Bimolecular fluorescence complementation (BiFC) was introduced a decade ago as a method to monitor alpha-synuclein (α-syn) oligomerization in intact cells. Since then, several α-syn BiFC cellular assays and animal models have been developed based on the assumption that an increase in the fluorescent signal correlates with increased α-syn oligomerization or aggregation. Despite the increasing use of these assays and models in mechanistic studies, target validation and drug screening, there have been no reports that 1) validate the extent to which the BiFC fluorescent signal correlates with α-syn oligomerization at the biochemical level; 2) provide a structural characterization of the oligomers and aggregates formed by the BiFC fragments; or 3) investigate the extent to which the oligomers of the fluorescent complex resemble oligomers formed on the pathway to α-syn fibrillization. To address this knowledge gap, we first analysed the expression level and oligomerization properties of the individual constituents of α-syn-Venus, one of the most commonly used BiFC systems, in HEK-293 & SH- SY5Y cells from three different laboratories using multiple approaches, including size exclusion chromatography, semiquantitative Western blot analysis, in-cell crosslinking, immunocytochemistry and sedimentation assays. Next, we investigated the biochemical and aggregation properties of α-syn upon co-expression of both BiFC fragments. Our results show that 1) the C-terminal-Venus fused to α-syn (a-syn-Vc) is present in much lower abundance than its counterpart with N-terminal-Venus fused to α-syn (Vn-a-syn) ; 2) Vn-a-syn exhibits a high propensity to form oligomers and higher-order aggregates; and 3) the expression of either or both fragments does not result in the formation of α-syn fibrils or cellular inclusions. Furthermore, our results suggest that only a small fraction of Vn-a-syn is involved in the formation of the fluorescent BiFC complex and that some of the fluorescent signal may arise from the association or entrapment of a-syn-Vc in Vn-a-syn aggregates. The fact that the N- terminal fragment exists predominantly in an aggregated state also indicates that one must exercise caution when using this system to investigate α-syn oligomerization in cells or in vivo. Altogether, our results suggest that cellular and animal models of oligomerization, aggregation and cell-to-cell transmission that are based on the a-syn BiFC systems should be thoroughly characterized at the biochemical level to ensure that they reproduce the process of interest and measure what they are intended to measure.

中文翻译：

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使用双分子荧光互补测定法监控α-突触核蛋白的低聚和聚集：您所看到的并不总是所得到的。

双分子荧光互补（BiFC）是十年前引入的一种方法，用于监测完整细胞中的α-突触核蛋白（α-syn）寡聚。从那时起，基于荧光信号的增加与增加的α-syn寡聚化或聚集相关的假设，开发了几种α-synBiFC细胞分析和动物模型。尽管在机械研究，靶标验证和药物筛选中越来越多地使用这些测定法和模型，但尚未有报道1）在生物化学水平上验证BiFC荧光信号与α-syn寡聚相关的程度；2）提供由BiFC片段形成的低聚物和聚集体的结构表征; 或3）研究荧光复合物的低聚物与在α-syn原纤维化途径中形成的低聚物相似的程度。为了解决这一知识鸿沟，我们首先分析了来自三个不同实验室的HEK-293和SH-SY5Y细胞中α-syn-Venus（最常用的BiFC系统之一）的各个成分的表达水平和寡聚特性，方法，包括体积排阻色谱，半定量蛋白质印迹分析，细胞内交联，免疫细胞化学和沉降分析。接下来，我们研究了两个BiFC片段共表达时α-syn的生化和聚集特性。我们的结果表明，1）与α-syn（a-syn-Vc）融合的C末端金星的丰度比与α-syn（Vn-a-syn）融合的N末端金星的对应物低得多）; 2）Vn-a-syn具有形成低聚物和高阶聚集体的高倾向性；3）任一或两个片段的表达都不会导致α-syn原纤维或细胞内含物的形成。此外，我们的结果表明，只有少量的Vn-a-syn参与荧光BiFC络合物的形成，并且某些荧光信号可能源于Vn-a中a-syn-Vc的缔合或截留。 -syn聚合。N-末端片段主要以聚集状态存在这一事实也表明，使用该系统研究细胞或体内α-syn寡聚时，必须谨慎行事。