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. 2023 Oct 16;19(10):e1010979. doi: 10.1371/journal.pgen.1010979

Fig 6. Arf1 and AP-1 regulate Rab6-compartment identity and the maturation of DCG compartments.

Fig 6

(A-E) Representative images of SCs expressing the CFP-Rab6 fusion gene from the endogenous Rab locus together with a control RNAi (A) or RNAis targeting Arf1 (RNAi #1; B), AP-1γ (C), AP-1μ (D) or AP-1σ (E). Cellular organisation is assessed through DIC imaging, CFP-Rab6 fluorescence and Lysotracker Red fluorescence, and a merged image is presented for each cell. Note the number of CFP-Rab6-positive compartments is reduced in all knockdown backgrounds, except AP-1γ, where a central cluster of small Rab6-positive compartments is often also observed, and in all knockdowns, few labelled compartments contain DCGs. (F, G) Representative SCs expressing the YFP-Rab1 fusion gene either alone (F) or together with an Arf1 RNAi #2 (G). Note that in control YFP-Rab1 SCs, no large non-acidic compartments are Rab1-positive. When SCs are subjected to Arf1 knockdown, by contrast, YFP-Rab1 does mark several large non-acidic compartments which do not contain DCGs. (H) Bar chart showing number of CFP-Rab6-labelled compartments in control SCs and following knockdown of Arf1 and AP-1. (I) Bar chart showing number of CFP-Rab6-compartments containing DCGs in these different genotypes. (J) Bar chart showing the percentage of CFP-Rab6-compartments, which fail to produce regularly shaped DCGs in these different genotypes. Approximate outlines of SCs are marked by dashed circles. Scale bars: 10 μm. For H-J, bars show mean ± SD; Control, n = 30; Arf1 #1, n = 39; Arf1 #2, n = 32; AP-1γ, n = 29; AP-1μ, n = 27; AP-1σ, n = 30. P<0.05: *, P<0.01: **, P<0.001: ***, P<0.0001: ****. Genotypes for images: (A) w1118; P{tub-GAL80ts}, TI{TI}Rab6CFP/P{ryTRiP.HMS02827}; dsx-GAL4/+; (B) w1118; P{tub-GAL80ts}, TI{TI}Rab6CFP/+; dsx-GAL4/P{Arf1GD12522}; (C) w1118; P{tub-GAL80ts}, TI{TI}Rab6CFP/+; dsx-GAL4/P{AP-1γTRiP.JF02684}; (D) w1118; P{tub-GAL80ts}, TI{TI}Rab6CFP/+; dsx-GAL4/P{AP-1μGD14206}; (E) w1118; P{tub-GAL80ts}, TI{TI}Rab6CFP/P{AP-1σKK108869}; dsx-GAL4/+; (F) w1118; +; TI{TI}Rab1CFP/+; (G) w1118; P{tub-GAL80ts}/P{Arf1KK101396}; dsx-GAL4/TI{TI}Rab1EYFP.