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Contributed by Nancy Pierce on 02/11/2011
Laura Murphy (Biology '11) and Ed Cluett presented, "Use of the Phospholipase A2 Inhibitor ONO RS-082 to Dissect the Transport Route of LDL-derived FreeCholesterol". American Society for Cell Biology, Philadelphia, PA. (Co-authored by Laura Murphy, Lauren Krisak (both Biology '11), and Edward B. Cluett.
Our research has focused on a possible role for membrane tubules in the intracellular trafficking of cholesterol. Phospholipase A2 (PLA2) activity is involved in the formation of some membrane tubules, and PLA2 inhibitors (PLAIs) disrupt structure and function in both the Golgi complex and endosomes. By fluorescence microscopy, treatment of HeLa cells with the PLAI, ONO-RS-082 (ONO), altered the distribution of rab11 and resulted in the accumulation of free cholesterol in the Endocytic Recycling Compartment (ERC) based on colocalization of filipin staining with immunolabeling of rab11 and the transferrin receptor. This accumulation was inhibited by treatment with cyclodextrin, filipin, or by growing cells in LDL-depleted serum. Addition of exogenous LDL induced accumulation of free cholesterol in the ERC in the absence of ONO. PLAI-mediated cholesterol redistribution was actin-dependent and low concentrations of ONO altered the localization of cdc42. The localization of rab5 and EEA1 was also altered by ONO treatment and was inhibited by wortmannin. Three isoforms of rab5 are associated with early endosomes, and recent evidence indicates that subsets of isoforms are involved in the trafficking of certain materials. We hypothesized that a subset of rab5 isoforms is specifically involved in cholesterol trafficking. We used siRNA to knockdown individual isoforms and monitored the effects on cholesterol distribution by filipin labeling and microscopy. Knockdowns of either rab5A or rab5B altered the localization of cholesterol compared to controls, but the pattern of filipin staining was markedly different for each isoform. In both cases, ONO-mediated redistribution of cholesterol did not occur. However, knockdown of rab5C did not appreciably alter the distribution of free cholesterol. These results suggest that membrane tubules play a role in the transport of cholesterol from the plasma membrane to endosomes and that a specific subset of rab5 isoforms may regulate that traffic.