Figure 1. Mass spectrometric analysis of C elegans embryo after
dlc-1 knock down:A-D) Immunostaining of control or
dlc-1(RNAi) embryos. P granule protein PGL-1 is red and DNA is labeled with DAPI (blue). (A, B) 4 cell embryos (C, D) ~40 cell embryos. Images were acquired using a confocal microscope. Scale bars are 10 μM. Dotted lines outline embryos, which are oriented with anterior to the left and posterior to the right in this and subsequent figures. P granules are larger and restricted to the posterior cell of the 4 cell embryo and perinuclear in the 40 cell embryo in control RNAi (A, C) but mis-localized, smaller and dispersed after
dlc-1(RNAi) (B, D). E) Volcano plot of mass spectrometric analysis of control RNAi vs.
dlc-1(RNAi) treated C. elegans embryos. &#
x2011;
log10(Padj) (y-axis) is plotted against the average
log2 fold change in protein level from 5 biological replicates (x-axis). Vertical black lines denote
log2-fold change of -1 and 1 while the horizontal black line indicates an adjusted P-value of 0.01. Blue or red color indicates a significant fold change: blue are significantly decreased 2 fold or more; Padj <0.01, red are significantly increased 2 fold or more; Padj <0.01. As expected, DLC-1 protein levels are significantly decreased in
dlc-1(RNAi) embryos and the PERM proteins were chosen for follow up analysis (enlarged blue points). Enlarged red points correspond to significantly increased proteins involved in stress response pathways, enlarged yellow points are P granule proteins and the enlarged green points are nucleoporin proteins. F) Relative protein levels of the core P granule proteins (corresponding to enlarged yellow dots in Figure 1E).
dlc-1(RNAi) values were scaled to the mean of the control (from 5 replicate values), which was set to 1. *-Padj<0.01. G) Relative protein levels of the nucleoporin proteins previously shown to be important for P granule integrity (Voronina and Seydoux 2010). *-Padj<0.01. H) Relative protein levels of PERM-1/2 proteins. *-Padj<0.01. I-K) Live imaging of control RNAi,
perm-2(RNAi) or
perm-4(RNAi) treated embryos. (Ii and Iii-Ki and Kii) 2-4 cell embryos. (Iiii and Iiv-Kiii and Kiv) ~40 cell embryos. (Ii and Iiii -Ki and Kiii) White dashed lines outline embryos, GFP-tagged P granule protein PGL-3 is green and DNA is labeled with Hoechst 33342 (blue). Scale bars are 10 μM. (Iii and Iiv-Kii and Kiv) Brightfield images of embryos outlined by black dashed lines. Scale bars are 10 μM. Notice that Hoechst 33342 is able to penetrate the nematode eggshell only after
perm-2 or
perm-4 knockdown (compare Ii to Ji and Ki and Iiii to Jiii and Kiii).