Plan apo 100x oil objective

For fluorescent imaging, all strains were grown to saturation in synthetic complete media with 2% glucose, diluted to OD₆₀₀ = 0.2, and induced for 6 hours with estradiol. All images were acquired with a Nikon Plan Apo 100x oil objective (NA 1.4) using a Nikon Eclipse Ti-E inverted microscope and a CCD camera (Andor technology). The fluorescent reporter strains were identical to those used in for the high content microscopy. Specifically, the Kar2_SS-msfGFP-HDEL gene was driven by the TDH3 promoter and integrated in single copy at the TRP1 locus. Both Kar2 and Scj1 were C-terminally fused to mNG at their endogenous locus. Total cellular GFP fluorescence was quantified manually using ImageJ. The fraction of cells with mislocalized GFP (i.e. GFP in the cytoplasm instead of the ER) was determined visually. For the experiments demonstrating STE24 rescue of the IAPP-induced phenotypes, STE24 was inserted with a single-copy integrating plasmid at the URA3 locus under the control of the pZ promoter. Due to the additional estradiol-inducible promoter binding site introduced by integrating STE24 in this manner, for the low toxicity experiments the concentration of estradiol used was double that of the screening experiments. For each data point, the fluorescence intensity of at least 100 cells was quantified using ImageJ. Experiment were performed in biological triplicate.

All strains were constructed in the using the W303 genetic background yeast strain. The EIFα-promoter driven ZEM was integrated at the YBR032W locus and pZ-driven SNCA or empty control vector was integrated at the HIS3 locus. The cells bearing an empty vector were differentiated from SNCA-expressing cells with the addition of a constitutively expressed blue fluorescent protein driven by the TDH3 promoter integrated at the LEU2 locus. For the co-culture experiments, cells harboring a single copy of SNCA were mixed at a 2:1 ratio with cells harboring a single copy of the empty plasmid. They were then grown for 6 hours from a starting density of OD600 = 0.08 in synthetically defined complete media containing 1 μM estradiol. All images were acquired with a Nikon Plan Apo 100x oil objective (NA 1.4) using a Nikon Eclipse Ti-E inverted microscope and a CCD camera (Andor technology).