Abstract 5166: Membrane phospholipid asymmetry mediates the selective packaging of protein factors in cancer microparticles.

Abstract

Abstract Objective: Growing evidence indicates that cancer cell-derived microparticles (MPs) play important regulatory roles on cellular and system levels. These activities are attributed to protein factors inside MPs. It has been known that contents in MPs are not a passive representation of the host cell, but a collection of factors packaged in a selective fashion. In the current study, we hypothesize that membrane phospholipid dynamics promotes the selective packaging of protein factors by modulating the electrostatic density inside MPs. Methods: The dynamics of phospholipid species across the membrane bilayer, including phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositides (PIs) are characterized using specific molecular probes. We used exogenous probes, Duramycin and bovine lactadherin, to stain the presence of PE and PS, respectively, on the outer surface of MP membrane. Endogenously expressed probes include GFP-tagged C2 domain of lactadherin (LacC2-GFP), and GFP-tagged 2PH-PLCdelta, are used to assess the relative density of PS and PI, respectively, in the inner leaflet of MP membranes. We examined the distribution of small Rho GTPases, including RhoA, Cdc42 and Rac1, using GFP-labeled reporters. To further examine the electrostatic interactions, we used RhoA mutants with the polybasic amino acids mutated to Gln. Additionally, we constructed a GFP-PBR to validate the electrostatic-dependent packaging mechanism. Results: PE is externalized to the outer surface of MPs. This is correlated with a greater density of negatively charged phospholipids, particularly PS, in the inner leaflet. The process is independent of apoptosis or the generation of apoptotic bodies. The redistribution of PE to the MP surface is correlated with an elevated presence of small GTPases in a PBR-dependent manner. Mutation of PBR abolished RhoA uptake in MPs. The incorporation of PBR to GFP significantly enhanced GFP localization in MPs. Conclusion: PE externalization to MP surface likely results in a greater density of negatively charged phospholipids, including PS, and to a lesser extent, PIs. An elevated density of anionic phospholipids provides an electrostatic attraction for membrane-associated proteins to MPs. This may provide a general mechanism for the selective sequestration of signal transduction factors in cancer cell-derived MPs. Citation Format: Songwang Hou, Carol Williams, Ming Zhao. Membrane phospholipid asymmetry mediates the selective packaging of protein factors in cancer microparticles. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5166. doi:10.1158/1538-7445.AM2013-5166