Fig 2: Separate OT organelle systems co-localize MCP, PylRS, tRNAPyl, and ribosomes, related to Figure 3(A) Overview cartoon of the constructs used. The lowercase “s” indicates that the PylRS and the MCP are expressed from two separate plasmids and not genetically fused.(B–D) HEK293T cells expressing the indicated OT organelle, tRNAPyl and a construct encoding NLS::EGFP39TAG::ms2 in the presence of the ncAA SCO-K. All images show a selected plane of a z stack. Scale bars: 20 μm.(B) IF staining against PylRS and FISH staining of tRNAPyl show strong tRNAPyl recruitment into membrane-associated OT organelles. Top to bottom: PylRS (magenta), tRNAPyl (yellow), merged (NLS::EGFP39TAG::ms2 in green).(C) IF staining against PylRS and RPL26L1 show that endogenous ribosomes co-localize with OT film-like organelles. Note that as RPL26L1 is an endogenous protein, also untransfected cells are stained. Top to bottom: PylRS (magenta), RPL26L1 (cyan), merged (NLS::EGFP39TAG::ms2 in green).(D) IF staining against PylRS and MCP (with an anti-HA antibody) show co-localization in OT organelles. Top to bottom: PylRS (magenta), MCP (yellow), merged (NLS::EGFP39TAG::ms2 in green).(E,F) HEK293T cells expressing the indicated OT organelle, tRNAPyl and a construct encoding EGFP39TAG::boxB in the presence of SCO-K. All images show a selected plane of a z stack. Scale bars: 20 μm.(E) IF staining against PylRS and λN22 (with an anti-Myc antibody) show co-localization in OT organelles. Left to right: PylRS (magenta), λN22 (yellow), merged (NLS::EGFP39TAG::boxB in green).(F) IF staining against PylRS and RPL26L1 show that endogenous ribosomes co-localize with membrane-associated OT organelles. Note that due to the abundance of ribosomes, a substantial staining outside of the OT organelles can be observed. Left to right: PylRS (magenta), RPL26L1 (cyan), merged (NLS::EGFP39TAG::boxB in green).

Fig 3: Synthetic OT film-like organelles efficiently recruit tRNAPyl and ribosomesHEK293T cells expressing the indicated OT organelle, tRNAPyl, and EGFP39TAG::ms2 in the presence of the ncAA SCO-K.(A) 3D-rendered images of IF staining against PylRS and FISH staining of tRNAPyl reveal strong tRNAPyl recruitment (see Figure S2A for raw images of individual planes). Top to bottom: PylRS (magenta), tRNAPyl (yellow), merged (NLS::EGFP39TAG::ms2 is shown in green in all panels). See Video S1 for corresponding animations. Scale bars, 10 μm.(B) Super-resolution microscopy confirms that tRNAPyl is localized to a fine layer at the respective membrane. Particularly for the PMPMCP system tRNAPyl appears restricted to a sub-resolution film of only a few tens of nanometers (FWHM, full width at half-maximum). Top to bottom: tRNAPyl stochastic optical reconstruction microscopy (STORM), magnified views of areas inside red boxes, and line scans corresponding to red lines. Scale bars, 1 μm.(C) 3D-rendered images of IF staining against PylRS and RPL26L1 (a ribosomal marker protein) showing endogenous ribosomes in MCP-based OT organelles (see Figure S2B for raw images of individual planes). Due to the abundance of ribosomes, staining outside of the OT organelles is also observed. Top to bottom: PylRS (magenta), RPL26L1 (cyan), merged (NLS::EGFP39TAG::ms2 is shown in green in all panels). Video S1 shows corresponding animations. Scale bars, 10 μm.See also Figures S3 and S4.

Fig 4: λN22-based synthetic organelles enrich tRNAPyl, mRNA::boxB, and cellular ribosomes, related to Figure 2CHEK293T cells expressing the indicated OT organelle, tRNAPyl and a construct encoding EGFP39TAG::boxB in presence of the ncAA SCO-K. All images show a selected plane of a z stack.(A) IF staining against PylRS and FISH staining of tRNAPyl shows strong recruitment of tRNAPyl into OT organelles. Left to right: PylRS (magenta), tRNAPyl (yellow), merged (NLS::EGFP39TAG::ms2 in green). Shown is a selected plane of a z stack. Scale bars: 20 μm.(B) 3D reconstruction corresponding to panel A. Left to right: PylRS (magenta), tRNAPyl (yellow), merged (NLS::EGFP39TAG::ms2 in green in all panels). See also Video S1 for the animated 3D reconstructions. Scale bars: 10 μm.(C) IF staining against PylRS and RPL26L1 shows that endogenous ribosomes co-localize with membrane-associated OT organelles. Note that due to the abundance of ribosomes, a substantial staining outside of the OT organelles can be observed. Left to right: PylRS (magenta), RPL26L1 (cyan), merged (NLS::EGFP39TAG::ms2 in green). Shown is a selected plane of a z stack. Scale bars: 20 μm.(D) 3D reconstruction corresponding to panel C. Left to right: PylRS (magenta), RPL26L1 (cyan), merged (NLS::EGFP39TAG::ms2 in green in all panels). See also Video S1 for the animated 3D reconstructions. Scale bars: 10 μm.(E) IF staining against PylRS and FISH staining of mRNA::boxB show recruitment of boxB-tagged mRNA into λN22-based OT organelles. Left to right: PylRS (magenta), mRNA::boxB (yellow), merged (NLS::EGFP39TAG::ms2 in green). Scale bars: 20 μm.