I would like to incorporate certain lipids (i.e. not fatty acids) into the membrane of living cells in culture. Is it possible to do it in a reliable and reproducible way, and if yes, how?
Thanks for your help.
Hi Peter,
What lipid are you trying to incorporate? It is possible that you can pre-load B-MCD with desired lipid. You would have to determine efficiency and dose but it is plausible to carry that out.
Michele
Thanks Michele for the response. The plan is to load DOPE (dioleoyl phosphoethanolamine) to the cell membrane without killing the cells.
Peter
I don't know about PE, but you can add PC in small amounts without killing cell. Adding lysoPC is more efficient, it will rapidly be converted to PC. Probably this will work for lysoPE as well. It will be difficult to specifically target PE to the PM by the way, it will probably end up predominantly in the endosomal membranes.
Thanks for the response. Could you refer me to a paper in which better loading with lyso-phospholipids and the accumulation of PE in endosomes is described?
We didn't really publish this observation I think. Some of it is reflected in this paper from our group. The CHO cell line mt58 has impaired PC synthesis at 40 degrees Celsius and rapidly goes into apoptosis. However, this can easily be rescued by the addition of lysoPC, which is converted to PC. I think this was not possible with PC.
J Lipid Res. 2009 Nov;50(11):2182-92. doi: 10.1194/jlr.M800660-JLR200. Epub 2009 May 20.
Depletion of phosphatidylcholine affects endoplasmic reticulum morphology and protein traffic at the Golgi complex. DOI: 10.1194/jlr.M800660-JLR200
The localization was based on an experiment with a fluorescent PC-probe (not published). It was incorporated in the cells after adding this to the medium, but the localization was mostly endosomal and in lipid droplets. LD-localization probably because of reincorporation of labelled FA into TAGs.
Hi Peter,
Did you find any references? Actually, DOPE, DPPE, SM and Cholesterol tend to incorporate in the plasma membrane very easily. One has to incubate the cells with these lipids for 15-20 min ( of course depending on the concentration -generally 100 ng/ml is enough). It is a very common practice in our lab to study lipid diffusion in the plasma membrane using this method. Please check the reference below:
Article Diffusion of lipids and GPI-anchored proteins in actin-free ...
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