Journal: Nature Communications

Article Title: Deterministic early endosomal maturations emerge from a stochastic trigger-and-convert mechanism

doi: 10.1038/s41467-023-40428-1

Figure Lengend Snippet: a FLIM of orientation with phosphoinositide lipid specificity. Scatter and box-and-whisker plots of PI(3)P staining intensity by endosome type; Rab5 positive and EEA1 negative ( magenta ), NT EEA1 bound ( blue ), or CT EEA1 bound ( green ). PI(3)P intensities are measured using 2xFYVE-GST labelling and normalised across each cell. Error bars on scatter plots indicate mean ± S.D. Statistical significance determined using one-way ANOVA, ** indicates p < 0.01, **** indicates p < 0.0001(EEA1 vs. NT EEA1 p = 0.0086, No EEA1 vs. CT EEA1 p = 1.32 × 10 −6 , NT EEA1 vs. CT EEA1 p = 3.45 × 10 −5 ) n = 18 cells. Single points indicate measured data, and the box plots correspond to the 25th to 75th percentile of events, with bars showing the total range. b SAR405-treated cells show reduced fusions between APPL1 and EEA1 endosomes, but not reduced APPL1 to EEA1 conversions. siRNA-INPP4A-treated cells show reduced fusions and conversions. Single points indicate measured data; the violin plots correspond to a normal distribution of all events; and the box plots correspond to the standard error of the mean, with bars showing the total range. Means are depicted by the open squares. Statistical significance was determined using one-way ANOVA (Conversions: untreated vs. SAR 405. p = 0.2936, Conversions: untreated vs SiRNA INPP4A. p = 1.45 × 10 −5 , Fusions: untreated vs SAR405 p = 0.36 × 10 −5 , Fusions: untreated vs SiRNA INPP4A. p = 0.85 × 10 −5 ; n = 5 cells per condition). c RPE1 wild-type cells and HeLa EEA1 knockout (KO) cell lines expressing wild-type EEA1 ( blue ) or N-terminal mutant deficient in binding Rab5 ( red ) were imaged using LLSM. d The total number of conversions and fusions were quantified for WT EEA1 (blue shade) and EEA1 Nt-mutant (Grey shade); these data indicate that the initial N-terminal of EEA1 is essential for endosomal conversions. ns indicates non-significant difference, * indicates p < 0.05; p = 0.0018. Each mean was compared against the others using an ordinary one-way ANOVA. In the case of HeLa EEA1 KO cells expressing EEA1 N-terminal Rab5 binding mutant, no events were detected by the analysis workflow or by visual inspection. Single points indicate measured data; the violin plots correspond to a normal distribution of all events; and the box plots correspond to the standard deviations of events, with bars showing the total range. Means are depicted by the open squares ( n = 6 cells per condition).

Article Snippet: Wild-type HeLa cells were transfected with pEGFPC1-human APPL1, a gift from Pietro De Camilli (Addgene plasmid #22198) ; EEA1 TagRFP-T, a gift from Silvia Corvera (Addgene plasmid #42635) ; EGFP-EEA1, a gift from Silvia Corvera (Addgene plasmid #42307) ; EGFP-Rab5, a gift from Marci Scidmore (Addgene plasmid #49888); and mRFP-Rab5, a gift from Ari Helenius (Addgene plasmid #14437) .

Techniques: Whisker Assay, Staining, Knock-Out, Expressing, Mutagenesis, Binding Assay