Global analysis of mRNA polarization in migrating endothelial cells

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DESCRIPTION (provided by applicant): Blood vessels are exposed to multiple stresses that can induce injury to the endothelium, which must undergo rapid repair to restore normal function of the vessel and the entire cardiovascular system. Endothelial cell (EC) migration has a critical role in this repair process, as well as in formation of new blood vessels. Polarization of multiple cell systems, including signaling, adhesion proteins, plasma membrane, secretion processes, cyto- skeletal arrangement, etc., is crucial for induction and control of cell migration. Recent studies have shown that 2-actin mRNA, and several other motility-related transcripts, accumulated in the front of moving cells, but little is known about the total ensemble of polarized mRNAs and their common localization elements. Our long-term goal is to understand the contribution of mRNA polarization to the development of asymmetry during EC movement, and the mechanisms by which mRNAs are polarized in planar moving cells. To accomplish this goal, we have initiated a comprehensive, global analysis of the mRNAs that become polarized during EC migration. We have applied the laser microdissection methodology to capture cell fragments from forward and rearward regions of planar-moving EC, a model representing the wound-healing process. We propose to couple this microdissection method with microarray technologies, followed by rigorous biochemical investigation to address the following hypothesis: cis-elements in mRNAs determine their localization in both the front and back of planar-migrating EC, and are responsible for targeting proteins to their site of function. We will test this hypothesis by pursuing the following specific aims: (1) identify novel mRNAs polarized in migrating EC, and (2) investigate RNA elements responsible for mRNA polarization during cell migration. Our studies will provide the first global analysis of mRNA localization during cell migration, and it will begin to fill important gaps in our understanding of cell polarization during migration. In particular, little is known about the RNA elements that induce mRNAs to accumulate in the cell front and virtually nothing is known about mRNAs (and their functional elements) that accumulate in the cell rear. In addition to the mechanistic contribution to our understanding of cell polarization, our work can reveal new insights into the regulation of cell movement, and has the potential of new interventions to alter motility. For example, identification of novel localization elements presents novel therapeutic targets for modulating mRNA localization and cell migration by RNA-based approaches, for example, by treatment with antisense RNA oligomers targeting the specific RNA element. Finally, our work is likely to provide and validate a technological advance that will permit global analysis of asymmetric mRNA in other systems. PUBLIC HEALTH RELEVANCE: Blood vessels are exposed to multiple stresses that can injure the endothelial cell (EC) lining of the vessel wall. Subsequent migration of EC is critical for vessel wall repair and restoration of normal blood vessel function. Polarization of the messenger RNAs (mRNA) encoding movement-related proteins is a newly discovered event that may contribute to cell directionality required for EC movement. Our studies will provide the first global analysis of mRNA polarization during planar cell migration, and it may reveal novel therapeutic targets for modulating localization of motility-related mRNAs and cell migration.