To study the localization and structure of Ephrin, a new technique was developed to transiently express proteins in living Drosophila embryos. Compared to the generation of stable transformants, which takes up to 6 weeks, transient expression enables protein localization and potential phenotypes to be studied after only a few hours (Bossing, 2002).
Syncytial blastoderm embryos were injected with plasmids in which expression is driven by a constitutive promoter (Polyubiquitin) or by the GAL4 UAS system. The injections result in expression in small cell clusters located near the site of injection. The time and cell type of expression can be selected by choosing the site of injections according to the embryonic fate map of Drosophila (Polyubiquitin plasmids) or by injecting into a GAL4 transformant strain with the desired expression pattern (UAS plasmids). Expression can be examined either 2 hours after the injection of Polyubiquitin plasmids or 3 hours after the onset of GAL4 expression (at 25°C). 80% of all embryos injected with the Polyubiquitin vector show expression. Expression of UAS plasmids depends on the GAL4 strain and varies between 40%-80% (Bossing, 2002).
The overall structure of Ephrin differs significantly from all other ephrins. The ephrin domain is not located at the N terminus but in the middle of the protein. Ephrin has no obvious signal peptide and an additional predicted transmembrane domain precedes the ephrin domain. To confirm Ephrin as a genuine member of the ephrin family the structure of the protein was studied in more detail (Bossing, 2002).
A polyclonal antibody was tested against the ephrin domain of Ephrin. This antibody binds to the cell surface of non-permeabilized Drosophila S2 cells in vivo, which express Ephrin endogenously. Incubation of S2 cells with doublestranded (ds) Ephrin RNA (Ephrin RNAi) reduces Ephrin expression and also diminishes the binding of anti-Ephrin to the cell surface. Thus, the binding of the antibody is specific for Ephrin and the ephrin domain is extracellular (Bossing, 2002).
The localization of the C terminus of Ephrin was studied. The C terminus was labelled with a GFP tag (Ephrin-GFP). If the C terminus is extracellular, proteinase K treatment of non-permeabilized Ephrin-GFP-expressing cells should digest the GFP tag and the extracellular ephrin domain. If the C terminus is cytoplasmic, the intact membrane should protect the GFP tag, while the extracellular ephrin domain should be destroyed. Ephrin-GFP-expressing cells were generated by plasmid injection into the syncytial blastoderm of wild-type embryos. Only cells strongly expressing Ephrin-GFP can be recognized by GFP fluorescence. In flat preparations of living and non-permeabilized embryos the C-terminal GFP tag is always protected from proteinase K digestion, but the ephrin domain is always destroyed. Without proteinase K digestion, anti-GFP and anti-Ephrin bind to the membrane of Ephrin-GFP-expressing cells. The anti-GFP signal overlaps the GFP fluorescence, whereas the anti-Ephrin signal is confined to the outside of the cell. This differential staining and the proteinase treatment strongly suggest the existence of a C-terminal cytoplasmic tail in Ephrin (Bossing, 2002).
Although Ephrin has no obvious signal peptide, the localization of Ephrin to the membrane depends on its N terminus. Full length Ephrin expressed in S2 cells and in embryos accumulates at the membrane and in cytoplasmic vesicles. Deletion of the N terminus (aa 1-202) results in a diffuse distribution of the truncated protein in the cytoplasm of S2 cells and embryos (Bossing, 2002).
Ephrin has three predicted transmembrane domains, one in the N terminus and two at the C terminus. If all domains are genuine membrane anchors, deletion of the C terminus of Ephrin should not interfere with membrane localization. A C-terminal truncation still carries the N-terminal sequences necessary for membrane localization and the predicted transmembrane domain preceding the ephrin domain. Expression of such a truncation (UAS-EphrinDeltaC-term, deletion of aa419-aa652) in S2 cells leads to an accumulation of the protein in the medium. In contrast, Ephrin can never be detected in the medium of S2 cells. It is concluded that EphrinDeltaCterm is secreted, suggesting that the protein is still sorted correctly to the membrane but the hydrophobic domain at the N terminus is not able to anchor the protein at the membrane. Anchoring at the membrane most likely requires the predicted transmembrane domains at the C terminus (Bossing, 2002).
