Mitochondrial dynamics, and indeed, many mitochondrial functions, are influenced by direct contacts between mitochondria and the endoplasmic reticulum (ER). The portions of the ER that form special contact regions with the mitochondria, called mitochondria-associated membranes, or MAMs, can be visualized using electron microscopy and fluorescence microscopy (Figure 1). Their lipid and protein composition differs somewhat from that of the rest of the ER. In yeast, a protein complex called ERMES (ER- mitochondria encounter structure) has been proposed to mediate the reversible tethering of MAMs to mitochondria. The ERMES complex is not present in mammals; the proteins that mediate the tethering of MAMs to mitochondria in com plex multicellular organisms are as yet unknown. Tethering proteins hold the MAM and the outer mitochondrial mem brane about 10–30 nm apart.
Fig1. Specialized regions of the endoplasmic reticulum called mitochondria-associated membranes (MAMs) directly contact mitochondria and influence mitochondrial shape, function, and sites of fission. (a) Transmission electron microscopic (EM) image of a section through rat brown adipose (fat) tissue. The lumen of the endoplasmic reticulum (ER) is false colored to show a MAM (yellow) and the non-MAM, bulk ER (blue). The MAM is closely apposed to the outer mitochondrial membrane. (b) Three-dimensional model of a segment of a mitochondrion (red, only outer membrane shown) and the adjacent MAM (yellow) determined from a line of cultured avian lymphoma cells using EM tomography (assembly of a three-dimensional image from consecutive individual sections). (c) A three-dimensional model of a mitochondrion (red) and adjacent MAMs (green) from a yeast cell using EM tomography. The two MAM domains are derived from ER tubules that in some cases can wrap around the mitochondrion, in the top case forming a clamp-like structure that appears to constrict the mitochondrion in preparation for fission. (d) Live cell fluorescence microscopic images of a Cos-7 monkey cell, showing a mitochondrion (white in the top panels, same mitochondrion in red in the bottom panels) and MAM (green in bottom panels), taken from a single field of view at 10-second intervals. The arrow points to the site of constriction and fission on the mitochondrion and to the MAM at the constriction/fission site. The MAM directs constriction and subsequent DRP1-mediated fission at this site. To visualize the mitochondria and ER, the Cos-7 cells were transfected with cDNA vectors encoding two fluorescent proteins that specifically accumulate in either the mitochondrion (red fluorescence) or the ER (green fluorescence). [Part (a) de Meis L., Ketzer, L. A., da Costa R. M., de Andrade I. R., Benchimol M. (2010) Fusion of the Endoplasmic Reticulum and Mitochondrial Outer Membrane in Rats Brown Adipose Tissue: Activation of Thermogenesis by Ca2+. PLoS ONE 5(3): e9439.doi:10.1371/journal.pone.0009439. Part (b) ©2006 Csordas et al. The Journal of Cell Biology. 174:915–921. doi:10.1083/jcb.200604016. Parts (c) and (d) republished with permission from AAAS, from Friedman, J. R., et al., “ER tubules mark sites of mitochondrial division,” Science, 2011, 334(6054):358-62; permission conveyed through the Copyright Clearance Center, Inc.]
MAMs contribute significantly to many cellular processes (see Table 1), including mitochondrial fission. MAM-mitochondrial contacts can initiate mitochondrial constriction and help recruit DPR1, which completes mem brane fission (see Figure2c). In yeast, MAM tubules have been seen to loop completely around mitochondria, forming a clamp that constricts the mitochondrion (Figure 1c). In mammalian cells, the MAMs contact the mitochondria at fission sites, but they have not been shown to loop fully around the mitochondria (Figure 1d).
