Pioneering biochemical studies have long forged the concept that the mitochondria are the 'energy powerhouse of the cell'. These studies, combined with the unique evolutionary origin of the mitochondria, led the way to decades of research focusing on the organelle as an essential, yet independent, functional component of the cell. Recently, however, our conceptual view of this isolated organelle has been profoundly altered with the discovery that mitochondria function within an integrated reticulum that is continually remodeled by both fusion and fission events. The identification of a number of proteins that regulate these activities is beginning to provide mechanistic details of mitochondrial membrane remodeling. However, the broader question remains regarding the underlying purpose of mitochondrial dynamics and the translation of these morphological transitions into altered functional output. One hypothesis has been that mitochondrial respiration and metabolism may be spatially and temporally regulated by the architecture and positioning of the organelle. Recent evidence supports and expands this idea by demonstrating that mitochondria are an integral part of multiple cell signaling cascades. Interestingly, proteins such as GTPases, kinases and phosphatases are involved in bi-directional communication between the mitochondrial reticulum and the rest of the cell. These proteins link mitochondrial function and dynamics to the regulation of metabolism, cell-cycle control, development, antiviral responses and cell death. In this review we will highlight the emerging evidence that provides molecular definition to mitochondria as a central platform in the execution of diverse cellular events.

Mitochondria and peroxisomes share a number of common biochemical processes, including the beta oxidation of fatty acids and the scavenging of peroxides. Here, we identify a new outer-membrane mitochondria-anchored protein ligase (MAPL) containing a really interesting new gene (RING)-finger domain. Overexpression of MAPL leads to mitochondrial fragmentation, indicating a regulatory function controlling mitochondrial morphology. In addition, confocal- and electron-microscopy studies of MAPL-YFP led to the observation that MAPL is also incorporated within unique, DRP1-independent, 70-100 nm diameter mitochondria-derived vesicles (MDVs). Importantly, vesicles containing MAPL exclude another outer-membrane marker, TOM20, and vesicles containing TOM20 exclude MAPL, indicating that MDVs selectively incorporate their cargo. We further demonstrate that MAPL-containing vesicles fuse with a subset of peroxisomes, marking the first evidence for a direct relationship between these two functionally related organelles. In contrast, a distinct vesicle population labeled with TOM20 does not fuse with peroxisomes, indicating that the incorporation of specific cargo is a primary determinant of MDV fate. These data are the first to identify MAPL, describe and characterize MDVs, and define a new intracellular transport route between mitochondria and peroxisomes.

Mitochondrial fusion in higher eukaryotes requires at least two essential GTPases, Mitofusin 1 and Mitofusin 2 (Mfn2). We have created an activated mutant of Mfn2, which shows increased rates of nucleotide exchange and decreased rates of hydrolysis relative to wild type Mfn2. Mitochondrial fusion is stimulated dramatically within heterokaryons expressing this mutant, demonstrating that hydrolysis is not requisite for the fusion event, and supporting a role for Mfn2 as a signaling GTPase. Although steady-state mitochondrial fusion required the conserved intermembrane space tryptophan residue, this requirement was overcome within the context of the hydrolysis-deficient mutant. Furthermore, the punctate localization of Mfn2 is lost in the dominant active mutants, indicating that these sites are functionally controlled by changes in the nucleotide state of Mfn2. Upon staurosporine-stimulated cell death, activated Bax is recruited to the Mfn2-containing puncta; however, Bax activation and cytochrome c release are inhibited in the presence of the dominant active mutants of Mfn2. The dominant active form of Mfn2 also protected the mitochondria against free radicalinduced permeability transition. In contrast to staurosporine-induced outer membrane permeability transition, pore opening induced through the introduction of free radicals was dependent upon the conserved intermembrane space residue. This is the first evidence that Mfn2 is a signaling GTPase regulating mitochondrial fusion and that the nucleotidedependent activation of Mfn2 concomitantly protects the organelle from permeability transition. The data provide new insights into the critical relationship between mitochondrial membrane dynamics and programmed cell death.

The mitochondrial protein apoptosis-inducing factor (AIF) translocates to the nucleus and induces apoptosis. Recent studies, however, have indicated the importance of AIF for survival in mitochondria. In the absence of a means to dissociate these two functions, the precise roles of AIF remain unclear. Here, we dissociate these dual roles using mitochondrially anchored AIF that cannot be released during apoptosis. Forebrain-specific AIF null (tel. AifDelta) mice have defective cortical development and reduced neuronal survival due to defects in mitochondrial respiration. Mitochondria in AIF deficient neurons are fragmented with aberrant cristae, indicating a novel role of AIF in controlling mitochondrial structure. While tel. AifDelta Apaf1(-/-) neurons remain sensitive to DNA damage, mitochondrially anchored AIF expression in these cells significantly enhanced survival. AIF mutants that cannot translocate into nucleus failed to induce cell death. These results indicate that the proapoptotic role of AIF can be uncoupled from its physiological function. Cell death induced by AIF is through its proapoptotic activity once it is translocated to the nucleus, not due to the loss of AIF from the mitochondria.

