Fig. 8: CaLB1 modulates autophagosome maturation. | Nature Communications

Fig. 8: CaLB1 modulates autophagosome maturation.

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

Fig. 8

a calb1-1 seedlings with CaLB1pro: CaLB1-GFP were treated for 2 days with 100 mM NaCl before the addition of 100 µM E-64d and autophagic bodies were stained with MDC. Rectangle: magnified region. The experiment was conducted twice, and one representative image is shown. Scale bars: 10 µm and 2 µm (magnification). b Seedlings expressing CaLB1pro: CaLB1-GFP and ALIXpro: GFP-ALIX were treated with either 150 mM NaCl or 150 mM NaCl and 100 µM E-64d for 2 h. Total extracts were subjected to immunoblotting with anti-GFP and anti-NBR1 antibodies. UGPase was used as loading control. The experiment was conducted three times, and one representative result is shown. Filled arrowhead: position of CaLB1-GFP or ALIX-GFP, open arrowhead: position of GFP. c The ratio of the intensities [NaCl+E-64d/NaCl] was calculated on three immunoblots conducted as in (b) and all values are shown. NBR1/CaLB1-GFP p = 0.0474 (*0.01p < 0.05), NBR1/GFP-ALIX p = 0.228 (ns: not significant, p > 0.05). d Seedlings expressing UBQ10pro: GFP-ATG8a were incubated with 150 mM NaCl for 2 h and root epidermis cells were analyzed using a confocal microscope. The experiment was conducted three times, and one representative image is shown. Scale bars: 10 µm. e The number of GFP-ATG8a-positive foci +/−NaCl in (d) is shown in a box plot. n = 35 NaCl-treated roots were analyzed. The number of autophagosomes after salt treatment was divided by the average of the number of autophagosomes in the untreated roots. Wild type/calb1-1 p = 0.00000479 (***p < 0.001). f Wild-type, calb1-1, calb1-1 with CaLB1pro: CaLB1-GFP, and atg10-1 seedlings were treated with 100 mM NaCl for 2 days and subsequently with 100 µM E-64d. Autophagic bodies were stained with MDC and root epidermis cells were imaged using a confocal microscope. Maximal projection images are shown. The experiment was conducted twice, and one representative image is shown. Scale bars: 10 µm. g Quantification of the results shown in (f). atg10-1 was omitted from the analysis as there was no accumulation of autophagic bodies. For each genotype, 15 seedlings (5 cells per seedling, n = 75) were analyzed. wild type/calb1-1 p = 0.000132 (***p < 0.001), wild type/calb1-1 with CaLB1pro:CaLB1-GFP p = 0.199 (ns: not significant). b, c, e, g Source data are provided as a Source Data file. c, e, g 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