Table 1.
Homologs of OST complex members in different species.
Figure 1.
Knockdown of different OST complex members shows similar phenotypes.
(A) Schematic representation of the membrane orientation of the C. elegans OST proteins in the ER membrane as calculated by toppred 0.01. (B) Embryonic lethality in OST complex member knockdown is similar in all four genes tested. Feeding was started at the L4 stage and carried on until worms stopped egg laying. dad-1(RNAi) was applied by dsRNA injection in young adult worms; as indicated by the asterisk. The ratio between total brood size and hatched larvae was determined. Error bars represent the standard deviation of at least 3 independent experiments. (C) Embryonic death did not occur at a specific stage in development upon knockdown of different OST complex subunits, but could happen any time before hatching. Early-arrested eggs showed characteristic rounded up cells as a consequence of the permeable eggshell and the slightly hyper-osmotic egg buffer. Late-arrested embryos could often twitch and showed distinct signs of morphogenesis like a pharynx, tail or gut, but also large vacuoles in the body as a sign of beginning necrosis. These phenotypes have been observed in more than 50% of the RNAied embryos in at least 3 independent experiments. (D) OSTD-1 knockdown embryos developed slower than WT embryos as shown by 4-cell stage embryos that were left to develop on a slide in egg buffer at RT. Every 30 min a Z-stack image was taken. If the embryo did not arrest before, usually after 1.5 hours it showed approx. 30% less nuclei than the WT. Representative data from 4 independent experiments are shown. Scale bars represent 10 µm.
Figure 2.
The amount of glycosylated proteins is reduced in oocytes but not embryos in ribo-1(RNAi).
(A) Downregulation of glycosylation cannot be detected in embryo lysates of ribo-1(RNAi)-treated worms. Both panels show the same gel loaded with 60 µg egg extract of either mock or ribo-1(RNAi)-treated embryos. Staining with ProQ Emerald 300 (Invitrogen) showed neither a reduction in staining nor an alteration of the band pattern in ribo-1(RNAi), while subsequent Coomassie staining confirmed that equal amounts of protein were loaded. Asterisks indicate the glycosylated bands in the CandyCane marker. n = 3 independent experiments. (B) Epifluorescence images of fixed embryos stained with Qdot®-wheat germ agglutinin (Invitrogen) show a similar amount of cytoplasmic granules in mock vs. ribo-1(RNAi), but the plasma membrane staining (arrows) is absent in ribo-1(RNAi) embryos. n = 3 independent experiments (C) Identically fixed and stained WT oocytes contain brightly fluorescent granules in the cytoplasm which are fewer or absent in most of the ribo-1(RNAi) oocytes. (D) Quantification was performed by categorizing images according to the examples shown below. Scale bars represent 10 µm.
Figure 3.
ER morphology is largely unaffected by N-glycosylation knockdown.
(A) Spinning disc confocal microscopy of fixed whole mount GFP::SP12 worms showed that except for a slightly stronger accumulation at the cell-cell boundaries (arrows), the structure of the endoplasmic reticulum was not much altered in (ribo-1)RNAi oocytes, neither in the periphery (upper panels) nor in the center of the cells (lower panels). Images show the first four oocytes adjacent to the spermatheca, which would be to the left in all images. (B) Fixed GFP::SP12 embryos have been imaged spinning disc confocal microscopy. The only marked difference between mock and (ribo-1)RNAi was the stronger cortical accumulation (arrows), while the overall structure as well as the cycling of the ER between dispersed and sheet state was not affected. Scale bars represent 10 µm. n = ≥4 independent experiments.
Figure 4.
Secretion is impaired in (ribo-1)RNAi.
