Journal: Nature Communications

Article Title: FERARI and cargo adaptors coordinate cargo flow through sorting endosomes

doi: 10.1038/s41467-022-32377-y

Figure Lengend Snippet: a Rab11 vesicles perform kiss-and-run dependent on FERARI. Movie stills showing kiss-and-run of endogenously GFP-tagged Rab11 vesicles (arrow) in ctr KO ( n = 41) and vipas39-KO ( n = 25) HeLa cells (see also Supplementary Movie ). Scale bar: 2 µm. b Rab11 vesicles dock on SNX1 networks with residence times in distinct intervals. Groups of vesicles with similar residence times appear as peaks (see also Supplementary Fig. for single-vesicle graph, mock : n = 41, vipas39-KO : n = 25). This pattern is abolished in vipas39 -KO cells (see Supplementary Fig. for ehd1-KO and rab11fip5-KO additional data). One-way multiple comparison ANOVA test P -values: mock-vipas39-KO P = 1.98E-09, mock-ehd1-KO P = 1.6E-10, mock-rab11fip5-KO P = 1.58E-09. c List of FERARI genes in C. elegans and H. sapiens . The human nomenclature is used throughout the manuscript. The model organism is indicated by a human or worm icon next to the data. d Movie stills showing kiss-and-run of Rab5 vesicles (arrow) in wild-type worms ( n = 53). Scale bar: 2 µm. e Movie stills showing kiss-and-run of Rab5 vesicles (arrow) in ehd1(RNAi) worms ( n = 33) (see also Supplementary Movie ). Scale bar: 2 µm. f Rab5 vesicles dock on SNX1 networks with residence times in distinct intervals (see also Supplementary Fig. for single-vesicle graph, n = 53). This pattern is abolished in ehd1 ( n = 33) and fip5 ( n = 35) knock-downs (see Supplementary Fig. for single-vesicle plots). One-way multiple comparison ANOVA test P -values: mock-ehd1 P = 4.63E-05, mock-fip5 P = 3.83E-05. g Residence times intervals in Rab5 vesicles docking to mCherry-EHD1 compartments ( wild-type from f as comparison, n = 63) (see Supplementary Fig. ). Unpaired two-tailed t -test: P = 0.8467. h Rab5 exhibits kiss-and-run behavior in HeLa cells stably expressing mApple-Rab5 and transiently expressing GFP-SNX1. Movie stills show representative vesicles for ctr KO ( n = 41) and vipas39- KO ( n = 23) cells (arrow) (see also Supplementary Movie ). Scale bars: 2 µm. i Binning of vesicles from (h) reveals distinct intervals of residence times for Rab5 vesicles ( mock : n = 41, vipas39-KO : n = 23). This pattern is abolished in vipas39-KO cells (see Supplementary Fig. for single-vesicle plots). Unpaired two-tailed t -test: P = 0.000118.

Article Snippet: The following commercially available plasmids were obtained: GFP-VIPAS39 (Sino.Bio; HG22032_ACG), GFP-Rabenosyn-5 (Addgene; 37538), turbo-GFP-SNX1 (Origene; RG201844), GFP-RAB11 (Addgene; 12674) sigma1-EGFP (Addgene; 53611).

Techniques: Two Tailed Test, Stable Transfection, Expressing

Journal: Nature Communications

Article Title: FERARI and cargo adaptors coordinate cargo flow through sorting endosomes

