The role of mitochondria in cell metabolism and survival is controlled by calcium signals that are commonly transmitted at the close associations between mitochondria and endoplasmic reticulum (ER). However, the physical linkage of the ER–mitochondria interface and its relevance for cell function remains elusive. We show by electron tomography that ER and mitochondria are adjoined by tethers that are ∼10 nm at the smooth ER and ∼25 nm at the rough ER. Limited proteolysis separates ER from mitochondria, whereas expression of a short “synthetic linker” (<5 nm) leads to tightening of the associations. Although normal connections are necessary and sufficient for proper propagation of ER-derived calcium signals to the mitochondria, tightened connections, synthetic or naturally observed under apoptosis-inducing conditions, make mitochondria prone to Ca2+ overloading and ensuing permeability transition. These results reveal an unexpected dependence of cell function and survival on the maintenance of proper spacing between the ER and mitochondria.

The mechanism during apoptosis by which cytochrome c is rapidly and completely released in the absence of mitochondrial swelling is uncertain. Here, we show that two distinct pathways are involved. One mediates release of cytochrome c across the outer mitochondrial membrane, and another, characterized in this study, is responsible for the redistribution of cytochrome c stored in intramitochondrial cristae. We have found that the "BH3-only" molecule tBID induces a striking remodeling of mitochondrial structure with mobilization of the cytochrome c stores (approximately 85%) in cristae. This reorganization does not require tBID's BH3 domain and is independent of BAK, but is inhibited by CsA. During this process, individual cristae become fused and the junctions between the cristae and the intermembrane space are opened.

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Mitochondria are complex organelles with a highly dynamic distribution and internal organization. Here, we demonstrate that mitofilin, a previously identified mitochondrial protein of unknown function, controls mitochondrial cristae morphology. Mitofilin is enriched in the narrow space between the inner boundary and the outer membranes, where it forms a homotypic interaction and assembles into a large multimeric protein complex. Down-regulation of mitofilin in HeLa cells by using specific small interfering RNA lead to decreased cellular proliferation and increased apoptosis, suggesting abnormal mitochondrial function. Although gross mitochondrial fission and fusion seemed normal, ultrastructural studies revealed disorganized mitochondrial inner membrane. Inner membranes failed to form tubular or vesicular cristae and showed as closely packed stacks of membrane sheets that fused intermittently, resulting in a complex maze of membranous network. Electron microscopic tomography estimated a substantial increase in inner:outer membrane ratio, whereas no cristae junctions were detected. In addition, mitochondria subsequently exhibited increased reactive oxygen species production and membrane potential. Although metabolic flux increased due to mitofilin deficiency, mitochondrial oxidative phosphorylation was not increased accordingly. We propose that mitofilin is a critical organizer of the mitochondrial cristae morphology and thus indispensable for normal mitochondrial function. INTRODUCTIONMitochondria are the center of cellular energy production and essential metabolic reactions. As double membranebound organelles, mitochondria from different species, tissues, and metabolic states are highly polymorphic in nature yet exhibit common structural features. The ultrastructural variations in mitochondrial architecture occur mainly due to the differences in the amount and shape of cristae, which derive from the infolded inner membrane in which protein complexes of oxidative phosphorylation and intermediate metabolism are embedded. Abundant cristae are found in mitochondria from tissues where energy demand is high. For example, mitochondria with densely packed cristae are observed in the flight muscle of the dragonfly, whereas the liver of a winter-starved frog displays mitochondria with sparse cristae (Ghadially, 1997). Furthermore, the inner membranes of isolated mitochondria undergo characteristic morphological changes in response to the metabolic state (Hackenbrock, 1966). Although little is known about the molecular mechanisms regulating cristae biogenesis and architecture, recent studies have implicated proteins resident in both the outer and inner membrane to have roles in this process.The mitochondrial fission and fusion machinery plays an essential role in the dynamics, division, distribution, and morphology of the organelle (Yaffe, 1999;Jensen et al., 2000;Griparic and van der Bliek, 2001;Shaw and Nunnari, 2002). Three evolutionarily conserved large GTPases, Dnm1/ Drp1/Dlp1, Fzo1/mitofusin, and Mgm1/MspI/OPA1, are...

Cryo-electron microscopy can provide high-resolution structural information about cells and organelles in the nearly native, frozen-hydrated state. Applicability, however, is limited by difficulties encountered in preparing suitably thin, vitreously frozen biological specimens. We demonstrate, by cryo-electron tomography of Escherichia coli cells, that a focused ion beam (FIB) can be used to thin whole frozen-hydrated cells in a convenient and essentially artifact-free way.