Fig. 7: CaLB1 and ALIX assemble into condensates. | Nature Communications

Fig. 7: CaLB1 and ALIX assemble into condensates.

From: Arabidopsis CaLB1 undergoes phase separation with the ESCRT protein ALIX and modulates autophagosome maturation

Fig. 7

a Predicted IDRs are highlighted (red) in the AlphaFold models. red line, IUPred2 (long); gray line, ANCHOR2. b 2 µM of labelled ALIX was mixed with 2 µM of labelled CaLB1 (FL) or CaLB1(C2) to analyze condensate formation. The experiment was conducted twice, and one representative image is shown. Mean circularity±standard deviation is shown. For ALIX/CaLB1(FL), 488 and 323 condensates were measured, respectively. In ALIX/CaLB1(C2), only ALIX condensates (n = 397) were measured as the number of CaLB1(C2) condensates were low. Scale bars: 10 µm. Number (c) and size (d) of condensates in two experiments (n = 10 images), except for the combination CaLB1(C2) 2 µM + ALIX 2 µM for which 13 images from two experiments were analyzed. All results are shown. c p-values for 0.5 µM ALIX, CaLB1 0.5 µM/2 µM p = 7.89×10−5 (***p < 0.001); for 2 µM ALIX, CaLB1(FL) 0.5 µM/2 µM p = 0.537(ns: not significant); for 2 µM ALIX, CaLB1(FL) 2 µM/CaLB1(C2) 2 µM p = 0.0158 (*0.01 < p < 0.05); for CaLB1(FL) 0.5 µM, ALIX 0.5 µM/2 µM p = 0.658 (ns); for CaLB1(FL) 2 µM, ALIX 0.5 µM/2 µM p = 0.402 (ns); for CaLB1(FL) or CaLB1(C2) 2 µM with ALIX 2 µM; CaLB1(FL)/CaLB1(C2) p = 0.00151 (**0.001 < p < 0.01). d p-values for 0.5 µM ALIX, CaLB1 0.5 µM/2 µM p = 0.285 (ns); for 2 µM ALIX, CaLB1 0.5 µM/2 µM p = 5.98 × 10−40(***p < 0.001); for 2 µM ALIX, CaLB1 2 µM/CaLB1(C2) 2 µM p = 2.23 × 10−72 (***p < 0.001); for CaLB1 0.5 µM, ALIX 0.5 µM/2 µM p = 0.174 (ns); for CaLB1 2 µM, ALIX 0.5 µM/2 µM p = 7.88 × 10−4 (***p < 0.001). e, f FRAP analysis of condensates containing labeled ALIX and CaLB1. Photobleaching was performed either on ALIX (e) or CaLB1 (f). Arrowhead, bleached condensate. s: seconds. The experiment was conducted three times, and representative images are shown. g, h Quantification of the results in (e, f). The line shows the mean value of all measured condensates and error bands indicate standard deviation. On the fit mean data (R Square: 0.99), the τ1/2 values (half time of recovery) and the proportion of the mobile fraction were calculated. s: seconds. 6 and 8 condensates were analyzed for ALIX and CaLB1, respectively. c, d, g, h Source data are provided as a Source Data file. c, d Box plot: center line, median; box limits, first and third quartiles; whiskers, 1.5x interquartile range. Two-tailed t-test, no equal variance.

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