doi: 10.1038/s41467-022-32377-y

Figure Lengend Snippet: a GFP-Rab10 compartments docking onto mCherry-SNX1 networks in worm intestine (see also 3D projection, Supplementary Movie ). Regions of co-localization are indicated by arrows. Quantification of co-localization by Mander’s coefficients ( n = 10). Data are presented as mean values +/– SD. M1 denotes the overlap between SNX1 and Rab10, and M2 between Rab10 and SNX1. One-way multiple comparison ANOVA test P -values: M1 SNX1-Rab10 vs. SNX1-DHS-3 P = 1.94E-08, M1 SNX1-Rab10 vs. SNX1-MANS P = 9.62E-09, M2 SNX1-Rab10 vs. SNX1-DHS-3 P = 4.37E-13, M2 SNX1-Rab10 vs. SNX1-MANS P = 4.43E-13. Scale bar: 2 µm. b Genetic interaction between FERARI subunits and Rab10. rab10(ok1494) causes accumulation of SNX1 compartments. RNAi of vps45 and vipas39 but not vps33B (CHEVI) show additional enlargement of SNX1 (arrowheads). n = 20 worms from 3 experiments (for quantification see Supplementary Fig. ). Scale bar: 10 µm. c Western blot of CRISPR–Cas9-mediated KO of Rab10 in HeLa cells ( n = 3 independent experiments). d Size of the endogenous SNX1 structure is enlarged in rab10 KO cells. SNX1 was detected by immunofluorescence. Violin plot showing the enlarged volume of the SNX1 structure in ctr and rab10 KO cells (volume of >14,700 particles was measured from each group from three independent experiments, median indicated by the red line). Unpaired two-tailed t -test: P = 1.46E-17. Scale bars 10 µm. e Movie stills for Rab10 vesicles (arrow) showing kiss-and-run in ehd1(RNAi) ( n = 40) and wild-type worms ( n = 51) (Supplementary Movie ). “N”: nucleus with high mCh-SNX1 signal. Scale bar: 2 µm. f Rab10 vesicles show residence times with quantal increases ( n = 51). This behavior is abolished in ehd1 ( n = 40) and fip5 ( n = 36) knock-downs (Supplementary Fig. for single-vesicle plots). One-way multiple comparison ANOVA test P -values: mock-ehd1 P = 7.23E-08, mock-fip5 P = 4.29E-10. g Kiss-and-run of Rab10 on sorting compartments is conserved in metazoans. Residence times of Rab10 vesicles exhibit characteristic intervals abolished in vipas39- KO cells (see Supplementary Fig. for single-vesicle plots). Unpaired two-tailed t -test: P = 0.000128. h Movie stills of mCherry-tagged Rab10 vesicles (arrow) showing kiss-and-run on SNX1 (GFP-tagged) compartment in ctr KO and vipas39- KO HeLa cells ( n = 3 independent experiments, mock : n = 53, vipas39-KO : n = 22 vesicles) (see Supplementary Movie ). Scale bar: 2 µm.

Article Snippet: The following commercially available plasmids were obtained: GFP-VIPAS39 (Sino.Bio; HG22032_ACG), GFP-Rabenosyn-5 (Addgene; 37538), turbo-GFP-SNX1 (Origene; RG201844), GFP-RAB11 (Addgene; 12674) sigma1-EGFP (Addgene; 53611).

Techniques: Western Blot, CRISPR, Immunofluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: FERARI and cargo adaptors coordinate cargo flow through sorting endosomes

doi: 10.1038/s41467-022-32377-y

Figure Lengend Snippet: a Co-localization between GFP-Rab11 and endogenous SNX1 is reduced in snx5 + 6 double knock out cells. Antibodies against GFP and SNX1 were used to detect Rab11 and SNX1 by immunofluorescence, respectively. Scale bars 10 µm; 5x magnified. Pearson’s coefficient from n = 65 and n = 71 cells from ctr KO and snx5 + 6 KO cells, respectively. Data are presented as single data points with median and min to max whiskers. Unpaired two-tailed t -test P = 2.5316E-07. b Size of the GFP-Rab11-positive structures is increased in snx5 + 6 KO cells in comparison with the ctr KO cells ( n = 3 independent experiments) Scale bars 10 µm; magnification 5x. c Volume of the GFP-Rab11-positive particles in both ctr and KO cells were determined (volume of >15,000 particles were measured from each group from three independent experiments, median is shown by the red line). Unpaired two-tailed t -test P = 3.05E-73. d CI-MPR trafficking is impaired in snx 5 + 6 KO cells. CI-MPR (red) is mostly localized in TGN (green) area in ctr KO cells and is dispersed in snx 5 + 6 KO cells ( n = 3 independent experiments, n = 150 cells per condition). Pearson’s coefficient measurements are shown on the right. Unpaired two-tailed t -test P = 1.97E-51. Scale bars 10 µm. e CI-MPR localization remain unchanged in vipas39 KO cells ( n = 3 independent experiments, n = 150 cells per condition). Scale bars 10 µm; magnification 5x. Co-localization was quantified by Pearsons’s coefficients. Unpaired two-tailed t -test P = 0.0536. f Immunoprecipitation data showing that endogenous SNX6 (left panel), but not SNX5 (right panel), binds to FERARI members Rabenosyn-5 and VIPAS39 in HEK-293 cells ( n = 3 independent experiments). g C. elegans SNX6 interacts with RABS-5 (but not with other FERARI subunits) in Y2H assays. Quantification is shown in Table ( n = 3 growth experiments with n = 6 independent yeast colonies each).

Article Snippet: The following commercially available plasmids were obtained: GFP-VIPAS39 (Sino.Bio; HG22032_ACG), GFP-Rabenosyn-5 (Addgene; 37538), turbo-GFP-SNX1 (Origene; RG201844), GFP-RAB11 (Addgene; 12674) sigma1-EGFP (Addgene; 53611).

Techniques: Knock-Out, Immunofluorescence, Two Tailed Test, Immunoprecipitation