On the influence of PI(4,5)P2 and PI(3,4,5)P3-enriched plasma membrane microdomains on exocytosis.

摘要

Although once thought to be a homogenous, randomly-oriented mixture of lipids and proteins, the cellular plasma membrane has recently been shown to contain highly-ordered and heterogenous domains which are enriched with specific proteins and lipids. Specific regions have also been shown to exist within the membrane which are preferred sites for exocytosis to take place. There has been strong evidence to support the idea that the organization of the lipids and protein machinery required for exocytosis into a subset of these domains is responsible for the occurrence of preferred exocytic sites, although the means by which these domains are organized, and the interactions between the different molecules involved in the exocytosis process remain to be fully understood. In this thesis, I present data from two studies which investigate the effects of different manipulations performed on the lipids present in these domains, and their resultant effect on the process of exocytosis. In the first study, we treated pheochromocytoma 12 (PC12) cells with the cholesterol sequestering drug methyl-ss-cyclodextrin (M-ss-CDX) (2 mM) with both short term (10 min.) and long term (2 day) treatment durations. This experiment was carried out in order to investigate whether removal of cholesterol from the membrane would result in disruption of organized lipid-enriched domains, as well as a resultant inhibition of exocytosis. Our results showed that both short and long-term treatment with M-ss-CDX led to an inhibition of exocytosis, however, short-term treatment did not result in a net decrease in membrane cholesterol levels. That we did not see any significant change in the morphology of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) or phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3)-enriched domains following treatment seems to imply that cholesterol might not be a requirement for the organization of these domains, as it is in others. Most significantly, that we saw a marked inhibition of exocytosis without commensurate cholesterol depletion following short-term M-ss-CDX treatment seems to imply a second, previously unknown interaction between M-ss-CDX and some component of the plasma membrane, aside from its previously established role as a cholesterol sequestering agent. In our second study, we expressed three different forms of fluorescently-tagged pleckstrin homology (PH) domains (PH-phospholipase C delta 1 (PLCd1), PH-Protein Kinase B (AKT), and PH-General Receptor for Phosphoinositides 1 (GRP1)), which are commonly used to label specific phospholipids which they bind with varying degrees of specificity in biological membranes. The purpose of this study was to examine the influence of plasma membrane pools of PI(4,5)P2 and PI(3,4,5)P3 on the localization of exocytic sites in the membrane. Our results showed that, of the PH domains tested, PH-PLCd1 is most specific for PI(4,5)P2, and PH-GRP1 is most specific for PI(3,4,5)P3. Furthermore, expression of all three PH domains led to significant inhibition of exocytosis and an increased occurrence of secretory vesicles dwelling at the membrane without the opening of a fusion pore. These results imply that expression of GFP-tagged PH domains effectively inhibits the exocytic machinery, regardless of specificity for either PI(4,5)P2 or PI(3,4,5)P3.