Figure 6.
Synergy between NBR1 and TAX1BP1 mitigates competition and optimizes flux. (A) Schematic of the tandem fluorescent (tf) reporter system. Throughout autophagosome formation, both RFP and GFP fluorescence are observed. Upon lysosomal fusion, the acidic lysosomal environment quenches GFP, whereas RFP is maintained. Therefore, an increasing red:green ratio corresponds with increased lysosomal delivery. (B) HeLa PentaKO cells were transfected with tf-p62, tf-NBR1, tf-TAX1BP1, tf-NDP52, or tf-Optineurin. After 24 h, cells were analyzed for red:green ratio by flow cytometry. Median values for each sample are identified by a black line within each violin. The red dotted line across all samples corresponds to red:green ratio of maximally inhibited conditions (e.g., BafA1 treatment) n > 10,000 cells per sample. (C) HeLa PentaKO cells were co-transfected with tf-NBR1 and indicated BFP-tagged receptors. After 24 h, cells were analyzed for red:green ratio by flow cytometry. Graphs represent mean ± SD from four independent experiments. n > 10,000 cells per sample. P values were determined using a one-way ANOVA (P = <0.0001). Multiple comparisons were made using Dunnett T3 correction. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. (D) HeLa PentaKO cells were co-transfected with tf-NBR1 (wild type or F740A) and BFP-TAX1BP1 (wild type or R441E). After 24 h, cells were analyzed for red:green ratio by flow cytometry (n > 10,000 cells per sample). Graphs represent mean ± SD from three independent experiments. P values were determined using a one-way ANOVA (P = <0.0001). Multiple comparisons were made using Tukey correction. ***, P < 0.001; ns, not significant. (E) HeLa pentaKO cells were co-transfected with tf-NBR1 (wild type or Y732A) and BFP-TAX1BP1 (wild type or V143S). After 24 h, cells were analyzed for red:green ratio by flow cytometry (n > 10,000 cells per sample). Graphs represent mean ± SD from three independent experiments. P values were determined using a one-way ANOVA (P = <0.0001). Multiple comparisons were made using Tukey correction. ***, P < 0.001; ****, P < 0.0001; ns, not significant. (F) HeLa pentaKO cells were co-transfected with tf-NBR1 and BFP-TAX1BP1 variants. After 24 h, cells were analyzed for red:green ratio by flow cytometry (n > 10,000 cells per sample). Graphs represent mean ± SD from four independent experiments. P values were determined using a one-way ANOVA (P = <0.0001). Multiple comparisons were made using Tukey correction. *, P < 0.05; ****, P < 0.0001; ns, not significant. Refer to the image caption for details.

Synergy between NBR1 and TAX1BP1 mitigates competition and optimizes flux. (A) Schematic of the tandem fluorescent (tf) reporter system. Throughout autophagosome formation, both RFP and GFP fluorescence are observed. Upon lysosomal fusion, the acidic lysosomal environment quenches GFP, whereas RFP is maintained. Therefore, an increasing red:green ratio corresponds with increased lysosomal delivery. (B) HeLa PentaKO cells were transfected with tf-p62, tf-NBR1, tf-TAX1BP1, tf-NDP52, or tf-Optineurin. After 24 h, cells were analyzed for red:green ratio by flow cytometry. Median values for each sample are identified by a black line within each violin. The red dotted line across all samples corresponds to red:green ratio of maximally inhibited conditions (e.g., BafA1 treatment) n > 10,000 cells per sample. (C) HeLa PentaKO cells were co-transfected with tf-NBR1 and indicated BFP-tagged receptors. After 24 h, cells were analyzed for red:green ratio by flow cytometry. Graphs represent mean ± SD from four independent experiments. n > 10,000 cells per sample. P values were determined using a one-way ANOVA (P = <0.0001). Multiple comparisons were made using Dunnett T3 correction. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. (D) HeLa PentaKO cells were co-transfected with tf-NBR1 (wild type or F740A) and BFP-TAX1BP1 (wild type or R441E). After 24 h, cells were analyzed for red:green ratio by flow cytometry (n > 10,000 cells per sample). Graphs represent mean ± SD from three independent experiments. P values were determined using a one-way ANOVA (P = <0.0001). Multiple comparisons were made using Tukey correction. ***, P < 0.001; ns, not significant. (E) HeLa pentaKO cells were co-transfected with tf-NBR1 (wild type or Y732A) and BFP-TAX1BP1 (wild type or V143S). After 24 h, cells were analyzed for red:green ratio by flow cytometry (n > 10,000 cells per sample). Graphs represent mean ± SD from three independent experiments. P values were determined using a one-way ANOVA (P = <0.0001). Multiple comparisons were made using Tukey correction. ***, P < 0.001; ****, P < 0.0001; ns, not significant. (F) HeLa pentaKO cells were co-transfected with tf-NBR1 and BFP-TAX1BP1 variants. After 24 h, cells were analyzed for red:green ratio by flow cytometry (n > 10,000 cells per sample). Graphs represent mean ± SD from four independent experiments. P values were determined using a one-way ANOVA (P = <0.0001). Multiple comparisons were made using Tukey correction. *, P < 0.05; ****, P < 0.0001; ns, not significant.

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