Figure 3.

Phase-separating assemblers are essential for the function of most OT film-like organelles

(A) Schematic overview of constructs that target PylRS and/or MCP with or without FUS/EWSR1 individually to the four membranes. The lowercase “s” indicates that PylRS and MCP are not genetically fused and expressed from separate plasmids.

(B) FFC analysis of HEK293T cells expressing the dual-color ms2 reporter (EGFP^39TAG, mCherry^190TAG::ms2), tRNA^Pyl, and a respective indicated OT organelle in presence of SCO-K. Figure S1A shows further controls. The black lines in the FFC dot plots indicate the EGFP signal cut-off used to identify transfected cells, the red dotted lines demarcate background expression (in absence of MCP) for each targeted membrane.

(C) Bar plot corresponding to FFC experiments shown in (B). The dark gray bars show the fold-change selectivity, the light gray bars represent the relative efficiency (see Figure 2B for the respective cytoplasmic PylRS control). Bar graphs show the mean value of at least three independent experiments; error bars represent the SD.

(D) FFC analysis of separate OT organelle constructs. HEK293T cells expressing the dual color ms2 reporter (EGFP^39TAG, mCherry^190TAG::ms2), tRNA^Pyl, a respective PylRS OT organelle construct, and either no MCP or MCP targeted to each respective membrane. The sums of at least three independent experiments are shown. The black lines in the FFC dot plots indicate the EGFP signal cut-off used to identify transfected cells.

(E and F) Heatmaps calculated from FFC data shown in (D) for visualizing the fold change selectivity (E) and the relative efficiency (F) of individual co-expressed constructs. The mean values of all experiments are shown. The upper halves of the heatmaps correspond to the data shown in (D). The lower halves of the heatmaps show corresponding data for the dual color boxB-reporter (EGFP^39TAG, mCherry^190TAG::boxB), tRNA^Pyl, a respective PylRS fusion and either no λ_N22 construct (row 10) or the λ_N22 peptides targeted to the indicated membrane (see Figures S1B for corresponding FFC dot plots and S4 for associated imaging experiments). White stars indicated when MCP/λ_N22 and PylRS are targeted to the same membrane.

See also Figure S6.