gamma-cyclodextrins greatly enhance translocation of hydrophobic fluorescent phospholipids from vesicles to cells in culture. Importance of molecular hydrophobicity in phospholipid trafficking studies.
Short-chain, fluorescent derivatives are commonly used to investigate intracellular phospholipid trafficking. However, their use can yield misleading results because they, unlike the native species, can rapidly distribute between organelles due to their low hydrophobicity. On the other hand, hydrophobic derivatives are very difficult to introduce to cells and thus have hardly been used. Here we show that carboxyethylated gamma-cyclodextrin (CE-gamma-CD) greatly enhances transfer of a variety of hydrophobic fluorescent phospholipid derivatives from vesicles to cultured cells. Several lines of evidence indicate that CE-gamma-CD enhances transfer of lipid molecules by increasing their effective concentration in the aqueous phase, rather than by inducing membrane fusion or hemifusion. Incubation with CE-gamma-CD and donor lipid vesicles does not extract cholesterol or phospholipids from the cells or compromise plasma membrane intactness or long term cell viability. Using CE-gamma-CD-mediated transfer, we introduced hydrophobic pyrene-labeled phosphatidylserine to the plasma membrane of fibroblast cells and followed their distribution with time. In contrast to what has been previously observed for other, less hydrophobic species, transport of this lipid to the Golgi apparatus or mitochondria was not detected. Rather, much of this fluorescent PS remained in the plasma membrane or was incorporated to various endocytotic compartments. These findings indicate that the native, typically hydrophobic phosphatidylserine molecules efflux only very slowly via the cytoplasm to intracellular organelles. This helps to explain how cells can maintain a very high concentration of phosphatidylserine in the inner leaflet of their plasma membrane. Furthermore, the present results underline the importance of using hydrophobic analogues when studying intracellular trafficking of many phospholipid classes.
Tanhuanpää
K
K
Institute of Biomedicine, Department of Medical Chemistry, University of Helsinki, Siltavuorenpenger 10 A, 00014 Helsinki, Finland.
Somerharju
P
P
D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D003505:Cyclodextrins / Q000494:pharmacology*; D005347:Fibroblasts; D005456:Fluorescent Dyes; D007700:Kinetics; D008561:Membrane Fusion / Q000502:physiology; D015388:Organelles / Q000187:drug effects / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D013329:Structure-Activity Relationship
D003505:Cyclodextrins; D005456:Fluorescent Dyes; D010743:Phospholipids