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. 2019 Jan 16;8:e41415. doi: 10.7554/eLife.41415

Figure 1. Constitutive PB formation upon Pat1 overexpression is Dhh1 dependent. .

(A) Overexpression (OE) of Pat1 leads to constitutive PB formation but only in the presence of Dhh1. Cells co-expressing the indicated PB components were grown in synthetic complete (SC) raffinose media to exponential growth phase after which Pat1 was overexpressed via addition of galactose. Cells in both raffinose and galactose were observed by fluorescence microscopy. In all Pat1 OE strains the endogenous promoter of Pat1 was replaced by the galactose 1–10 promoter (p-GAL-PAT1). Scale bar: 5 µm. (B) Quantification of images in A depicting number of PBs/cell. N = 3 biological replicates with >800 cells/replicate. Error bars: SEM. (C) Pat1-NC (AA 5–79 + 456–587) is functional in vivo. Cartoon of the Pat1-NC construct (see text for details). (D) PAT1-NC induces PB formation upon stress in vivo. Cells co-expressing the indicated the PB components in PAT1 full length or PAT1-NC background were grown in synthetic complete (SCD) media to exponential growth phase then shifted to glucose-rich or glucose-starvation conditions for 30 min and observed by fluorescence microscopy. PBs induced in the PAT1-NC background were dissolved by addition of 2% glucose demonstrating reversibility. Scale bar: 5 µm. (E) Quantification of images shown in A depicting number of PBs/cell. Bars: SEM. N = 3 biological replicates with >800 cells/replicate. (F) PAT1-NC (AA 5–79 + 456–587) OE leads to constitutive PB induction. p(GAL)-PAT1 or p(GAL)-PAT1-NC cells expressing Dhh1-GFP were grown in SC raffinose media to exponential growth phase after which Pat1 OE was induced with galactose. Scale bar: 5 µm. (G) Graph depicts Dhh1 PBs/cell, SEM. N = 3 biological replicates with >800 cells/replicate. [Diatrack 3.05 and cell segmentation using in house Matlab code was used for the quantification of PB and cell numbers respectively for all the experiments shown in the manuscript].

Figure 1.

Figure 1—figure supplement 1. Controls for Pat1 OE related to Figure 1 and growth rescue experiments of Pat1-NC.

Figure 1—figure supplement 1.

(A) Controls for the Pat1 OE, relating to Figure 1. Upper panel: PAT1 (endogenous promoter) cells expressing Dhh1-GFP were grown in (SC) raffinose media to exponential growth phase after which galactose was added and cells were imaged by fluorescence microscopy. Lower Panel: p(GAL)-HA pat1Δ (Hemagglutinin tag) OE does not lead to constitutive PB formation. Scale bar: 5 µm. (B) PAT1-NC rescues the pat1Δ growth phenotype. PAT1, pat1Δ and PAT1-NC cells were grown overnight in SCD media to saturation and the next day the different strains were inoculated to OD 0.1 in SCD. Absorbance based growth measurements were recorded for 24 hrs. The growth curve depicts O.D. 600 nm versus time. (C) PAT1-NC rescues the thermosensitive growth phenotype of pat1Δ. Cells expressing PAT1-NC, PAT1 and cells lacking PAT1 were grown to saturation in SCD media overnight and the next day spotted on YPD plates at the indicated temperatures. Growth was monitored after 2–3 days of incubation. The phenotype was confirmed with three biological replicates. (D) PAT1-NC fully rescues the growth phenotype in pat1Δ edc3Δ scd6Δ cells at 37°C. Cells expressing PAT1 edc3Δ scd6Δ, pat1Δ edc3Δ scd6Δ and PAT1-NC edc3Δ scd6Δ were grown to saturation in SCD media overnight and the next day spotted on YPD plates at the indicated temperatures. Growth was monitored after 2–3 days of incubation. The phenotype was confirmed with three biological replicates. (E) Pat1-NC localizes to PBs upon stress in vivo. Cells co-expressing Pat1-NC-GFP and Dcp2-mCherry as an additional PB marker were grown in SCD media to exponential growth phase then shifted to glucose-rich or glucose-starvation conditions for 30 min and observed by fluorescence microscopy. After visualization 2% final concentration of glucose was added for one hour to demonstrate reversibility and dissolution of Pat1-NC PBs. Scale bar: 5 µm.