Superresolution Imaging of Human Cytomegalovirus vMIA Localization in Sub-Mitochondrial Compartments
Shivaprasad Bhuvanendran,
Kyle Salka,
Kristin Rainey,
Sen Chandra Sreetama,
Elizabeth Williams,
Margretha Leeker,
Vidhya Prasad,
Jonathan Boyd,
George H. Patterson,
Jyoti K. Jaiswal,
Anamaris M. Colberg-Poley
Affiliations
Shivaprasad Bhuvanendran
Research Center for Genetic Medicine, Children's Research Institute, Children's National Health System, 111 Michigan Avenue, NW, Washington, DC 20010, USA
Kyle Salka
Research Center for Genetic Medicine, Children's Research Institute, Children's National Health System, 111 Michigan Avenue, NW, Washington, DC 20010, USA
Kristin Rainey
Section on Biophotonics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892, USA
Sen Chandra Sreetama
Research Center for Genetic Medicine, Children's Research Institute, Children's National Health System, 111 Michigan Avenue, NW, Washington, DC 20010, USA
Elizabeth Williams
Research Center for Genetic Medicine, Children's Research Institute, Children's National Health System, 111 Michigan Avenue, NW, Washington, DC 20010, USA
Margretha Leeker
Research Center for Genetic Medicine, Children's Research Institute, Children's National Health System, 111 Michigan Avenue, NW, Washington, DC 20010, USA
Vidhya Prasad
Research Center for Genetic Medicine, Children's Research Institute, Children's National Health System, 111 Michigan Avenue, NW, Washington, DC 20010, USA
Jonathan Boyd
Life Science Division, Leica Microsystems, Inc., 1700 Leider Lane, Buffalo Grove, IL 60089, USA
George H. Patterson
Section on Biophotonics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892, USA
Jyoti K. Jaiswal
Research Center for Genetic Medicine, Children's Research Institute, Children's National Health System, 111 Michigan Avenue, NW, Washington, DC 20010, USA
Anamaris M. Colberg-Poley
Research Center for Genetic Medicine, Children's Research Institute, Children's National Health System, 111 Michigan Avenue, NW, Washington, DC 20010, USA
The human cytomegalovirus (HCMV) viral mitochondria-localized inhibitor of apoptosis (vMIA) protein, traffics to mitochondria-associated membranes (MAM), where the endoplasmic reticulum (ER) contacts the outer mitochondrial membrane (OMM). vMIA association with the MAM has not been visualized by imaging. Here, we have visualized this by using a combination of confocal and superresolution imaging. Deconvolution of confocal microscopy images shows vMIA localizes away from mitochondrial matrix at the Mitochondria-ER interface. By gated stimulated emission depletion (GSTED) imaging, we show that along this interface vMIA is distributed in clusters. Through multicolor, multifocal structured illumination microscopy (MSIM), we find vMIA clusters localize away from MitoTracker Red, indicating its OMM localization. GSTED and MSIM imaging show vMIA exists in clusters of ~100–150 nm, which is consistent with the cluster size determined by Photoactivated Localization Microscopy (PALM). With these diverse superresolution approaches, we have imaged the clustered distribution of vMIA at the OMM adjacent to the ER. Our findings directly compare the relative advantages of each of these superresolution imaging modalities for imaging components of the MAM and sub-mitochondrial compartments. These studies establish the ability of superresolution imaging to provide valuable insight into viral protein location, particularly in the sub-mitochondrial compartments, and into their clustered organization.
