Fig. 6: CaLB1 foci are resolved upon 1,6-hexanediol treatment. | Nature Communications

Fig. 6: CaLB1 foci are resolved upon 1,6-hexanediol treatment.

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

Fig. 6

a Localization of CaLB1-GFP under the native promoter in root epidermis cells treated with 1,6-hexanediol or 1,2,6-hexanetriol. The experiments were conducted three times, and representative images are shown. Scale bars: 10 µm. b Number of CaLB1-GFP-positive foci in (a). The experiments were conducted three times, and the mean for each experiment is shown [(n = 23 seedlings (untreated); n = 26 seedlings (1,6-hexanediol); n = 28 seedlings (1,2,6-hexanetriol)). untreated/1,6-hexandiol p = 0.000892 (***p < 0.001), untreated/1,2,6-hexanetriol p = 0.0333 (*0.01 < p < 0.05). Seedlings co-expressing UBQ10pro:mRFP-ATG8i and CaLB1pro: CaLB1-GFP (c) or ALIXpro: GFP-ALIX (d) were analyzed under a confocal microscope upon NaCl or NaCl and 1,6-hexanediol (1,6-HD) treatment. The experiment with CaLB1 and ALIX were conducted two and three times, respectively, and representative images are shown. Scale bars: 10 µm. e, f Quantification of the results in (c) and (d). p = 1.50 × 10−4, ***p < 0.001 (CaLB1-GFP) and p = 0.00186, **0.001 < p < 0.01 (GFP-ALIX), p = 0.998 (c, ATG8i) and 0.430 (d, ATG8i), not significant (ns, p > 0.05)]. In total, for +NaCl/+NaCl+1,6-HD, 249/128 foci in 4 roots were analyzed for CaLB1-GFP and ATG8i, 178/269 foci in 8 roots for CaLB1-GFP and ATG8i, for GFP-ALIX and ATG8i, 485/477 foci in 8 roots, and for GFP-ALIX and ATG8i, 220/570 foci in 8 roots. g CRISPR target [CRISPR(CaLB1)] and mutated target [CRISPR(mutCaLB)] sequences. h Protein extracts of protoplasts transformed with CaLB1pro:mRFP-CaLB1 alone or with CaLB1pro:mRFP-CaLB1 and CRISPR(CaLB1)/3xFLAG-Cas9 or CRISPR(mutCaLB)/3xFLAG-Cas9 were subjected to anti-FLAG and anti-RFP immunoblotting. Histon H3 was used as loading control. The experiment was conducted twice, and one representative result is shown. i Representative images of Arabidopsis root cell culture-derived protoplasts transformed with the CRISPR constructs shown in (g) co-transformed with GFP-ALIX. Scale bar: 10 µm. j Violin plot of the number of GFP-ALIX-positive foci per cell in (i) [n = 120 CRISPR(CaLB1), n = 111 CRISPR (mutCaLB1)]. CRISPR(CaLB1)/CRISPR(mutCaLB1): p = 5.69*10−7 (***p < 0.001). (b, e, f, h, j) Source data are provided as a Source Data file. b, e, f, j Box plot: center line, median; box limits, first and third quartiles; whiskers, 1.5x interquartile range. Two-tailed t-test, no equal variance.

Back to article page