In Western blots of S2 cell lysates, anti-Ephrin reveals two prominent bands at ~50 kDa and frequently a weaker band at ~75 kDa, the predicted size of Ephrin. In embryonic lysates, anti-Ephrin detects a band at ~51 kDa and ~75 kDa. Since the Ephrin antisera was generated against the ephrin domain, these bands represent different forms of Ephrin that all contain the ephrin domain. cDNA analysis revealed only one Ephrin transcript. Therefore the two different isoforms of Ephrin might either result from protein cleavage or from alternative initiation of translation (Bossing, 2002).
To examine the possibility of protein cleavage GFP fusions to the N terminus (GFP-Ephrin) and the C terminus (Ephrin-GFP) were generated. Cleavage of the protein at either terminus should separate the GFP from Ephrin (Bossing, 2002).
Expression of GFP-Ephrin in S2 cells or embryos results in a different subcellular distribution of GFP and Ephrin. GFP is mainly found in the cytoplasm, whereas Ephrin concentrates at the membrane. Interestingly, the GFP tag at the N terminus does not interfere with the membrane localization of Ephrin. No GFP band could be detected in Western blots of lysates taken from GFP-Ephrin-expressing S2 cells or embryos. The absence of GFP might indicate a degradation of the N-terminal cleavage product. In contrast, GFP and Ephrin always co-localize in S2 cells or embryos expressing Ephrin-GFP. Western blots of lysates taken from Ephrin-GFP-expressing S2 cells confirm the absence of cleavage at the C terminus (Bossing, 2002).
It was noted that the Ephrin mRNA has a translation initiation consensus in front of the methionine doublet at position +544. A start of translation at this site would result in a 50 kDa protein with a signal peptide. To test this possibility the first 630 bp of the Ephrin mRNA was fused to GFP (NtermEphrin-GFP). If translation can start at the beginning and in the middle of the Ephrin mRNA, it would be expected that transfection of Schneider cells would result in two proteins of different sizes. However, S2 cells only produce one protein migrating at around 51 kDa, a size expected from a translational initiation at the first methionine (24 kDa of Ephrin + 27 kDa of GFP) (Bossing, 2002).
It is concluded that the two different isoforms of Ephrin result from N-terminal cleavage of the protein. This cleavage depends on the full length molecule; no cleavage of the EphrinDeltaCterm truncation or the NtermEphrin-GFP fusion could be detected. The cleavage of Ephrin yields a band of about 51 kDa in embryonic lysates. The doublet around 50 kDa in S2 cells might indicate different phosphorylation or glycosylation states of Ephrin (Bossing, 2002).
To test if Ephrin is able to bind to axons, the secreted EphrinDeltaC-term truncation, which has an intact receptor binding domain, was expressed. Expression of EphrinDeltaC-term in muscles overlying the CNS (GAL4 line 24B) results in a specific accumulation of the truncated protein on axons. In vertebrates, injection of secreted forms of ephrins give a dominant negative phenotype. However, expression of EphrinDeltaC-term in CNS or muscles (elav-GAL4, sim-GAL4 and GAL424B) failed to cause any obvious defects. This lack of phenotypes is most likely due to insufficient levels of expression (Bossing, 2002).
The accumulation of EphrinDeltaC-term around axons suggests that Ephrin may bind to Eph. The binding between Ephrin and Eph was confirmed in cell culture. Drosophila S2 cells were transfected with a UAS construct encoding the extracellular part of Eph fused to GFP (Ephex-GFP). After incubation with Ephex-GFP-containing medium, non-permeabilized Schneider cells can be labelled with anti-GFP. Hence, Ephex-GFP can bind onto the surface of S2 cells that express endogenous Ephrin. To confirm that Ephex-GFP binds to Ephrin the level of Ephrin expression was lowered by incubation of S2 cells with dsEphrin mRNA. Indeed, Ephrin RNAi treatment of S2 cells diminishes the binding of Ephex-GFP. Control incubation of Schneider cells with dsGFP RNA (GFP RNAi) or dsEph RNA (Eph RNAi) does not interfere with the binding (Bossing, 2002).
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