Reference: Bircham PW, et al. (2011) Secretory pathway genes assessed by high-throughput microscopy and synthetic genetic array analysis. Mol Biosyst 7(9):2589-98

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Abstract


We developed a procedure for automated confocal microscopy to image the effect of the non-essential yeast gene deletion set on the localisation of the plasma membrane GFP-labelled protein Mrh1p-GFP. To achieve this it was necessary to devise an expression system expressing Redstar2 RFP-fluorescence specifically in the nucleus, mCherry RFP at a lower intensity in the cytoplasm and Mrh1p-GFP in the plasma membrane. This fluorescence labelling scheme utilising specifically designed image analysis scripts allowed automated segmentation of the cells into sub-regions comprising nuclei, cytoplasm and cell-surface. From this high-throughput high content screening approach we were able to determine that gene deletions including emc1?, emc2?, emc3?, emc4?, emc5? and emc6?, caused intracellular mislocalisation at the ER of a plasma membrane protein Mrh1p-GFP. CPY processing patterns were unaffected in these mutants and collectively our data suggest a transport role for the EMC genes within the early secretory pathway. HAC1 is central to the unfolded protein response (UPR) and in its absence, i.e. the absence of UPR, emc1?-, emc3?-, emc4?-, emc5?-hac1? double mutants were specifically hypersensitive to ER-stress (tunicamycin) lending credence to the usefulness of the high content microscope screening for discovery of functional effects of single mutants.

Reference Type
Journal Article
Authors
Bircham PW, Maass DR, Roberts CA, Kiew PY, Low YS, Yegambaram M, Matthews J, Jack CA, Atkinson PH
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