ort Cathepsin K Synonyms membrane profiles in optical mid sections and as a network in cortical sections. In contrast, estradiol-treated cells had a peripheral ER that predominantly consisted of ER sheets, as evident from long membrane profiles in mid sections and strong membrane MAO-A list places in cortical sections (Fig 1B). Cells not expressing ino2 showed no change in ER morphology upon estradiol therapy (Fig EV1). To test no matter whether ino2 expression causes ER stress and may possibly in this way indirectly result in ER expansion, we measured UPR activity by suggests of a transcriptional reporter. This reporter is based onUPR response components controlling expression of GFP (Jonikas et al, 2009). Cell treatment with the ER stressor DTT activated the UPR reporter, as expected, whereas expression of ino2 did not (Fig 1C). Moreover, neither expression of ino2 nor removal of Opi1 altered the abundance from the chromosomally tagged ER proteins Sec63-mNeon or Rtn1-mCherry, although the SEC63 gene is regulated by the UPR (Fig 1D; Pincus et al, 2014). These observations indicate that ino2 expression doesn’t result in ER pressure but induces ER membrane expansion as a direct outcome of enhanced lipid synthesis. To assess ER membrane biogenesis quantitatively, we developed three metrics for the size in the peripheral ER in the cell cortex as visualized in mid sections: (i) total size of the peripheral ER, (ii) size of person ER profiles, and (iii) quantity of gaps among ER profiles (Fig 1E). These metrics are less sensitive to uneven image high quality than the index of expansion we had applied previously (Schuck et al, 2009). The expression of ino2 with distinctive concentrations of estradiol resulted inside a dose-dependent improve in peripheral ER size and ER profile size and a decrease in the number of ER gaps (Fig 1E). The ER of cells treated with 800 nM estradiol was indistinguishable from that in opi1 cells, and we made use of this concentration in subsequent experiments. These results show that the inducible system permits titratable control of ER membrane biogenesis without causing ER tension. A genetic screen for regulators of ER membrane biogenesis To identify genes involved in ER expansion, we introduced the inducible ER biogenesis system and the ER marker proteins Sec63mNeon and Rtn1-mCherry into a knockout strain collection. This collection consisted of single gene deletion mutants for many of the around 4800 non-essential genes in yeast (Giaever et al, 2002). We induced ER expansion by ino2 expression and acquired pictures by automated microscopy. According to inspection of Sec63mNeon in mid sections, we defined six phenotypic classes. Mutants were grouped in accordance with whether or not their ER was (i) underexpanded, (ii) properly expanded and hence morphologically typical, (iii) overexpanded, (iv) overexpanded with extended cytosolic sheets, (v) overexpanded with disorganized cytosolic structures, or (vi) clustered. Fig 2A shows two examples of each class. To refine the search for mutants with an underexpanded ER, we applied the threeFigure 1. An inducible technique for ER membrane biogenesis. A Schematic of your manage of lipid synthesis by estradiol-inducible expression of ino2. B Sec63-mNeon pictures of mid and cortical sections of cells harboring the estradiol-inducible technique (SSY1405). Cells had been untreated or treated with 800 nM estradiol for six h. C Flow cytometric measurements of GFP levels in cells containing the transcriptional UPR reporter. WT cells containing the UPR reporter (SSY2306), cells addition

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