Figure 1.
mRuby-PKR forms clusters in response to synthetic and natural inputs. (A) Representative time-lapse micrographs showing discrete mRuby-PKR clusters in H4 cells transfected with poly I:C. mRuby-PKR in clusters comprises 5.06% ± 0.2 of the total amount of protein (see Materials and Methods for details). Scale bar: 10 µm. (B) Violin plots showing the maximum number of mRuby-PKR clusters per cell in H4 cells transfected with poly I:C (N = 5 experiments, n > 200 cells). (C) Representative live-imaging time-lapse micrographs showing merging and segregation of mRuby-PKR clusters. Scale bar: 2 µm. (D) Representative immunofluorescence image showing that phosphorylated mRuby-PKR is enriched in clusters. Scale bar: 10 µm. (E) Representative immunofluorescence images of wild-type H4 cells treated with poly I:C and immunostained for endogenous PKR. (F) Representative micrograph showing formation of mRuby-PKR clusters in measles-infected (strain MVvac-CKO-GFP) H4 cells. The inset corresponds with the outlined cell in the high-magnification image. Note that the uninfected cell in the top left corner shows no mRuby-PKR clusters. Scale bar: 10 µm. (G) Quantification of de novo mRuby-PKR clustering frequency (red bars) and MVvac-CKO-GFP replication (GFP MFI and 95% confidence interval bands) as function of time. Red trace, nonlinear curve fit of the mRuby-PKR clustering frequency data. The percentage of infected cells that formed mRuby-PKR clusters was, on average, 45 ± 5% (N = 3 experiments, n > 400 cells). (H) Quantification of normalized mRuby-PKR cluster fluorescence intensity in 1,6-hexandiol–treated H4 cells. T0 corresponds to 60 min of poly I:C treatment. The data were binned and are shown as the mean and 95% confidence interval bands (n = 60 cells). The micrographs show representative images of mRuby-PKR cluster dissolution by 1,6-hexandiol treatment. Scale bar: 10 µm. (I) FRAP analysis showing the recovery of normalized fluorescence intensity of mRuby-PKR clusters. The data are shown as in H (N = 3 experiments, n = 30 cells). The micrographs show representative images of a single mRuby-PKR cluster photobleached with a 561-nm laser beam.

mRuby-PKR forms clusters in response to synthetic and natural inputs. (A) Representative time-lapse micrographs showing discrete mRuby-PKR clusters in H4 cells transfected with poly I:C. mRuby-PKR in clusters comprises 5.06% ± 0.2 of the total amount of protein (see Materials and Methods for details). Scale bar: 10 µm. (B) Violin plots showing the maximum number of mRuby-PKR clusters per cell in H4 cells transfected with poly I:C (N = 5 experiments, n > 200 cells). (C) Representative live-imaging time-lapse micrographs showing merging and segregation of mRuby-PKR clusters. Scale bar: 2 µm. (D) Representative immunofluorescence image showing that phosphorylated mRuby-PKR is enriched in clusters. Scale bar: 10 µm. (E) Representative immunofluorescence images of wild-type H4 cells treated with poly I:C and immunostained for endogenous PKR. (F) Representative micrograph showing formation of mRuby-PKR clusters in measles-infected (strain MVvac-CKO-GFP) H4 cells. The inset corresponds with the outlined cell in the high-magnification image. Note that the uninfected cell in the top left corner shows no mRuby-PKR clusters. Scale bar: 10 µm. (G) Quantification of de novo mRuby-PKR clustering frequency (red bars) and MVvac-CKO-GFP replication (GFP MFI and 95% confidence interval bands) as function of time. Red trace, nonlinear curve fit of the mRuby-PKR clustering frequency data. The percentage of infected cells that formed mRuby-PKR clusters was, on average, 45 ± 5% (N = 3 experiments, n > 400 cells). (H) Quantification of normalized mRuby-PKR cluster fluorescence intensity in 1,6-hexandiol–treated H4 cells. T0 corresponds to 60 min of poly I:C treatment. The data were binned and are shown as the mean and 95% confidence interval bands (n = 60 cells). The micrographs show representative images of mRuby-PKR cluster dissolution by 1,6-hexandiol treatment. Scale bar: 10 µm. (I) FRAP analysis showing the recovery of normalized fluorescence intensity of mRuby-PKR clusters. The data are shown as in H (N = 3 experiments, n = 30 cells). The micrographs show representative images of a single mRuby-PKR cluster photobleached with a 561-nm laser beam.

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