Endomembrane systems are reorganized by ORF3a and Membrane (M) of SARS-CoV-2

The endomembrane reticulum (ER) is largely reorganized by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 ORF3a and membrane (M) protein expression affects ER-derived structures including cubic membrane and double membrane vesicles in coronavirus-infected cells; however, the molecular mechanisms underlying ER remodeling remain unclear. We introduced a 'plug and playable' proximity labeling tool (TurboID-GBP) for interactome mapping of GFP-tagged SARS-CoV-2 ORF3a and M proteins. Through mass spectrometric identification of the biotinylated lysine residue (K+226 Da) on the viral proteins using Spot-TurboID workflow, 117 and 191 proteins were robustly determined as ORF3a and M interactomes, respectively, and many, including RNF5 (E3 ubiquitin ligase), overlap with the mitochondrial-associated membrane (MAM) proteome. RNF5 expression was correlated to ORF3a ubiquitination. MAM formation and secreted proteome profiles were largely affected by ORF3a expression. Thus, SARS-CoV-2 may utilize MAM as a viral assembly site, suggesting novel anti-viral treatment strategies for blocking viral replication in host cells.

Evolution of cubic membranes as antioxidant defence system

Possibly the best-characterized cubic membrane transition has been observed in the mitochondrial inner membranes of free-living giant amoeba ( Chaos carolinense ). In this ancient organism, the cells are able to survive in extreme environments such as lack of food, thermal and osmolarity fluctuations and high levels of reactive oxygen species. Their mitochondrial inner membranes undergo rapid changes in three-dimensional organization upon food depletion, providing a valuable model to study this subcellular adaptation. Our data show that cubic membrane is enriched with unique ether phospholipids, plasmalogens carrying very long-chain polyunsaturated fatty acids. Here, we propose that these phospholipids may not only facilitate cubic membrane formation but may also provide a protective shelter to RNA. The potential interaction of cubic membrane with RNA may reduce the amount of RNA oxidation and promote more efficient protein translation. Thus, recognizing the role of cubic membranes in RNA antioxidant systems might help us to understand the adaptive mechanisms that have evolved over time in eukaryotes.

A look through ‘lens’ cubic mitochondria

Cell membranes may fold up into three-dimensional nanoperiodic cubic structures in biological systems. Similar geometries are well studied in other disciplines such as mathematics, physics and polymer chemistry. The fundamental function of cubic membranes in biological systems has not been uncovered yet; however, their appearance in specialized cell types indicates a role as structural templates or perhaps direct physical entities with specialized biophysical properties. The mitochondria located at the inner segment of the retinal cones of tree shrew ( Tupaia glis and Tupaia belangeri ) contain unique patterns of concentric cristae with a highly ordered membrane arrangement in three dimensions similar to the photonic nanostructures observed in butterfly wing scales. Using a direct template matching method, we show that the inner mitochondrial membrane folds into multi-layered (8 to 12 layers) gyroid cubic membrane arrangements in the photoreceptor cells. Three-dimensional simulation data demonstrate that such multi-layer gyroid membrane arrangements in the retinal cones of a tree shrew's eye can potentially function as: (i) multi-focal lens; (ii) angle-independent interference filters to block UV light; and (iii) a waveguide photonic crystal. These theoretical results highlight for the first time the significance of multi-layer cubic membrane arrangements to achieve near-quasi-photonic crystal properties through the simple and reversible biological process of continuous membrane folding.

Cubic membranes: a structure-based design for DNA uptake

Cubic membranes are soft three-dimensional crystals found within cell organelles in a variety of living systems, despite the aphorism of Fedorov: ‘crystallization is death’. They consist of multi-bilayer lipid–protein stacks, folded onto anticlastic surfaces that resemble triply periodic minimal surfaces, forming highly swollen crystalline sponges. Although cubic membranes have been observed in numerous cell types and under different pathophysiological conditions, knowledge about the formation and potential function(s) of non-lamellar, cubic structures in biological systems is scarce. We report that mitochondria with this cubic membrane organization isolated from starved amoeba Chaos carolinense interact sufficiently with short segments of phosphorothioate oligonucleotides (PS-ODNs) to give significant ODNs uptake. ODNs condensed within the convoluted channels of cubic membrane by an unknown passive targeting mechanism. Moreover, the interaction between ODNs and cubic membrane is sufficient to retard electrophoretic mobility of the ODN component in the gel matrix. These ODN–cubic membrane complexes are readily internalized within the cytoplasm of cultured mammalian cells. Transmission electron microscopic analysis confirms ODNs uptake by cubic membranes and internalization of ODN–cubic membrane complexes into the culture cells. Cubic membranes thus may offer a new, potentially benign medium for gene transfection.

Cubic membranes: a legend beyond the Flatland* of cell membrane organization

Cubic membranes represent highly curved, three-dimensional nanoperiodic structures that correspond to mathematically well defined triply periodic minimal surfaces. Although they have been observed in numerous cell types and under different conditions, particularly in stressed, diseased, or virally infected cells, knowledge about the formation and function of nonlamellar, cubic structures in biological systems is scarce, and research so far is restricted to the descriptive level. We show that the “organized smooth endoplasmic reticulum” (OSER; Snapp, E.L., R.S. Hegde, M. Francolini, F. Lombardo, S. Colombo, E. Pedrazzini, N. Borgese, and J. Lippincott-Schwartz. 2003. J. Cell Biol. 163:257–269), which is formed in response to elevated levels of specific membrane-resident proteins, is actually the two-dimensional representation of two subtypes of cubic membrane morphology. Controlled OSER induction may thus provide, for the first time, a valuable tool to study cubic membrane formation and function at the molecular level.