Fig. 4: CC-LLPS condensates from a single polypeptide chain type comprising homodimeric CC modules of the opposite pairing orientation.
From: Programmable de novo designed coiled coil-mediated phase separation in mammalian cells
a Two pairs of 3-heptad CC homodimers. APHh3 (orange) pairs in an antiparallel orientation, while GCNh3 (green) pairs in a parallel orientation. Right, representations of polypeptide chains of different lengths and arrangements of CCs connected by gs linkers. b–d HEK293T cells, expressing protein composed of mCitrine fused to different numbers and arrangements of antiparallel (A) and parallel (P) segments, with gs linkers. Scale bar, 10 μm. b APPAP arrangement of 5 CC, (c) segregated arrangement, six P followed by four As, and (d) APPAPAPPAP arrangement comprising 10 CC segments. e–g Snapshot at 20 μs from simulation for (e) APPAP arrangement, (f) PPPPPPAAAA and, (g) APPAPAPPAP arrangement at 310. h FRAP analysis of mCitrine-APPAPAPPAPh3 protein forming condensates. Relative recovery ratio, represented by the normalized recovery (n = 17 condensates from different cells examined over 3 independent experiments), of fluorescence intensity following photobleaching. The bleached region of the condensate was imaged every 1.3 s, and the plot shows the mean ± standard deviation of the normalized recovery over time. Confocal microscopy images of the bleached condensate are presented beneath the plot, with the acquisition time indicated. Scale bar 5 µm. Source data are provided as a Source Data file. i Fluorescence confocal images of condensates undergoing fusion, insets from Supplementary Movie 3. The red arrows indicate the locations of fusions. Scale bar, 10 μm. j Mean squared displacement analysis of simulated APPAPAPPAP molecules. Lines are mean, and the shaded regions are the standard deviation (n = 3 simulation replicates).
