RNAi screening of Drosophila (Sophophora) melanogaster S2 cells for ricin sensitivity and resistance

Abstract

The ribosome-inhibiting toxin ricin binds exposed β1→4 linked galactosyls on multiple glycolipids and glycoproteins on the cell surface of most eukaryotic cells. After endocytosis, internal cell trafficking is promiscuous, with only a small proportion of ricin proceeding down a productive (cytotoxic) trafficking route to the endoplasmic reticulum (ER). Here, the catalytic ricin A chain traverses the membrane to inactivate the cytosolic ribosomes, which can be monitored by measuring reduction in protein biosynthetic capacity or cell viability. Although some markers have been discovered for the productive pathway, many molecular details are lacking. To identify a more comprehensive set of requirements for ricin intoxication, the authors have developed an RNAi screen in Drosophila S2 cells, screening in parallel the effects of individual RNAi treatments alone and when combined with a ricin challenge. Initial screening of 806 gene knockdowns has revealed a number of candidates for both productive and nonproductive ricin trafficking, including proteins required for transport to the Golgi, plus potential toxin interactors within the ER and cytosol.

Fig. 1.

Flies and S2 cells are sensitive to ricin. (A) Starved adult flies were fed with one meal of ricin at a range of concentrations in 1% sucrose, then fed daily with a maintenance diet of 1% sucrose, and scored for survival. (B). Dose response of S2 cells to a 24-h exposure to a range of concentrations of ricin. (C) Dose responses of S2 cells to a 24-h exposure to a range of concentrations of ricin in the presence of increasing concentrations of galactose.

Fig. 2.

Establishing screening conditions. (A) Mean viabilities of cells grown on the outer wells (outer, n = 36), remaining wells (inner, n = 60), or inner wells (n = 60) of a plate whose outer wells were occupied with water (inner plus moat). Bars, ±1 SD. (B) S2 cells were grown for 3 days in the presence or absence of RNAi against Tango7 and then treated for 24 h with a range of concentrations of ricin prior to the MTS assay. (C) Relative MTS signals of S2 cells treated with RNAi only.

Fig. 3.

Modeling the screen to determine ricin dose. Upper: S2 cells were treated with a range of ricin concentrations, and viability was measured by the MTS assay, generating a cytotoxicity curve (open circles). This curve was then shifted to the right or left to model likely results from 2-, 5-, or 10-fold shifts in sensitivity (2XS, 5XS, and 10XS sensitizing shifts; 2XP, 5XP, and 10XP protective shifts). Lower: Vertical slices (dotted lines, upper panel) were used to model expected results of treating with a range of concentrations of ricin. Arrows, magnitude of maximal protective (P) or sensitizing (S) effects.

Fig. 4.

Screening results. (A) Left panel: MTS signals from initial screening of 96 RNAi treatments are displayed as pairs of bars (white, RNAi treatment alone; black, RNAi treatment with subsequent ricin challenge). A number of candidate hits are highlighted (arrows). Right panel: Corresponding ricin-treated controls (no RNAi treatment). (B) Signals from A plotted in scatter format, defining the sectors into which protective and sensitizing hits are likely to fall. (–), cells alone; (+), cells treated with ricin. (C) Standard z scores plotted versus relative growth of RNAi-treated cells for all 768 RNAi treatments tested. Gray circles: treatments rejected because the RNAi signal alone was 25% or less that of non-RNAi-treated cells. White circles: treatments rejected that lie within 1 SD from the mean value. Black circles: 45 remaining candidate treatments that may influence ricin cytotoxicity. (D) Testing alternative RNAi treatments: upper panels, RNAi treatments against PDI and β′-COP taken from B (RNAi 1, left) and from different RNAi treatments targeted against the same genes (RNAi 2, right); lower panels, RNAi 1 (left panel) from B against archipelago (arch) and an alternative RNAi 2 against the same gene (right panel). (E) Upper panel: Cell extracts (10 µg) of S2 cells treated or not (ctl) with torp4a RNAi were electrophoresed, immunoblotted, and probed for torp4a protein. *, cross-reacting protein. Approximate migration of size markers is shown on the left. Lower graph: S2 cells treated or not with torp4a RNAi were subsequently treated with ricin, and viabilities were determined by the MTS assay.