Abstract
The internalization of fluorescent analogs of phosphatidylserine and phosphatidylethanolamine following their insertion into the plasma membrane of cultured Chinese hamster fibroblasts was examined. When liposomes containing the fluorescent lipid 1,2-(palmitoyl-N-4-nitrobenzo-2-oxa-1,3-diazole-amino-caproyl) phosphatidylserine [palmitoyl-C6-NBD)-PS), were incubated with monolayer cell cultures at 2 degrees C, spontaneous transfer of the fluorescent lipid from the liposomes to the cells occurred, resulting in prominent labeling of the plasma membrane. However, if the cells were washed and warmed to 7 degrees C for 30 min, the (palmitoyl-C6-NBD)-PS also labeled numerous intracellular membranes. Evidence is presented suggesting that this internalization was not due to endocytosis, but was the result of transmembrane movement of the (palmitoyl-C6-NBD)-PS at the plasma membrane followed by translocation of lipid monomers from the plasma membrane to internal membranes. This transmembrane movement was reversibly inhibited by depletion of cellular ATP levels and was blocked by treatment with structural analogs of the lipid or by pretreatment of cells with glutaraldehyde or N-ethyl-maleimide. A fluorescent analog of phosphatidylethanolamine [palmitoyl-C6-NBD)-PE), which also exhibits transmembrane movement at the plasma membrane at 7 degrees C (Sleight, R. G., and Pagano, R. E. (1985) J. Biol. Chem. 260, 1146-1154), was further studied. Its transmembrane movement was also inhibited by depletion of cellular ATP levels, or by pretreatment of cells with N-ethylmaleimide. The transmembrane movement of the fluorescent phosphatidylserine and phosphatidylethanolamine analogs was inhibited when the unnatural D-isomers of these lipids were used, further suggesting that this process was stereospecific and therefore likely to have been protein-mediated.
Highlights
The internalization of fluorescent analogs of phos- mitoyl-Cfi-NBD)-PS)’ andphosphatidylethanolamine in livphatidylserine and phosphatidylethanolamine follow- ing cultured fibroblasts and show that these lipids were caing their insertion into the plasma membrane of cul- pable of rapid transbilayer movement at the plasma memtured Chinese hamsterfibroblastswas examined. brane
Uptake and Internalization of (Palmitoyl-C6-NBD)-PsWhen cells were incubated with DOPC vesicles containing NRh-PE and-PSfor 30 min at 2 "C, a significant amount (-50 pmollpg DNA) of the NBD lipid was transferred to cells
The pattern of et al, 1983).TLC analysis indicated that theonly fluorescent intracellular fluorescence wasvirtually identical to thatprelipid present in the cells or medium after the 2 "C treatment viously observedusing a fluorescent analog of phosphatidylwas(palmitoyl-C6-NBD)-PS
Summary
The internalization of fluorescent analogs of phos- mitoyl-Cfi-NBD)-PS)’ andphosphatidylethanolamine in livphatidylserine and phosphatidylethanolamine follow- ing cultured fibroblasts and show that these lipids were caing their insertion into the plasma membrane of cul- pable of rapid transbilayer movement at the plasma memtured Chinese hamsterfibroblastswas examined. brane. The internalization of fluorescent analogs of phos- mitoyl-Cfi-NBD)-PS)’ andphosphatidylethanolamine in livphatidylserine and phosphatidylethanolamine follow- ing cultured fibroblasts and show that these lipids were caing their insertion into the plasma membrane of cul- pable of rapid transbilayer movement at the plasma memtured Chinese hamsterfibroblastswas examined. That this internalization was not due to endocytosis, but was the resulotf transmembrane movement of the (palmitoyl-C6-NBD)-PSart the plasma membrane followed by translocation of lipid monomers from the plasma membrane to internal membranes. A fluorescent analog of phosphatidylethanolamine ((palmitoyl-C6-NBD)-PE),which exhibits transmembrane movement at the plasma membrane at7 “C Fluorescent Phospholipids-Fluorescent P S and P E were synthesized from (palmitoyl-C6-NBD)-PCby transphosphatidylation (Comfurius and Zwaal, 1977)using phospholipase D and L-serine or ethanolamine, respectively, and purified by preparative TLC using chlo-
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
