Superresolution Imaging, Trafficking, and Clustering of a Human Cytomegalovirus Immediate Early Protein on the Outer Mitochondrial Membrane
Open AccessThe human cytomegalovirus (HCMV) is a beta-herpesvirus that is widespread across the world and is believed to have infected 50-80% of individuals in the U.S. and developing countries. The HCMV UL37 exon 1 (UL37x1) codes for an anti-apoptotic protein - the viral mitochondrial-localized inhibitor of apoptosis (vMIA). vMIA’s anti-apoptotic functions are critical for the production of viral progeny. Additionally, vMIA induces Ca2+ efflux from the ER, decreases ATP production in the mitochondria, regulates lipid synthesis, and disrupts actin cytoskeleton polymerization. vMIA traffics sequentially from the endoplasmic reticulum (ER) to the mitochondria-associated membrane (MAM) and outer mitochondrial membrane (OMM) where it sequesters the pro-apoptotic protein Bax and prevents Bax mediated OMM permeabilization. vMIA is N-terminally anchored to the ER, MAM, and OMM and localizes in cholesterol enriched lipid domains (rafts) in the MAM. The mechanism of vMIA trafficking and its distribution on the OMM is not well understood. Through use of imaging techniques including confocal imaging, superresolution imaging (multifocal structured illumination microscopy (MSIM), gated stimulated emission depletion (GSTED), and photoactivated localization microscopy (PALM)), as well as fluorescence lifetime microscopy (FLIM) we have shown here that vMIA localizes to the OMM in clusters sizes of ~100-150 nm. We utilized two vMIA mutants, a cholesterol binding domain mutant (CBDII) and a high hydrophobicity (HHB) leader mutant, in order to determine their roles in vMIA clustering. The CBDII mutant is not able to localize to lipid rafts of the MAM; however, it is still able to traffic to the OMM while the HHB mutant is mislocalized to the ER and secretory apparatus. Using MSIM and FLIM, we showed that these mutants were defective in their ability to cluster, suggesting the importance of cholesterol binding in vMIA clustering at the OMM. We also investigated the role of cellular proteins mitofusin 1 (MFN1) and mitofusin 2 (MFN2), which tether the mitochondria at the MAM and facilitate mitochondrial fusion on the localization and distribution of vMIA on the mitochondria. Knockout of both mitofusins did not affect the trafficking of vMIA to the OMM and did not affect its ability to cluster on the OMM. Overexpression of MFN2 also did not affect the localization of vMIA to the OMM. These results suggest that trafficking of vMIA from the MAM to OMM does not require these mitofusin tethers, which bridge these two organelles together. Above results identify the ability of vMIA to form clusters and show that vMIA trafficking and clustering is affected by its hydrophobic and lipid interactions and not by the mitofusin-based tethering of ER and mitochondria.
- All rights reserved
Notice to Authors
If you are the author of this work and you have any questions about the information on this page, please use the Contact form to get in touch with us.