Table1. Multiple Functions of Mitochondria
Fig2. Mitochondria undergo rapid fusion and fission. (a) A human HeLa cell labeled with a mitochondrion-specific fluorescent dye (MitoTracker Green) was imaged using three-dimensional structured illumination fluorescence microscopy (a 6.1-μm-thick section through the cell is shown). The network of fused and branched mitochondria is seen in the cytoplasm, with only a few mitochondria observed above or below the nucleus (unstained central dark oval). The identity of the striations seen within the mitochondria is not known. The mitochondria are shown in artificial colors to indicate their positions relative to the surface to which the cell is attached (blue is closest to and red farthest from the surface). (b) Mitochondria labeled with a fluorescent protein in a live normal mouse embryonic fibroblast were observed using time-lapse fluorescence microscopy. Several mitochondria undergoing fusion (top) or fission (bottom) are artificially highlighted in blue and with arrows. (c) Mitochondrial fusion (top) and fission (bottom) are mediated by a set of GTPase enzymes (MFN1, MFN2, OPA1, and DRP1). The integral membrane proteins MFN1 and MFN2 (MFN1/2) mediate outer mitochondrial mem brane (OMM) fusion, which is followed by fusion of the inner mitochondrial membranes (IMM) mediated by the integral membrane protein OPA1. The matrix and inner membrane space (IMS) remain distinct. The soluble cytosolic GTPase DRP1 is recruited to a constricted site on the surface of a mitochondrion, where DRP1 polymers sever the membrane, resulting in fission. A variety of post-translational modifications of DRP1 regulate fission. (d) (Left) Rat liver cells (hepatocytes) one day after being removed from the liver and placed in cell culture, are stressed and depolarized (lack some of the morphological and biochemical properties of epithelial cells), have low levels of oxidative phosphorylation and ATP production, and have fragmented mitochondria (visualized by staining with MitoTracker Green). (Right) After growth in culture for six days, the hepatocytes become polarized, their mitochondria fuse, forming an extensive network, and the cells exhibit high levels of oxidative phosphorylation and ATP production. Insets show higher-magnification views of the mitochondria. [(a) Reprinted by permission from Macmillan Publishers Ltd: Shao et al., “Super-resolution 3D microscopy of live whole cells using structured illumination,” Nature Methods, 8:12, 1044-1046, Fig. S4, 2011, courtesy of Mats Gustafsson. (b) Republished with permission from Elsevier. Modified from Chan D. C., “Mitochondria: Dynamic Organelles in Disease, Aging, and Development,” Cell, 2006, 125(7):1241–52. Permission conveyed through Copyright Clearance Center, Inc. (c) Information from P. Mishra and D. C. Chan, 2014, Nat. Rev. Mol. Cell Biol. 15:634–646. (d) From Proc. Natl. Acad. Sci. USA 2013. 110(18):7288-7293, Fig. 3 Day 1 and Day 6. “Coordinated elevation of mitochondrial oxidative phosphorylation and autophagy help drive hepatocyte polarization,” by Fu, D. et al. Courtesy Jennifer Lippincott-Schwartz.]
MAMs also play an integral role in intracellular calcium and energy metabolism. Variations in the concentrations of calcium ions in intracellular compartments—cytosolic calcium ([Ca2+]c), mitochondrial calcium ([Ca2+]m), and calcium in the ER ([Ca2+]er)—are employed to control a wide variety of activities within cells, a process called calcium signaling. Calcium is also important for extra cellular processes, such as the activity of some blood-clotting proteins. Intramitochondrial calcium ions play an important role in controlling mitochondrial function, and MAMs mediate this control by delivering calcium from the ER to mitochondria. For example, an increase in [Ca2+]m in the matrix can increase mitochondrial production of ATP. Increased [Ca2+]m directly increases the activities of three mitochondrial enzymes that produce NADH from NAD+: pyruvate dehydrogenase and α-ketoglutarate and isocitrate dehydrogenases. As we shall see later in this chapter, NADH provides high- energy electrons for ATP synthesis. Thus continuous low-level release of Ca2+ from MAMs into mitochondria is necessary for ATP synthesis when cells are in a basal, or resting, state. Increased delivery of Ca2+ via MAMs can occur when cells require more ATP—for example, when muscle cells are stimulated to contract. Strikingly, calcium signaling is used both to induce muscle contraction and coordinately to increase mitochondrial ATP synthesis to provide the energy to fuel that contraction. When [Ca2+]m is elevated, mitophagy can be induced. Indeed, mitochondrial calcium overload can activate regulated cell death pathways. Thus the control of [Ca2+]m can literally control the life and death of cells.
