Mechanisms and Intracellular Components in Exocytosis, Membrane Assembly and Repair, and Parasite Replication

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Linked publications, trials & patents

Abstract

Living cells are open non-equilibrium systems. To exist, a cell requires precisely controlled maintenance of gradients in the chemical potential, between the extracellular environment, the cytoplasm, and the lumen of organelles, of many constituents. The amphiphilic nature of lipid molecules, self-assembling into lipid bilayers, provides an extremely low permeability barrier to both electrolytes and large nonelectrolytes. For the processes of life, a continuous exchange of matter must occur across all membranes. For example, uptake of large molecules and compounds from the outside occurs via endocytosis, phagocytosis, and macro and micro pinocytosis, secretion occurs via exocytosis, and intracellular protein trafficking via transport vesicles between endoplasmic reticulum, Golgi apparatus, endosomes, and lysosomes. The movement of such membrane-bound cargo dictates membrane recycling, or cells and organelles would be incapable of maintaining their volumes and shapes. These ubiquitous and multifarious events, plus the accommodation of lipid bilayers to membrane proteins and transient pores, all require the lipid bilayer to change its topology. Physical models of these topological changes, essential to life, must take into account the energy of membrane deformations within the framework of an adequate theory of elasticity. All of these biophysical parameters are equally important for communication between cells by extracellular vesicles. 1. We examined the mechanisms by which CD47 directly or indirectly regulates which RNAs are packaged into T cell EVs. Mass spectrometry, biochemistry, light and electron microscopy, pharmacological manipulation, and genetic analyses were used to parse the mechanism by which CD47 controls the loading of EVs with specific RNAs into EVs. It was demonstrated that CD47 regulates nuclear/cytoplasmic transport of m7G-capped RNAS and their abundance in EVs via a physical interaction of CD47 and its cytoplasmic signaling adapter ubiquilin-1 with exportin-1 and several regulators of its nuclear export complex. the CD47/ubiquilin-1 complex regulates intracellular trafficking of capped miRNAs and mRNAs and their trafficking into EVs. These results establish TSP1/CD47 signaling as a regulator of nuclear/cytoplasmic RNA trafficking and the subsequent packaging and release of a subset of 5-7-methylguanosine-modified (m7G) RNAs in EVs. The relevance of m7G-cap-dependent RNA trafficking to the physiological functions of CD47 in cardiovascular disease, aging, cancer, and infection remain to be investigated. 2. In this study, we compared the effects on Human Umbilical Vein Endothelial cell (HUVEC) gene expression of CD63+ and MHC-I+ EV subsets derived from WT T cells and CD47-KO T cells. The goal was to identify several functional gene families in HUVECs that are differentially regulated by the two EV populations. The present study uses biochemical approaches, genetic sequencing, and electron microscopy to evaluate the EVs isolated from the cell types. The study reveals that CD63+ and MHC-I+ EVs from CD47-KO T cells are enriched in small non-coding RNAs relative to EVs from wild-type T cells. CD47-deficient T cells secrete more CD63+ and MHC-I+ EVs, but MHC-I+ EVs are selectively taken up more by HUVECs. Transcriptomics analysis of endothelial cells treated with CD63+ or MHC-I+ EVs showed surface marker- and CD47-dependent changes in gene expression in the target cells. Gene set enrichment analysis identified effects of T cell EVs on VEGF and inflammatory signaling, cell cycle, and lipid and cholesterol metabolism. Thus, subsets of T cell EVs differentially regulate endothelial cell metabolism and inflammatory and angiogenic responses. Future studies will focus on identifying mechanisms by which CD63 and MHC-I regulate EV uptake and the distinct functional effects of MHC-I+ EVs and CD63+ EVs on endothelial cells and other types of target cells. These studies may identify additional functions of EVs produced by T cells that regulate physiological angiogenic and inflammatory pathways and antitumor immunity.