(A) Epifluorescence images of live whole mount RME-2::GFP worms showed that the yolk receptor was no longer strongly accumulated at the plasma membrane of the oocytes in (ribo-1)RNAi. Depicted are the -2 and -3 oocytes adjacent to the spermatheca, which would be to the left in all images. (B) Quantification of the RME-2::GFP signal by ImageJ. A 50-pixel wide band was drawn over the center of the cells where the nuclei are located, and the mean gray value of every 50 pixel-column was plotted over the length of two cells. These line plots clearly illustrate the reduced plasma membrane accumulation of the yolk receptor in ribo-1(RNAi) oocytes. (C) VIT-2::GFP accumulated in the body cavity of (ribo-1)RNAi worms (open arrow heads and middle panels) while the oocytes were practically devoid of VIT-2::GFP staining (dashed lines and middle panels). Moreover, the lowest panels show that yolk protein was not even efficiently secreted from the gut cells, as they appeared much brighter in the (ribo-1)RNAi worms, pointing towards a general defect in secretion. These phenotypes have been observed in more than 80% of the RNAied worms, in 3 independent experiments. The two very bright gut cells right next to the pharynx (filled arrow heads in upper panel) can be found in many GFP worm lines. (D) Immunoblots of worm lysates developed with an anti-GFP antibody detect an increased electrophoretic mobility of RME-2::GFP protein upon ribo-1(RNAi). This phenotype was present in lysates from isolated gonads as well as in total worm lysate from RME-2::GFP-tagged worms, while the absence of a similar band in the N2-lysate demonstrates its specificity. Also, RME-2::GFP has a calculated mass of approximately 130 kDa, while the bands we detected run at around 160 kDa, indicating that RME::2 is probably modified. Upon OST knockdown, these modifications are altered, leading to a different electrophoretic mobility. (E) CAV-1::GFP secretion is not impaired in ribo-1(RNAi) worms. Live imaging CAV-1::GFP tagged worms demonstrate that there is not a general block in secretion upon knockdown of the OST complex. The CAV-1::GFP staining in mock treated and ribo-1(RNAi) oocytes is comparable. Scale bars in all panels represent 10 µm, if not annotated differently.
Figure 5.
N-Glycosylation knockdown causes severe defects in chromosome segregation and cytokinesis.
(A) Live permeabilized H2B::GFP embryos stained with FM4-64 showed an increased number of nuclei accumulating in cells of the (ribo-1)RNAi embryos. (B) Quantification of the phenotypes shown in A, C, D and E. n = >3 independent experiments with >30 embryos/experiment were counted. Only embryos earlier than 16-cell stage were analyzed for the cytokinesis defect. For the analysis of trailing DNA pieces only cells in ana- or telophase of embryos in the 1–8 cell stage were taken into account (n = >3 independent experiments), and a DNA filament had to be visible between the two DNA masses to be classified positive in the sense of the phenotype. As DNA fragments we classified small DAPI- or GFP-stained spots in the cytoplasm at a marked distance away from the nuclei, as shown in (E) and (F) (arrows). As multiple nuclei we counted cells that showed accumulations of two or more DNA masses of similar size next to each other. (C) Fixed tbb-2::GFP embryos stained with DAPI. The accumulated nuclei in the anterior cell of the (ribo-1)RNAi embryo divided simultaneously, the spindle microtubules interconnecting several centrosomes to form an extended spindle network throughout the cell. (D) Fixed 2-cell stage embryos stained with DAPI, showing the AB cell in anaphase. In the (ribo-1)RNAi embryo, the clearly visible DNA thread connecting the two DNA masses indicated that a chromosome had been attached to microtubules from both spindle poles during Metaphase and now has been pulled apart. (E) Fixed tbb-1::GFP embryos stained with DAPI. Arrows point to two DNA fragments next to a metaphase nucleus, which seem not to be arranged on the metaphase plate. These might be the remnants of a previous mis-segregation event. (F) MAN-1::GFP embryos stained with anti-GFP and DAPI. Arrows point to a small piece of DNA that attracted nuclear envelope components and thus formed a micronucleus. The content of the dashed-line box was magnified. Scale bars, if not differently annotated, represent 10 µm.