Fig. 2: The influence of valency and linker type on LLPS and liquid properties of CC-LLPS condensates in mammalian cells.
From: Programmable de novo designed coiled coil-mediated phase separation in mammalian cells
a NIH-3T3 cells expressing pairs of proteins with different numbers of CC repeats. From left to right, 6 CC, followed by 4, and 2 CC modules tagged with Cit. The second protein is labeled with TagBFP, with repeats of S2 and S4, interspersed with gs linker; from top to bottom 6, 4, and 2 CC segments. b Schematic representation of CC segments needed in each chain for the formation of condensates, green dots—condensates, empty circles—no condensates. c NIH-3T3 cells expressing mCit with three repeats of interchanged S1 and S3 CC, connected with SAH linkers and TagBFP with segregated three repeats of S2, and S4 CC, connected with a GS linker (same as in a) top left), separately for each channel and merged. d Fluorescence intensity profile showing colocalization of both fluorescent signals (mCit and BFP). Line scan of the red line in cE) from point A to B. e Fluorescence confocal images of condensates undergoing fusion. The red arrows indicate the locations of fusions. Insets from Supplementary Movie 1. Merge of the cyan and yellow channel. Scale bars, 10 μm. f FRAP analysis of the two proteins required for condensate formation. The plots show the normalized recovery after photobleaching for each protein mCit-(S1-S3)3-SAH left yellow and TagBFP-(S2)3(S4)3-gs right blue graph. Data are presented as mean ± SD (n = 12 for mCit and n = 8 for BFP). Source data are provided as a Source Data file. Fluorescence confocal images of bleached condensate are presented under the plot, with the acquisition time. g, h Cells expressing a combination of proteins, both containing a gs linker (g) or a SAH linker (h), each with 6 CC modules in the same arrangement as a) top left. Merge of both channels. Scale bars, 10 μm.
