Journal: Nature Communications

Article Title: ISGylation controls exosome secretion by promoting lysosomal degradation of MVB proteins

doi: 10.1038/ncomms13588

Figure Lengend Snippet: ( a ) Confocal microscopy analysis of the endosome markers HRS (red) and CD63 (green) in non-transfected HEK293 cells or HEK293 cells co-transfected with plasmids encoding the ISGylation machinery and the functional (ISG15WT) or mutated ISG15 (ISG15MUT). Scale bar, 10 μm. Right graph: quantification of HRS + and CD63 + average particle size per cell ( n =20). Each dot represents the average particle size from individual cells and mean is indicated in red lines. ( b ) Electron microscopy images showing representative fields with MVBs (red arrows) and lysosomes/autophagosomes (blue arrows) in HEK293 transfected as in a . Right graph: quantification of MVB numbers in more than 25 fields per condition. Each dot represents the number of MVBs per section and mean is indicated in red lines. Scale bar (insets), 500 nm. ( c ) Confocal microscopy analysis of HRS in WT or ISG15KO BMDMs left untreated or treated with 1,000 U ml −1 IFN for 16 h. Scale bar, 10 μm. Right graph: quantification of the number of HRS + particles per cell. Each dot represents the number of HRS + particles from individual cells and mean is indicated in red lines ( n =28). ( d ) Electron microscopy images showing MVBs (red arrows) in WT or ISG15KO BMDMs treated with 1,000 U ml −1 IFN for 16 h. Upper graph, quantification of MVB numbers in 20 fields per condition. Each dot represents the number of MVBs per section and mean is indicated in red lines. Lower graph, quantification of ILV numbers per MVB. Each dot represents the number of ILVs per MVB and mean is indicated in red lines ( n ≥24). Scale bar (insets), 500 nm. ( e ) Confocal microscopy analysis of CD63 (red) in Rab5-Q79L-GFP + endosomes (green). HEK293 cells were transfected with Rab5-Q79L-GFP and treated 16 h with 1,000 U ml −1 IFN or left untreated. ( f ) Confocal microscopy analysis of CD63 (red) in Rab5-Q79L-GFP + endosomes (green). HEK293 cells were co-transfected with Rab5-Q79L-GFP mutant, the ISGylation machinery and ISG15WT or ISG15MUT. Images from e , f : Scale bar, 10 μm. Right graphs: each dot represents the percentage of CD63-filled endosomes per individual cell and mean is indicated in red lines; t -test; NS: P -value>0.05 and *** P -value<0.0001.

Article Snippet: When indicated, cells were previously transfected with GFP-Rab5CA (Q79L) plasmid (gift from Sergio Grinstein, Addgene plasmid 35140) or LC3-GFP-RFP tandem construct (gift from Tamotsu Yoshimori, Addgene plasmid 21074).

Techniques: Confocal Microscopy, Transfection, Functional Assay, Electron Microscopy, Mutagenesis