Fig 11. SifA-dependent lysosome clustering requires both SKIP and Vps39.

a-b) Representative confocal micrographs of HeLa cells co-transfected with Myc-SifA and GFP-LAMP1 plasmids. The cells were fixed and co-stained using anti-Myc (blue) and anti-Vps18 (a, red) or anti-Vps41 (b, red) antibodies. Different panels represent a higher magnification of the boxed areas and HOPS subunits enrichment on clustered lysosomes is indicated by the arrowheads. c-e) HeLa cells treated with indicated siRNA were transfected with Myc-SifA expressing plasmid. The cells were fixed and co-stained using anti-Myc (green) and anti-LAMP1 (red) antibodies. Insets show higher magnification of the boxed areas clustered lysosomes induced by SifA expression, which is dependent upon Vps39 and SKIP expression. Bars: (main) 10 μm; (insets) 5 μm. f) Average size of LAMP1-positive compartments calculated in cells treated with indicated siRNA and transfected with Myc-SifA plasmid. Data represent mean ± S.D. of ~25–30 transfected per experiment over three independent experiments (**, P < 0.01; Student’s t test). g) Schematic depicting the molecular machinery required for SCV fusion with late endosomes and lysosomes. Multisubunit tethering factor-HOPS complex is a target for Salmonella effector SifA, which associates with its known binding partner-SKIP to recruit HOPS complex to SCV membranes, thereby enabling SCV fusion with Arl8b-positive lysosomes.