Superresolution imaging of human cytomegalovirus vMIA localization in sub-mitochondrial compartments.
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.
Research Center for Genetic Medicine, Children's Research Institute, Children's National Health System, 111 Michigan Avenue, NW, Washington, DC 20010, USA. SBhuvanendran@childrensnational.org.
Shivaprasad
S
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@gmail.com.
Kyle
K
Salka
Section on Biophotonics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892, USA. kristin.rainey@nih.gov.
Kristin
K
Rainey
Research Center for Genetic Medicine, Children's Research Institute, Children's National Health System, 111 Michigan Avenue, NW, Washington, DC 20010, USA. Sreetama.SenChandra@childrensnational.org.
Sen Chandra
SC
Sreetama
Research Center for Genetic Medicine, Children's Research Institute, Children's National Health System, 111 Michigan Avenue, NW, Washington, DC 20010, USA. elizabeth.anne.williams.umd@gmail.com.
Elizabeth
E
Williams
Research Center for Genetic Medicine, Children's Research Institute, Children's National Health System, 111 Michigan Avenue, NW, Washington, DC 20010, USA. maggie.leeker@gmail.com.
Margretha
M
Leeker
Research Center for Genetic Medicine, Children's Research Institute, Children's National Health System, 111 Michigan Avenue, NW, Washington, DC 20010, USA. VPrasad@childrensnational.org.
Vidhya
V
Prasad
Life Science Division, Leica Microsystems, Inc., 1700 Leider Lane, Buffalo Grove, IL 60089, USA. Jonathan.Boyd@Leica-Microsystems.com.
Jonathan
J
Boyd
Section on Biophotonics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892, USA. kristin.rainey@nih.gov.
George H
GH
Patterson
Research Center for Genetic Medicine, Children's Research Institute, Children's National Health System, 111 Michigan Avenue, NW, Washington, DC 20010, USA. jkjaiswal@childrensnational.org.
Jyoti K
JK
Jaiswal
Research Center for Genetic Medicine, Children's Research Institute, Children's National Health System, 111 Michigan Avenue, NW, Washington, DC 20010, USA. acolberg-poley@childrensnational.org.
Anamaris M
AM
Colberg-Poley
D003587:Cytomegalovirus / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D017874:Immediate-Early Proteins / Q000032:analysis*; D008853:Microscopy / Q000379:methods; D051336:Mitochondrial Membranes / Q000737:chemistry*; D061848:Optical Imaging / Q000379:methods*
D017874:Immediate-Early Proteins; C420323:UL37 protein, Human herpesvirus 5