Fig. 9: CaLB1 modulates autophagosome maturation. | Nature Communications

Fig. 9: CaLB1 modulates autophagosome maturation.

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

Fig. 9

a Colocalization of ESCRT-III with autophagosomes. 5-day-old wild-type and calb1-1 seedlings expressing UBQ10pro:GFP-ATG8a and VPS2.1pro:VPS2.1-3xFLAG-mRFP were incubated in 1/2 MS media containing 150 mM NaCl for two hours. Root epidermis cells were analyzed using a confocal microscope. Rectangle: magnified region. Scale bars: 10 µm and 1 µm (magnification). b Quantification of open and closed autophagosomes at the ultrastructure level in wild-type or calb1-1 seedling roots using transmission electron microscopy. 5-day-old seedlings expressing were treated for 2 h in 1/2 MS supplemented with 150 mM NaCl before cryofixation. The analysis was conducted on sections taken from 4 different roots (wild-type, n = 68 autophagosomes; calb1-1, n = 70 autophagosomes). Wild type/calb1-1 p = 0.0174 (*0.01 < p < 0.05), two-tailed two proportion z-test. c Autophagosomes were isolated from wild-type and calb1-1 seedlings and treated with Proteinase K or Proteinase K and Triton X-100 and subjected to immunoblotting using and anti-GFP antibody. The experiment was conducted four times, and one representative result is shown. d Box plot shows the quantification of the results in (c). The intensity of the non-proteinase K sample was set at 100%. 4 replicates were analyzed, and all results are shown. Wild type/calb1-1 (+Proteinase K) p = 0.0453 (*0.01 < p < 0.05). e GFP cleavage assay. 7-day-old seedlings were incubated in 1/2 MS, or 1/2 MS supplemented with either 150 mM NaCl or 150 mM NaCl and 100 µM E-64d for two hours. GFP-ATG8a and free GFP in total extracts of wild type and calb1-1 were detected by immunoblotting along with NBR1. UGPase was used as loading control. The experiment was conducted five times, and one representative result is shown. f Box plot shows the quantification of the results in (e). Five replicates were analyzed, and all results are shown. wild type/calb1-1 (untreated) p = 0.00610 (**0.001 < p < 0.01), wild type/calb1-1 (+NaCl) p = 0.0282 (*0.01 < p < 0.05), wild type/calb1-1 (+NaCl+E-64d) p = 0.0352 (*0.01 < p < 0.05). b, c, d, e, f Source data are provided as a Source Data file. d, f 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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