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Fig. 5
Pharmacological disruption of IDR-interactions leads to partially fragmented Buc-GFP aggregates.
(A-C) Balbiani body of living Buc-GFP transgenic oocytes, either before (A), after a 30 min treatment with 5% 1,6-hexanediol (HD) (B), or 30 min after washout of the drug (lateral view, animal to the top). Arrowheads in B and B' indicate Buc-GFP granule outside the Balbiani body. Scale bar (A-C): 20 μm (A'-C'): 2 μm. (D-K) Germ plasm of transgenic Buc-GFP embryos after hexanediol treatment (HD). (D, E) lateral view of living 2-cell embryo as shown in boxed area of icon. Control embryos show unfragmented Buc-GFP aggregates (green) (D arrowhead), whereas 5% hexanediol for 30 min leads to fragmentation(arrowheads). (F) Quantification of embryos with unfragmented Buc-GFP in control (Co; 100±0%; n = 20) and embryos treated for 30 min with hexanediol (HD; 35.0±0.8%; n = 20; p = 0.0065). Student's t-test; P-value: **<0.01. (G, H) lateral view of living 4-cell embryos. Control embryo with unfragmented BucGFP (green, arrowhead), whereas Buc-GFP stays fragmented 30 min after washout of hexanediol (green; arrowheads). Scale bar (D-H): 100 μm. (I-K) Buc-GFP aggregates in 3 hpf embryos transgenic for Buc-eGFP. (I) The morphology of control (Co) and hexanediol-treated embryos (HD). Lateral view, animal to the top. (I', I'') Fragmented Buc-GFP aggregates (white arrowheads) persist until 3 hpf (I') lateral view, (I'') animal view. (J) Treatment with hexanetriol (HT) also leads to fragmented germ plasm (right embryo in J; animal view). Scale bar (I-J): 500 μm. (K) Quantification of germ plasm fragmentation (more than four puncta) at 3 hpf in controls (Co; 2.2±3.9%; n = 45), hexanetriol (HT; 26.3±11.5; n = 45) and hexanediol (HD; 75.5±3.9; n = 45; p = 1.9e-08). Error bars represent standard deviation of the mean. Student’s t-test; P-value: ***<0.001. (L-N) Protein aggregates upon transfection of HEK cells with (L) Buc(aa1-601)-GFP (50.32±2.95%; n = 70 percentage of transfected cells showing aggregated GFP signal), (M) Buc(aa1-361)-GFP (77.9±8.8%; n = 89) and (N) GFP (0%; n = 81). Scale bar (L-N): 10μm. (O-Q) Buc aggregation in zebrafish embryos. Embryos at 3 hpf after injection of mRNA encoding wt Buc(aa1-639)-eGFP (O), Buc(aa1-601)-eGFP (P) or eGFP(Q) at the one cell stage (lateral view, animal to the top). Scale bar (O-Q): 200 μm. Note the aggregation of wt Buc (aa1-639) and Buc (aa1-601) compared to GFP (insets; 25x magnification of stippled box). (R-U) IDRs are not sufficient for germ cell induction. Embryos form germ cells (white arrowheads) after injection with wt buc mRNA (aa 1–639) (R; 76.6±2.3%; n = 60), but less with mutant Buc (K; aa1-601) containing most IDRs (S; 35.9±2.6%; n = 60; p = 0.04) or an unrelated IDP (human FUS; T; 0±0; n = 26). Scale bar (J-L): 200 μm. (I) Quantification of injection results (three independent experiments for each RNA). Error bars represent standard deviation of the mean. Student’s t-test; P-value: *<0.05.