Abstract
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.
Highlights
The mitochondria-associated membrane (MAM) sub-compartment of the endoplasmic reticulum (ER) plays critical roles in ER-mitochondrial cross-talk by allowing efficient transfer of calcium (Ca2+)from the ER to mitochondria without elevating cytosolic Ca2+ levels [1,2,3]
human cytomegalovirus (HCMV) viral mitochondria-localized inhibitor of apoptosis (vMIA) imaging by confocal/deconvolution microscopy indicated its outer mitochondrial membrane (OMM) localization, which was confirmed by gated STED (GSTED), MSIM, and PALM imaging
These imaging modalities all demonstrate that at the OMM vMIA is present in clusters, indicating nanoscale localization of HCMV vMIA at the mitochondrial periphery, away from intermembrane space (IMS) and matrix
Summary
The mitochondria-associated membrane (MAM) sub-compartment of the endoplasmic reticulum (ER) plays critical roles in ER-mitochondrial cross-talk by allowing efficient transfer of calcium (Ca2+). We have previously found that enhanced green fluorescent protein (EGFP) tagged vMIA partially co-localizes with MAM, lipid raft, and mitochondrial markers [20,21,23]. Multiple approaches have been developed to resolve fluorescent signals below diffraction limit and these include structured illumination (e.g., structured illumination microscopy, SIM; multifocal SIM, MSIM), reduction of point spread function by grounding emissions outside of the excitation center (e.g., stimulated emission depletion, STED; gated STED, GSTED) or activation of single fluorophores (e.g., photoactivated localization microscopy, PALM; stochastic optical reconstruction microscopy, STORM). Each of these approaches has its own strengths and weaknesses. Similar to MSIM, PALM provides the ability to image multiple colors and better resolution than confocal, but slower imaging speed
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