Of the GTPases involved in the regulation of the fusion machinery, mitofusin 2 (Mfn2) plays an important role in the nervous system as point mutations of this isoform are associated with Charcot Marie Tooth neuropathy. Here, we investigate whether Mfn2 plays a role in the regulation of neuronal injury. We first examine mitochondrial dynamics following different modes of injury in cerebellar granule neurons. We demonstrate that neurons exposed to DNA damage or oxidative stress exhibit extensive mitochondrial fission, an early event preceding neuronal loss. The extent of mitochondrial fragmentation and remodeling is variable and depends on the mode and the severity of the death stimuli. Interestingly, whereas mitofusin 2 loss of function significantly induces cell death in the absence of any cell death stimuli, expression of mitofusin 2 prevents cell death following DNA damage, oxidative stress, and K ؉ deprivation induced apoptosis. More importantly, whereas wild-type Mfn2 and the hydrolysis-deficient mutant of Mfn2 (Mfn2 RasG12V ) function equally to promote fusion and lengthening of mitochondria, the activated Mfn2 RasG12V mutant shows a significant increase in the protection of neurons against cell death and release of proapoptotic factor cytochrome c. These findings highlight a signaling role for Mfn2 in the regulation of apoptosis that extends beyond its role in mitochondrial fusion.It has been recently demonstrated that the apoptotic program includes the regulated induction of mitochondrial fragmentation (1, 2). In addition, it has been shown that the rate of mitochondrial fusion is reduced early in the apoptotic program (3, 4), which together with the activation of the fission machinery leads to a morphological shift of the mitochondria to the fragmented state. The regulatory mechanisms and functional importance of these events during death remain unclear. For example, it is not clear whether the inhibition of mitochondrial fusion is an essential step in apoptosis or if fragmentation is promoted mainly because of the increase in fission. In addition, it has been shown that the loss of either Drp1 or hFis1 delayed, but did not block cell death, questioning the importance of the mitochondria morphological shift in the apoptotic cascade (5, 6). In this context, there is an emerging emphasis on the examination of mitochondrial fusion in the context of cell death.Mitochondrial fusion is regulated by at least three essential GTPases, the outer membrane-anchored proteins mitofusin 1 (Mfn1), 3 and mitofusin 2 (Mfn2) along with the intermembrane space GTPase, Opa1 (7,8). Although the functions of Mfn2 overlap with Mfn1 in the process of mitochondrial fusion (9), there are clear distinctions between these two GTPases. Perhaps most informative of their distinct function is their biochemical difference in nucleotide binding and hydrolysis properties, where Mfn1 has a faster GTPase hydrolysis rate and higher affinity for nucleotide relative to Mfn2 (10, 11). In addition, Mfn1, but not Mfn2, has been shown to geneticall...

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Mitochondrial morphology with its two membrane system, intricate cristae architecture and numerous links to the endoplasmic reticulum and other organelles has been well characterized by electron microscopists throughout the ages. Studies initially directed towards the dissection of mitochondrial division ultimately led to the identification of small mitochondrial fragments which appeared to carry only select proteins. Given the previous assumption that mitochondrial protein import should be equal in all organelles, the appearance of structures demonstrating only certain specific proteins was unusual. Ultrastructural analysis of these structures in intact cells revealed previously uncharacterized, vesicular profiles emanating from the sides of otherwise intact mitochondria. The formation of these vesicles did not depend on the known mitochondrial fission machinery, consistent with a novel machinery. Indeed, the edges of these structures appeared electron dense, suggesting some coat-related structure, and were of uniform size, between 70-120 nm in diameter. Pre-embedded immunogold labeling for the mitochondrial marker protein MAPL (mitochondria-anchored protein ligase), shown to transport in vesicles to the peroxisome, demonstrated that many of the vesicles carry both single and double membranes. Further experimentation revealed that vesicles that did not contain MAPL instead carry oxidized proteins that are subsequently delivered to the lysosomes for degradation. In vitro generation of mitochondrial derived vesicles allowed their separation by sucrose density gradients, and subsequent ultrastructural analysis of the fractions demonstrated the enrichment of single vs. double membrane structures at unique buoyant densities. Taken together, the combination of confocal, EM and biochemical reconstitution has revealed the presence of a novel vesicular transport route between the mitochondria, peroxisomes and lysosomes.References:[1] Neuspiel, M., Schauss, A.C., Braschi, E., Zunino, R., Rippstein, P., Rachubinski, R.A., Andrade-Navarro, M.A., and McBride, H.M. Cargo-selected transport from the mitochondria to peroxisomes is mediated by vesicular carriers. Current Biology. 18(2), 102-108, 2008.[2] Zunino, R., Rippstein, P., Andrade-Navarro, M., and McBride, H.M. The SUMO protease SenP5 is required to maintain mitochondrial morphology and function. . The SUMO protease SenP5 is required to maintain mitochondrial morphology and function.