Uptake of Extracellular Double-Stranded RNA by SID-2 Deborah L. McEwan, Alexandra S. Weisman, Craig P. Hunter  Molecular Cell  Volume 47, Issue 5, Pages 746-754 (September 2012) DOI: 10.1016/j.molcel.2012.07.014 Copyright © 2012 Elsevier Inc. Terms and Conditions

Molecular Cell 2012 47, 746-754DOI: (10.1016/j.molcel.2012.07.014) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 SID-2 Is Required to Internalize Environmental dsRNAs into C. elegans cy5-labeled dsRNA (500 bp) fed to C. elegans could only be visualized intracellularly in sid-2(+) animals [observed in 6/15 sid-2(+) animals, 0/15 sid-2(–) animals]. Molecular Cell 2012 47, 746-754DOI: (10.1016/j.molcel.2012.07.014) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 SID-2 Expression Is Sufficient for Efficient dsRNA Transport into Drosophila S2 Cells at an Acidic pH (A) cy5-labeled dsRNA is internalized into and has extensive colocalization with SID-2::GFP expressed in S2 cells in media at pH 5 (42/57 cells contain at least three points of colocalization) but not with cells expressing Cbr-SID-2::GFP (n = 48) or with SID-2::GFP expressed in cells at pH 7 (n = 53). All images were taken at the same time using the same camera settings. (B) At pH 5, S2 cells expressing SID-2 internalize more 32P-labeled dsRNA than cells expressing Cbr-SID-2 or mock-transfected cells. Results shown are an average of 20 (SID-2, Cbr-SID-2B pH 5), nine (Cbr-SID-2A pH 5), or eight (remaining conditions) biological replicates. (C) Unlike SID-2, SID-1-dependent transport of 32P-labeled dsRNA is inhibited in media at pH 5 compared to pH 7. Results shown are an average of six biological replicates. (D) dsRNA internalized by SID-1 or SID-2 is processed into functional siRNAs as indicated by reduced luciferase expression in these cells after the addition of luciferase dsRNA to media at pH 5 (SID-2 and SID-1) or pH 7 (SID-1) as compared to cells not exposed to dsRNA. Results shown are an average of five to eight biological replicates. ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 0.0005, ∗∗∗∗p < 1 × 10−6. Error bars represent 1 standard deviation. p values determined with a two-tailed paired t test. The scale bar represents 10 μm. See also Figure S1 and Table S1. Molecular Cell 2012 47, 746-754DOI: (10.1016/j.molcel.2012.07.014) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 SID-2-Mediated Transport Is Selective for Long dsRNA (A) The addition of unlabeled dsRNA competitor blocks the uptake of 32P-labeled dsRNA into cells expressing SID-2, while unlabeled dsDNA has only a moderate effect. Results shown are an average of five biological replicates. (B–D) Internalization of 32P-labeled substrates into SID-2-expressing, Cbr-SID-2-expressing, or mock-transfected S2 cells. Results shown are an average of nine (100 bp, 25 bp SID-2 and Cbr-SID-2), three (25 bp mock), or six (remaining conditions) biological replicates. Cells expressing Cbr-SID-2 were indistinguishable from mock-transfected cells for all tested substrates. Error bars represent one standard deviation. Starred conditions accumulated at least 1.4-fold more substrate than mock-transfected cells with ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 5 × 10−4. p values determined with a two-tailed paired t test. See also Table S1. Molecular Cell 2012 47, 746-754DOI: (10.1016/j.molcel.2012.07.014) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Domain Swaps Identify a Critical Role for the SID-2 Extracellular Domain in Environmental RNAi (A) sid-2(gk505) deletion worms expressing SID-2, Cbr-SID-2, or hybrids of the C. elegans/C. briggsae SID-2 proteins were scored as rescued if exposure to ingested pal-1 dsRNA caused >95% embryonic lethality. Only proteins containing the extracellular SID-2 domain could rescue the environmental RNAi defect. (B) S2 cells expressing mutagenized SID-2 constructs internalize less 32P-labeled dsRNA than the wild-type (WT) SID-2 control. An exception to this is the triple histidine-to-arginine mutant, which internalizes more 32P-labeled dsRNA than WT. Results shown are an average of five biological replicates. Error bars represent one standard deviation. See also Figure S2 and Table S1. Molecular Cell 2012 47, 746-754DOI: (10.1016/j.molcel.2012.07.014) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 Vesicle Trafficking Is Required for SID-2 to Take Up dsRNA (A) In S2 cells, SID-2-dependent uptake of 32P-labeled dsRNA is a continuous process. Results shown are an average of eight (100 min) or five (remaining conditions) biological replicates. (B) SID-2-dependent uptake of 32P-labeled dsRNA is strongly reduced by exposing the S2 cells to oligomycin (inhibits ATP synthase), latrunculin A (latA; inhibits actin polymerization), and bafilomycin A1 (bafA; inhibits vesicle maturation); SID-2-expressing cells exposed to these inhibitors internalize less than 2-fold more dsRNA than relevant control cells. Results shown are an average of five (SID-2) or four (Cbr-SID-2) biological replicates. (C) Endocytosis inhibitors do not prevent SID-1-dependent internalization of 32P-labeled dsRNA; SID-1-expressing cells exposed to these inhibitors internalize between 7.5- and 37-fold more dsRNA than relevant control cells. Results shown are an average of five biological replicates. (D) LatA inhibits cytoplasmic SID-2::GFP structures and reduces the internalization of cy5-labeled dsRNA (7/50 cells contain at least three points of colocalization between cy5-dsRNA and SID-2::GFP). The scale bar represents 10 μm. ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 5 × 10−5. Error bars represent one standard deviation. p values were determined with a two-tailed paired t test. Molecular Cell 2012 47, 746-754DOI: (10.1016/j.molcel.2012.07.014) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 Model of SID-2 and SID-1 Coordinated Uptake of Ingested dsRNA in C. elegans dsRNA is internalized from the intestinal lumenal space by SID-2-mediated endocytosis. dsRNA is retained in the vesicle until directly transported into the cytoplasm by SID-1 (left) or released in the pseudocoelomic fluid for subsequent cellular import via SID-1 (right). Molecular Cell 2012 47, 746-754DOI: (10.1016/j.molcel.2012.07.014) Copyright © 2012 Elsevier Inc. Terms and Conditions