Fig. 5: CaLB1 and ALIX localize on salt-induced autophagosomes. | Nature Communications

Fig. 5: CaLB1 and ALIX localize on salt-induced autophagosomes.

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

Fig. 5

Confocal microscopy of NaCl-treated root epidermal cells expressing CaLB1pro: CaLB1-GFP (a), UBQ10pro: GFP-ATG8a (b), or ALIXpro: GFP-ALIX (c). Scale bars: 10 µm. The experiment was conducted six times, and one representative image is shown. d Quantification of (a–c). CaLB1-GFP −/+ NaCl p = 0.0289 (*0.01 < p < 0.05), GFP-ATG8a −/+ NaCl p = 2.32×10−5 (***p < 0.001), ALIX-GFP −/+ NaCl p = 0.0370 (*0.01 < p < 0.05). The analysis was conducted six times, and the average of all experiments is shown. Seedlings expressing ALIXpro: GFP-ALIX and CaLB1pro: CaLB1-mRFP (e) or CaLB1pro: CaLB1-mRFP and UBQ10pro:mRFP-ATG8i (f) were treated with 150 mM NaCl for 2 h before confocal imaging. 54% of CaLB1 foci (n = 506 from 12 seedlings) were positive for the GFP-ALIX. Scale bars: 10 µm. g Analysis of time-lapse images of NaCl-treated CaLB1pro:CaLB1-GFP/UBQ10pro:mRFP-ATG8a seedlings. n = 36 autophagosomes (16 seedlings). h Immuno-electron microscopy on CaLB1-GFP using an anti-GFP antibody after treatment with 150 mM NaCl for 2 h. Arrowhead: gold particle. Scale bar: 200 nm. Three seedling roots were analyzed, and one representative image is shown. i Membrane fraction (P4) was analyzed by immunoblotting along with the total extract using anti-GFP, anti-NBR1, anti-ATG8, and anti-UGPase antibodies. j Seedlings expressing CaLB1pro: CaLB1(F64A)-GFP and UBQ10pro:mRFP-ATG8i were treated with 150 mM NaCl for 2 h before confocal imaging. Scale bars 10 µm. k Number of ATG8a-, ATG8e-, or ATG8i foci with CaLB1-GFP or CaLB1-GFP(F64A) signals. ATG8i with CaLB1-GFP/CaLB1-GFP(C2mut) p = 0.00416 (**0.001 < p < 0.01). The analysis was conducted twice, and all results are shown. For CaLB1-GFP, n = 728 autophagosomes in 14 seedlings for ATG8a, n = 506 (15 seedlings) for ATG8e, n = 586 (12 seedlings) for ATG8i, and for CaLB1-GFP(F64A) and ATG8i n = 1150 (24 seedlings) were analyzed. l Seedlings expressing ALIXpro: GFP-ALIX and UBQ10pro:mRFP-ATG8i were treated with 150 mM NaCl for 2 h before confocal imaging. scale bars: 10 µm. 25.7% of the mRFP-ATG8i marked autophagosomes (n = 1036 from 11 roots) were positive for GFP-ALIX signals. Rectangle: magnified region. Scale bar in the magnification: 1 µm. m Classification of GFP-ALIX signals on autophagosomes. Images taken as in (l) were analyzed (n = 11 roots). d, g, k, I, m Source data are provided as a Source Data file. d, k Box plot: center line, median; box limits, first and third quartiles; whiskers, 1.5x interquartile range. Two-tailed t-test, no equal variance. e, f, i, j, l Experiments were conducted three times and one representative result is shown.

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