Review Structure and dynamics of the mitochondrial inner membrane cristae Carmen A. Mannella ��� Resource for Visualization of Biological Complexity, Wadsworth Center, Empire State Plaza, Albany, NY 12201-0509, USA Received 3 April 2006 received in revised form 6 April 2006 accepted 10 April 2006 Available online 20 April 2006 Abstract Three-dimensional images of mitochondria provided by electron tomography reveal that the micro-compartments (cristae) defined by the inner membrane are connected to the periphery of this membrane by narrow tubular junctions, which likely restrict diffusion. The tomograms also strongly suggest that inner membrane topology represents a balance between membrane fusion and fission processes. The hypothesis being developed is that inner membrane topology is a regulated property of mitochondria. This review summarizes the evidence about how inner membrane shape influences mitochondrial function and, conversely, what is known about the factors that determine this membrane's topology. �� 2006 Elsevier B.V. All rights reserved. Keywords: Mitochondria Electron microscopy Electron tomography Membrane topology Bioenergetics Cardiolipin The first images of the internal structure of mitochondria were provided over 50 years ago by transmission electron microscopy (TEM) of thin sections of chemically fixed and plastic-embedded specimens [1]. It was soon apparent that the mitochondrial inner membrane can assume an astounding variety of morphologies [2], even though it has the same two basic functions in every cell: to serve as the scaffold for the assembly and operation of the respiratory chain complexes, and to provide the permeability barrier across which the respiratory machinery generates its chemiosmotic gradient. In the last 10 years, the technique of electron tomography ��� employ- ing advanced, higher voltage electron microscopes and computer-based reconstruction algorithms ��� has afforded biologists true three-dimensional images of mitochondria, first in thick plastic sections [3���8] and more recently in frozen- hydrated suspensions [8���10] or tissue sections [11,12]. While the results have underscored the tremendous pleomorphism of the mitochondrial inner membrane, they also point to conserved design principles and to an important role for membrane dynamics in maintaining membrane morphology. Moreover, these studies suggest that the diversity in inner membrane shape and the changes observed under certain conditions might not be random, but rather might reflect a novel regulatory mechanism. The growing implication is that certain mitochondrial functions are affected (or even effected) by changes in mitochondrial membrane topology, e.g., by altering the diffusion of metabo- lites and proteins between internal compartments [4,8,10]. This review is intended to highlight recent findings relevant to the influence of inner membrane shape on mitochondrial function and the identification of factors that, in turn, regulate this membrane's topology. 1. Morphology and implied dynamics of mitochondrial inner membranes The mitochondrion is structurally defined, in large measure, by its two membranes: a limiting outer membrane that enwraps the energy-transducing inner membrane, which in turn encloses a dense, protein-rich matrix. The outer membrane is topolog- ically simple, varying in shape depending on whether the mitochondrion is in a cell (usually tubular when attached to the cytoskeleton, sometimes reticulated) or isolated in suspension (ellipsoidal or spherical). The inner membrane, which has a larger surface area than the outer membrane, contains features referred to as cristae (literally, crests) which have long been represented as simple infoldings of this membrane [1,7]. Although this ���baffle��� interpretation was not universally accepted (e.g., [13]), it rapidly found its way into the textbooks. Subsequent electron tomographic analyses of a variety of Biochimica et Biophysica Acta 1763 (2006) 542 ��� 548 http://www.elsevier.com/locate/bba ��� Tel.: +1 518 474 2462 fax: +1 518 402 5381. E-mail address: carmen@wadsworth.org. 0167-4889/$ - see front matter �� 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2006.04.006

mitochondria, isolated and in situ, have provided overwhelming evidence that cristae are not simply random folds in the inner membrane but rather internal compartments formed by invagination of the membrane (Figs. 1 and 2). These invaginations originate at narrow neck-like segments, called pediculi cristae (crista feet) when first detected by conventional TEM [13] and later crista junctions [5]. Rat-liver mitochondria display highly variable crista mor- phology, even within a single organelle. In the frozen-hydrated mitochondrion shown in Fig. 1, the inner membrane comprises curved, tubular regions that co-exist or merge with flattened lamellar segments. The overall impression is one of a highly fluid, interconnected membrane continuum. Hackenbrock [14] defined two morphologic states in chemically fixed mitochon- dria, condensed and orthodox, which differed primarily in the relative states of expansion or contraction of the matrix and intracristal compartments. Three-dimensional analyses of rat- liver mitochondria in these states indicate major differences in inner membrane topology (Fig. 2). Cristae in the orthodox (matrix expanded) state tend to be tubes or short flat lamellae with one or two openings (junctions) in the peripheral region of the inner membrane. Condensed (matrix compacted) mitochon- dria (Fig. 2, also Fig. 1) have larger internal compartments with multiple tubular connections to the peripheral region and to each other. Interconversion of these states can only be achieved by the inner membrane undergoing fusion and fission [8]. Matrix compression is accompanied by fusion of individual cristae into larger compartments, while matrix expansion requires fissioning of the large cisternae into individual tubes or lamellae and (with extreme matrix swelling) recruitment of membrane from the interior to the periphery. Analyses of a variety of mitochondria reveal inner membrane morphologies that appear to represent extreme states of membrane fusion or fission (Fig. 3). Thus, the ���typical��� crista morphology represented by mitochondria like those of Figs. 1 and 2 likely represents a balance between intra-mitochondrial membrane fusion and fission processes [10]. As discussed below, this raises the possibility that inner membrane morphology is regulated, at least in part, by the same types of proteins that control inter-mitochondrial fission and fusion. 2. Sites of inner membrane invagination: crista junctions Tubular connections between cristae and the peripheral or boundary region of the mitochondrial inner membrane have been observed in mitochondria from protozoa (amoeba), fungi, and metazoa from nematodes to flies to mammals [3��� 9,15���17]. The junctions have been shown to form spontane- ously in yeast mitochondria after extreme osmotic swelling and re-contraction of the inner membrane, indicating that their formation is energetically favored [8]. However, while crista junctions are ubiquitous, they are not structurally constant. Perkins et al. reported that crista junctions in neuronal mitochondria have uniformly circular cross-sections with diameters of ���28 nm [5]. However, in rat-liver mitochondria the cross-section of the junctions can vary from circular to elliptical (short slots) with openings typically in the range 20���50 nm [3,4,8]. The crista junctions in Neurospora mitochondria have been reported to be exclusively slot-like Fig. 1. Model derived from cryo-electron tomography of the membranes in an intact, frozen-hydrated mitochondrion isolated from rat liver. This mitochon- drion has a diameter of 700 nm. Fig. 2. Change in mitochondrial inner membrane topology associated with the orthodox-condensed transition. Model on left reproduced from [4] with permission of Elsevier Science. Model on right reproduced from C.A. Mannella, (2004) Mitochondrial Membranes, Structural Organization, in Encyclopedia of Biological Chemistry (W.J. Lennarz and M.D. Lane, eds), vol. 2, pp. 720���724, with permission of Elsevier Science. Mitochondria (left to right) have diameters of 1500 nm and 500 nm. 543 C.A. Mannella / Biochimica et Biophysica Acta 1763 (2006) 542���548