Goat antibodies against unique regions of both XveloFL and Splice.

“These antibodies proved ineffective in western blots of endogenous proteins, although they did detect over-expressed fusion proteins. The antibodies did work well in immunofluorescence staining of oocytes. We find that both isoforms of the endogenously expressed Xvelo protein are localised in the germ plasm RNPs of full-grown oocytes and fertilised eggs. The XveloFL is also localised to the Balbiani body of pre-vitellogenic oocytes.”

“The specificity of the XveloSV antibody was confirmed by depleting XveloSV RNA and using the antibody to see if the XveloSV protein signal was reduced. The reduction was very marked. This approach was not possible for XveloFL, because the protein is very stable, both in its native and GFP tagged forms.”

Published.

Figure 1. Interaction of in vitro-translated Xvelo1 isoforms with Hermes. (A) eGFP-XveloFL, eGFP-XveloSV and HA-Hermes fusion proteins were prepared in vitro by coupled transcription–translation in the presence of 35S-methionine. Translated proteins were resolved by SDS-PAGE and Figure 1. Interaction of in vitro-translated Xvelo1 isoforms with Hermes. (A) eGFP-XveloFL, eGFP-XveloSV and HA-Hermes fusion proteins were prepared in vitro by coupled transcription–translation in the presence of 35S-methionine. Translated proteins were resolved by SDS-PAGE and visualized by autoradiography. (B) In pull down experiments translated proteins were added as indicated and immunoprecipitated with a mouse anti-HA monoclonal antibody. Immunoprecipitated proteins were resolved by SDS-PAGE and visualized by autoradiography. The predicted molecular weight for the individual fusion proteins is indicated on the right and marker sizes on the left.

Figure 2. Distribution of GFP-tagged Hermes interacting candidates in the germ plasm region of stage VI oocytes. The vegetal pole of oocytes was injected with Cy5-nanos1 and mRNAs encoding GFP-tagged proteins as indicated. After culturing in OCM for 48 hours the cortex of live oocytes was examined by confocal microscopy. Overlays are shown on the right. Xvelo proteins localise strongly to germ plasm RNPs, but Rbm42b and particularly Rbm24b localise more diffusely in the general vicinity of the germ plasm. Control RNA encoding GFP protein is shown in F.

Figure 3. Demonstration of in vivo protein-protein interactions of Hermes and its partners by BiFC analysis. Candidate interacting proteins were expressed in stage VI oocytes by injecting mRNAs expressing the C-terminal (VC) and N-terminal (VN) fragments of Venus fused to the N-terminus of the two candidate proteins. Oocytes were then cultured for 48 hours in OCM, after which the vegetal cortex of live oocytes was examined by confocal microscopy. Constant gain settings were used in all instances, and with these settings non-interacting controls, such as Poc1B/Hermes (Panel G) or other negative combinations (panel F) gave no significant signal. In all cases Cy5-nanos1 was also co-injected and was coincident with the green fluorescence, but this is shown only in A.

Figure 4. Sub-cellular localisation of GFP fusion proteins and of the sites of where they interact. Stage VI oocytes were injected with mRNA encoding the constructs indicated, and cultured in OCM for 48 hours. Oocytes were then fixed with methanol/1% formaldehyde at -20°C overnight, rehydrated and sectioned in an animal/vegetal plane by hand. Low power fluorescent stereo microscope images are shown.

Figure 5. Confocal analysis of BiFC constructs co-injected with labelled nanos1 mRNA. Sectioned stage VI oocytes, prepared as in Figure 4, were imaged in the confocal microscope. Sections made by hand with a scalpel are quite irregular, so Z-stacks are shown. Panel (A) shows an oocyte injected with Cy5-nanos1 RNA alone.

Figure 6. Expression of endogenous Xvelo isoforms during oogenesis. Oocytes, isolated with collagenase treatment to remove follicles, were fixed and stained with affinity-purified antibodies prior to confocal microscopy. (A–B) Previtellogenic oocytes stained for XveloFL and SV respectively. (C–F) The germ plasm region of stage VI oocytes and fertilized eggs. In (C) Hermes and XveloFL were stained and we show coincidence in an overlay in the right hand panel. In (D–F) only the overlays of Hermes (green) and Xvelo proteins (red) are shown. C) and (E) show stage VI oocytes and (D) and (F) show fertilized eggs. Alexa Fluor 488 and Dylight 594 conjugates were used as secondary antibodies for Hermes and Xvelo protein-stained oocytes respectively. Pre-immune serum for both XveloFL and SV is shown in panels G–H. The data was identical when secondary antibodies alone were used (not shown).

Figure 7. Localisation of Xvelo mutant proteins. (A) Schematic diagram of GFP-tagged Xvelo mutant constructs. Hatched boxes represent amino acid residues present in both XveloFL and XveloSV proteins. Clear and grey boxes represent amino acid residues specific to XveloFL and XveloSV proteins respectively. The position of a putative Dynein light chain 8 (DLC-8) interacting motif, identified using the ELM software (elm.eu.org) database is also shown. (B) Stage VI oocytes were co-injected with Cy5-nanos1 RNA and RNA encoding the indicated mutant Xvelo protein. Oocytes were cultured for 48 hours in OCM and localisation was then assessed by confocal microscopy.

Figure 8. Depletion of endogenously expressed XveloSV protein by anti sense (AS) DNA oligos. Stage VI oocytes were co-injected with Cy5-nanos1 RNA and 11 ng of the indicated AS-oligo. Following culture in OCM for 48 hours, oocytes were fixed, and stained with purified anti XveloSV antibodies using an Alexa Fluor 488 conjugated secondary antibody. The germ plasm region was then examined by confocal microscopy. The co-injected AS-oligo is indicated in each panel.

Figure 9. Effect of RNA depletion on the size of germ plasm islands and the localisation of YFP-Hermes. Stage VI oocytes were injected with RNA encoding YFP-Hermes plus 11 ng of each antisense oligo. After culturing for 48 hours in OCM, mitochondria were stained with TMRE and the germ plasm region examined by confocal microscopy. Overlays of YFP-Hermes (green) and mitochondria (red) are shown, and the AS oligos used indicated on each panel.

Figure 10. RNA depletion by antisense oligos does not affect the distribution of microtubules in the germ plasm region. Stage VI oocytes were co-injected with Cy5-nanos1 RNA and 11 ng of each of the AS oligos indicated. Oocytes were then cultured in OCM for 48 hours, fixed and stained with a rabbit anti-a-Tubulin polyclonal antibody #2144 (Cell Signalling Technology) and a goat anti rabbit Alexa Fluor 488 conjugated secondary antibody, prior to confocal microscopy. Panels show Cy5-nanos1 (red) left, and an overlay of this with microtubules (green) right. Scale Bar, 10 mm.