Journal: PLOS Biology

Article Title: Polyphosphate modulates the stress-responsive formation of functional RNA-protein condensates in bacteria and mammalian cells

doi: 10.1371/journal.pbio.3003775

Figure Lengend Snippet: (A) Brightfield images of Hfq condensates reconstituted with the indicated Hfq concentrations in the presence or absence of 1× polyP-300 (in Pi units). Samples were prepared in 50 mM NaCl and 20 mM HEPES pH 7.0. (B) Phase diagram of Hfq and polyP-300 in the same buffer as (A). Formation of condensates was computationally determined by quantitative image analysis as detailed in the Methods section. Lines corresponding with specific polyP:Hfq ratios are highlighted. (C) Fluorescence and brightfield images of condensates reconstituted with 50 µM Hfq (supplemented with 4% Cy3-Hfq S65C) in the presence or absence of 1 µM FAM-labeled rA 30 or 100 µM AF647-labeled polyP-300. Samples were prepared in the same buffer as (A). (D) Electrophoretic mobility shift assays (EMSA). Lanes 1–6: 25 µM Hfq with 0, 0.5, 1, 2, 3, 4 µM FAM-rA 30 . Lanes 7–10 and lanes 11–14: 25 µM Hfq with 1 µM or 4 µM FAM-rA 30 , respectively, +25, 62.5, 125, 250 µM AF647-polyP300. Lane 15: 25 µM Hfq + 125 µM AF647-polyP300. A representative gel ( n = 2) is shown. Hfq was stained with Coomassie blue. Hfq-polyP-RNA complexes indicated by vertical arrowheads. (E) Left panel: Native western blot of bacterial lysates from MG1655 hfq::hfq-mCherry WT and Δ ppk at N+ and N-24 using antibodies against mCherry. Right panel: UV-bleached DAPI stain of native gel shown in left panel. PolyP is shown as dark areas. (F) Left panel: Native western blot of cell lysates from N-24 MG1655 hfq::hfq-mCherry WT (MG1655) using antibodies against mCherry. Lysates were treated with the indicated enzymes for 2 h prior to electrophoresis. Right panel: UV-bleached DAPI stain of native gel as shown in left panel. (G) Native western blot of N+ and N-24 lysates from hfq::hfq-mCherry rne::rne-mTQ2 WT (MG1655) relative to Δ ppk using antibodies against mCherry (Hfq) and GFP (RNase E). N-24 samples were left untreated (−) or digested with Ppx for 2 h (+) prior to electrophoresis. (H) Subcellular localization of Hfq-mCherry and RNase E-mTQ2 in N24 WT (MG1655) or Δ ppk . Scale bars: 5 µm. (I) Distribution of log2 ratios of RNase E intensities in Hfq foci (see Methods for details) vs. cellular background. Each data point shows a separate imaging field taken across two separate biological replicates. Scale bars: 5 µm. All underlying data can be found in .

Article Snippet: HfqS65C was labeled with Cy3 Maleimide Mono-Reactive Dye (Amersham) following the manufacturer’s protocol.

Techniques: Fluorescence, Labeling, Electrophoretic Mobility Shift Assay, Staining, Western Blot, Electrophoresis, Imaging