neuronal development and neuron function.
Highlights
Lrrk2 encodes a large multidomain protein (ϳ286 kDa) belonging to the ROCO protein superfamily [4], comprising a characteristic combination of a ROC (Ras of complex protein) domain adjacent to a COR (C-terminal of ROC) domain [11], 1 The abbreviations used are: PD, Parkinson’s disease; ANOVA, analysis of variance; co-IP, co-immunoprecipitation; DA, dopaminergic; day in vitro (DIV), days in vitro; F-actin, filamentous actin; G-actin, globular actin; Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; Lrrk2, leucine-rich repeat kinase 2; Oas1, 2Ј-5Ј-oligoadenylate synthetase; P:A ratio, perimeter to area ratio; PIE, physical interaction enrichment; protein-protein interactions (PPIs), protein-protein interaction; QUICK, quantitative immunoprecipitation combined with knockdown; RNAi, RNA interference; SB, storage buffer; shRNA, short hairpin RNA; SILAC, stable isotope labeling by amino acids in cell culture; TH, tyrosine hydroxylase; TH-ir, TH-immunoreactive; ventral mesencephalic (VM), ventral mesencephalon; wt, wildtype
Lrrk2 Interacts with Components of the Actin Cytoskeleton—To identify Lrrk2 interacting proteins we conducted the QUICK approach [22], which combines SILAC, RNAi-induced knockdown, co-IP and quantitative MS to screen for endogenous PPIs
To test whether the knockdown of Lrrk2 itself would lead to global changes in the proteome of NIH3T3 cells, whole cell lysates were prefractionated by SDSPAGE, proteins were subjected to tryptic in-gel cleavage and the resulting peptides were analyzed by Liquid Chromatography (LC)-MS/MS (Supplemental Table 1)
Summary
Physiological relevance of the hypothesis was verified by both F-actin cosedimentation assays, determining the ability of Lrrk2 to bind F-actin and to modulate its assembly in vitro, and cellular assays, analyzing the morphology of NIH3T3 cells and DA neurons within primary VM cultures upon lentiviralmediated knockdown of Lrrk2. To test whether the knockdown of Lrrk2 itself would lead to global changes in the proteome of NIH3T3 cells, whole cell lysates were prefractionated by SDSPAGE, proteins were subjected to tryptic in-gel cleavage and the resulting peptides were analyzed by LC-MS/MS (Supplemental Table 1).
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