| Autor: |
Bircham PW; Department of Biological Sciences, Victoria University of Wellington, Kelburn Parade, Wellington, New Zealand., Maass DR, Roberts CA, Kiew PY, Low YS, Yegambaram M, Matthews J, Jack CA, Atkinson PH |
| Jazyk: |
angličtina |
| Zdroj: |
Molecular bioSystems [Mol Biosyst] 2011 Sep; Vol. 7 (9), pp. 2589-98. Date of Electronic Publication: 2011 Jul 06. |
| DOI: |
10.1039/c1mb05175j |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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