Department of Cell and Molecular Biology, University of Hawaii at Manoa, 651 Ilalo Street, Honolulu, HI 96813, USA.
Molecules. 2020 May 31;25(11):2561. doi: 10.3390/molecules25112561.
Mutations within the gene are the most common genetic cause of autosomal and sporadic Parkinson's disease (PD). LRRK2 is a large multidomain kinase that has reported interactions with several membrane proteins, including Rab and Endophilin, and has recently been proposed to function as a regulator of vesicular trafficking. It is unclear whether or how the spatiotemporal organization of the protein is altered due to LRRK2 activity. Therefore, we utilized fluctuation-based microscopy along with FLIM/FRET to examine the cellular properties and membrane recruitment of WT LRRK2-GFP (WT) and the PD mutant G2019S LRRK2-GFP (G2019S). We show that both variants can be separated into two distinct populations within the cytosol; a freely diffusing population associated with monomer/dimer species and a slower, likely vesicle-bound population. G2019S shows a significantly higher propensity to self-associate in both the cytosol and membrane regions when compared to WT. G2019S expression also resulted in increased hetero-interactions with Endophilin A1 (EndoA1), reduced cellular vesicles, and altered clathrin puncta dynamics associated with the plasma membrane. This finding was associated with a reduction in transferrin endocytosis in cells expressing G2019S, which indicates disruption of endocytic protein recruitment near the plasma membrane. Overall, this study uncovered multiple dynamic alterations to the LRRK2 protein as a result of the G2019S mutation-all of which could lead to neurodegeneration associated with PD.
基因内的突变是常染色体和散发性帕金森病（PD）的最常见遗传原因。LRRK2 是一种大型多功能激酶，据报道与几种膜蛋白（包括 Rab 和 Endophilin）相互作用，最近被提议作为囊泡运输的调节剂。尚不清楚由于 LRRK2 活性，蛋白质的时空组织是否以及如何改变。因此，我们利用基于波动的显微镜以及 FLIM/FRET 来检查 WT LRRK2-GFP（WT）和 PD 突变体 G2019S LRRK2-GFP（G2019S）的细胞特性和膜募集。我们表明，这两种变体都可以在细胞质中分为两个不同的群体；与单体/二聚体物种相关的自由扩散群体和较慢的，可能与囊泡结合的群体。与 WT 相比，G2019S 在细胞质和膜区域中都表现出更高的自缔合倾向。G2019S 的表达还导致与 Endophilin A1（EndoA1）的异源相互作用增加，细胞内囊泡减少以及与质膜相关的网格蛋白斑点动力学改变。这一发现与表达 G2019S 的细胞中转铁蛋白内吞作用减少有关，这表明质膜附近的内吞蛋白募集受到破坏。总的来说，这项研究揭示了 G2019S 突变导致 LRRK2 蛋白的多种动态改变-所有这些都可能导致与 PD 相关的神经退行性变。
