"
Team:MIT/GFP
From 2013.igem.org
| (14 intermediate revisions not shown) | |||
| Line 15: | Line 15: | ||
<h1>Overview</h1> | <h1>Overview</h1> | ||
| - | <p> | + | <p>In 2011 Steve Gould’s lab showed that fusing GFP to the TyA protein and adding an N-terminal acylation tag led to protein oligomerization and selective targeting to the protein membrane, facilitating exosome mediated export of GFP. (Shen, 2011). We hope to engineer sender cells that export proteins of interest, by fusing them to Acyl-Tya. Thus we attempted to replicate Shen's experiment and created our own Acyl-Tya-eGFP fusion protein.</p> |
<p><b>Method:</b><br/> | <p><b>Method:</b><br/> | ||
| - | Previous research on biogenesis of exosomes has shown the rhodamine phosphatidylethanolamine (Rh-PE) preferentially labels the endosome-like-domain (ELD), which is believed to be the exosomal budding site on cell membrane. 24 hours after Jurkat T cells were nucleofected with CMV_Acyl-TyA-eGFP construction, the cells were stained with Rh-PE. The colocalization of the stain and green fluorescence was observed by confocal for 24 hrs.</p> | + | Previous research on biogenesis of exosomes has shown the rhodamine phosphatidylethanolamine (Rh-PE) preferentially labels the endosome-like-domain (ELD), which is believed to be the exosomal budding site on cell membrane (Booth, 2006). 24 hours after Jurkat T cells were nucleofected with CMV_Acyl-TyA-eGFP construction, the cells were stained with Rh-PE. The colocalization of the stain and green fluorescence was observed by confocal for 24 hrs.</p> |
<p><b>Results:</b><br/> | <p><b>Results:</b><br/> | ||
Rh-PE was seen to localize at the ELD immediately after staining procedure. Over the course of 24 hrs, exosomes budded out with stain. 48 hrs post nucleofection, the green fluorescence was observed to localize on cell membrane.</p> | Rh-PE was seen to localize at the ELD immediately after staining procedure. Over the course of 24 hrs, exosomes budded out with stain. 48 hrs post nucleofection, the green fluorescence was observed to localize on cell membrane.</p> | ||
| Line 23: | Line 23: | ||
<div align="center"> | <div align="center"> | ||
<img src="https://static.igem.org/mediawiki/2013/8/84/AcyTyA_membraneLocalizationDigram.png" width="400" height="550"> | <img src="https://static.igem.org/mediawiki/2013/8/84/AcyTyA_membraneLocalizationDigram.png" width="400" height="550"> | ||
| + | </div | ||
| + | <br><br> | ||
| + | Jurkat T cells were transfected with pCMV_Acyl-TyA-GFP and after 24 hours stained with Rh-PE and NucBlue nuclear stain. The following images were taken on the confocal microscope 12 hours later as sequential focal planes. The planes are then stacked together to generate the 3D image. | ||
| + | <div align="center"> | ||
| + | <img src="https://static.igem.org/mediawiki/2013/f/f5/Projection_ChampionRB.gif" width="45%"> | ||
| + | <img src="https://static.igem.org/mediawiki/2013/f/ff/Projection_ChampionGB.gif" width="45%"> | ||
</div> | </div> | ||
| - | <img src="https://static.igem.org/mediawiki/2013/ | + | <br> |
| - | <img src="https://static.igem.org/mediawiki/2013/ | + | Here, we overlay both the fluorescence of the green channel and the red channel to show the colocalization of Acyl-TyA-GFP and Rh-PE, indicating that our Acyl-TyA tag is localizing at the ELD (the site of exosome biogenesis). |
| + | <br> | ||
| + | <div align="center"> | ||
| + | <img src="https://static.igem.org/mediawiki/2013/4/45/Projection_ChampionRGB.gif" width="45%"> | ||
| + | <img src="https://static.igem.org/mediawiki/2013/5/57/ChampionRGB-1.png" width="45%"><br><br> | ||
| + | <b>Conclusion:</b></br></div> | ||
| + | |||
| + | From the following microscopy data, we can conclude that the Acyl-TyA tag is targeting our GFP protein to the membrane and to the site of exosome biogenesis. These results inform later protein fusion experiments, strengthening our confidence that the protein signals fused to Acyl-TyA are making their way into exosomes. | ||
| + | <p> <p> | ||
| + | Booth, Amy et al. Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane. J Cell Biol. (2006) <br><br> | ||
| + | Shen, B et al. Protein targeting to exosomes/microvesicles by plasma membrane anchors. J Biol Chem. (2011) | ||
</div> <!--End col_left--> | </div> <!--End col_left--> | ||
</body> | </body> | ||
</html> | </html> | ||
Latest revision as of 02:28, 29 October 2013
Overview
In 2011 Steve Gould’s lab showed that fusing GFP to the TyA protein and adding an N-terminal acylation tag led to protein oligomerization and selective targeting to the protein membrane, facilitating exosome mediated export of GFP. (Shen, 2011). We hope to engineer sender cells that export proteins of interest, by fusing them to Acyl-Tya. Thus we attempted to replicate Shen's experiment and created our own Acyl-Tya-eGFP fusion protein.
Method:
Previous research on biogenesis of exosomes has shown the rhodamine phosphatidylethanolamine (Rh-PE) preferentially labels the endosome-like-domain (ELD), which is believed to be the exosomal budding site on cell membrane (Booth, 2006). 24 hours after Jurkat T cells were nucleofected with CMV_Acyl-TyA-eGFP construction, the cells were stained with Rh-PE. The colocalization of the stain and green fluorescence was observed by confocal for 24 hrs.
Results:
Rh-PE was seen to localize at the ELD immediately after staining procedure. Over the course of 24 hrs, exosomes budded out with stain. 48 hrs post nucleofection, the green fluorescence was observed to localize on cell membrane.
Jurkat T cells were transfected with pCMV_Acyl-TyA-GFP and after 24 hours stained with Rh-PE and NucBlue nuclear stain. The following images were taken on the confocal microscope 12 hours later as sequential focal planes. The planes are then stacked together to generate the 3D image.
Here, we overlay both the fluorescence of the green channel and the red channel to show the colocalization of Acyl-TyA-GFP and Rh-PE, indicating that our Acyl-TyA tag is localizing at the ELD (the site of exosome biogenesis).
Conclusion:
Booth, Amy et al. Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane. J Cell Biol. (2006)
Shen, B et al. Protein targeting to exosomes/microvesicles by plasma membrane anchors. J Biol Chem. (2011)
