化学
细胞生物学
人口
蛋白质聚集
生物物理学
酿酒酵母
蛋白质稳态
绿色荧光蛋白
细胞
α-突触核蛋白
抗坏血酸
酵母
生物化学
生物
基因
病理
医学
帕金森病
疾病
环境卫生
食品科学
作者
João Fernandes,Sandra Tenreiro,Andreia Gameiro,V. Chu,Tiago F. Outeiro,J.P. Conde
出处
期刊:Lab on a Chip
[The Royal Society of Chemistry]
日期:2014-01-01
卷期号:14 (20): 3949-3957
被引量:33
摘要
Parkinson's disease (PD) is a common age-associated neurodegenerative disorder. The protein α-synuclein (aSyn) is a key factor in PD both due to its association with familial and sporadic cases and because it is the main component of the pathological protein aggregates known as Lewy bodies. However, the precise cellular effects of aSyn aggregation are still elusive. Here, we developed an elastomeric microfluidic device equipped with a chemical gradient generator and 9 chambers containing cell traps to study aSyn production and aggregation in Saccharomyces cerevisiae. This study involved capturing single cells, exposing them to specific chemical environments and imaging the expression of aSyn by means of a GFP fusion (aSyn-GFP). Using a galactose (GAL) gradient we modulated aSyn expression and, surprisingly, by tracking the behavior of single cells, we found that the response of individual cells in a population to a given stimulus can differ widely. To study the combined effect of environmental factors and aSyn expression levels, we exposed cells to a gradient of FeCl3. We found a dramatic increase in the percentage of cells displaying aSyn inclusions from 27% to 96%. Finally, we studied the effects of ascorbic acid, an antioxidant, on aSyn aggregation and found a significant reduction in the percentage of cells bearing aSyn inclusions from 87% to 37%. In summary, the device developed here offers a powerful way of studying aSyn biology with single-cell resolution and high throughput using genetically modified yeast cells.
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