Journal
LAB ON A CHIP
Volume 14, Issue 20, Pages 3949-3957Publisher
ROYAL SOC CHEMISTRY
DOI: 10.1039/c4lc00756e
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Funding
- Fundacao para a Ciencia e Tecnologia (FCT) through the Associated Laboratory IN
- Fundacao para a Ciencia e Tecnologia (FCT) through the Associated Laboratory IMM
- FCT [SFRH/BD/73908/2010, SFRH/BPD/35767/2007]
- EMBO Installation Grant
- DFG Center for Nanoscale Microscopy and Molecular Physiology of the Brain
- [PTDC/BIA-BCM/117975/2010]
- Fundação para a Ciência e a Tecnologia [PTDC/BIA-BCM/117975/2010, SFRH/BPD/35767/2007, SFRH/BD/73908/2010] Funding Source: FCT
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Parkinson's disease (PD) is a common age-associated neurodegenerative disorder. The protein a-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 microftuldic 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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