Secretory pathway genes assessed by high-throughput microscopy and synthetic genetic array analysis Article

Bircham, PW, Maass, DR, Roberts, CA et al. (2011). Secretory pathway genes assessed by high-throughput microscopy and synthetic genetic array analysis . 7(9), 2589-2598. 10.1039/c1mb05175j

cited authors

  • Bircham, PW; Maass, DR; Roberts, CA; Kiew, PY; Low, YS; Yegambaram, M; Matthews, J; Jack, CA; Atkinson, PH

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 emc1Δ, emc2Δ, emc3Δ, emc4Δ, emc5Δ and emc6Δ, 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, emc1Δ-, emc3Δ-, emc4Δ-, emc5Δ- hac1Δ 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. © The Royal Society of Chemistry 2011.

publication date

  • September 1, 2011

Digital Object Identifier (DOI)

start page

  • 2589

end page

  • 2598

volume

  • 7

issue

  • 9