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The X-linked disease Barth syndrome (BTHS) is caused by mutations in TAZ; TAZ is the main determinant of the final acyl chain composition of the mitochondrial-specific phospholipid, cardiolipin. To date, a detailed characterization of endogenous TAZ has only been performed in yeast. Further, why a given BTHS-associated missense mutation impairs TAZ function has only been determined in a yeast model of this human disease. Presently, the detailed characterization of yeast tafazzin harboring individual BTHS mutations at evolutionarily conserved residues has identified seven distinct loss-of-function mechanisms caused by patient-associated missense alleles. However, whether the biochemical consequences associated with individual mutations also occur in the context of human TAZ in a validated mammalian model has not been demonstrated. Here, utilizing newly established monoclonal antibodies capable of detecting endogenous TAZ, we demonstrate that mammalian TAZ, like its yeast counterpart, is localized to the mitochondrion where it adopts an extremely protease-resistant fold, associates non-integrally with intermembrane space-facing membranes and assembles in a range of complexes. Even though multiple isoforms are expressed at the mRNA level, only a single polypeptide that co-migrates with the human isoform lacking exon 5 is expressed in human skin fibroblasts, HEK293 cells, and murine heart and liver mitochondria. Finally, using a new genome-edited mammalian BTHS cell culture model, we demonstrate that the loss-of-function mechanisms for two BTHS alleles that represent two of the seven functional classes of BTHS mutation as originally defined in yeast, are the same when modeled in human TAZ.

mtDNA sequence alterations are challenging to generate but desirable for basic studies and potential correction of mtDNA diseases. Here, we report a new method for transferring isolated mitochondria into somatic mammalian cells using a photothermal nanoblade, which bypasses endocytosis and cell fusion. The nanoblade rescued the pyrimidine auxotroph phenotype and respiration of ρ0 cells that lack mtDNA. Three stable isogenic nanoblade-rescued clones grown in uridine-free medium showed distinct bioenergetics profiles. Rescue lines 1 and 3 reestablished nucleus-encoded anapleurotic and catapleurotic enzyme gene expression patterns and had metabolite profiles similar to the parent cells from which the ρ0 recipient cells were derived. By contrast, rescue line 2 retained a ρ0 cell metabolic phenotype despite growth in uridine-free selection. The known influence of metabolite levels on cellular processes, including epigenome modifications and gene expression, suggests metabolite profiling can help assess the quality and function of mtDNA-modified cells.

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Defects in mitochondrial fusion or fission are associated with many pathologies, raising the hope that pharmacological manipulation of mitochondrial dynamics may have therapeutic benefit. This approach assumes that organ physiology can be restored by rebalancing mitochondrial dynamics, but this concept remains to be validated. We addressed this issue by analyzing mice deficient in Mff, a protein important for mitochondrial fission. Mff mutant mice die at 13 wk as a result of severe dilated cardiomyopathy leading to heart failure. Mutant tissue showed reduced mitochondrial density and respiratory chain activity along with increased mitophagy. Remarkably, concomitant deletion of the mitochondrial fusion gene Mfn1 completely rescued heart dysfunction, life span, and respiratory chain function. Our results show for the first time that retuning the balance of mitochondrial fusion and fission can restore tissue integrity and mitochondrial physiology at the whole-organ level. Examination of liver, testis, and cerebellum suggest, however, that the precise balance point of fusion and fission is cell type specific.

None of the 28 identified point mutations in tafazzin (Taz1p), which is the mutant gene product associated with Barth syndrome (BTHS), has a biochemical explanation. In this study, endogenous Taz1p was localized to mitochondria in association with both the inner and outer mitochondrial membranes facing the intermembrane space (IMS). Unexpectedly, Taz1p does not contain transmembrane (TM) segments. Instead, Taz1p membrane association involves a segment that integrates into, but not through, the membrane bilayer. Residues 215-232, which were predicted to be a TM domain, were identified as the interfacial membrane anchor by modeling four distinct BTHS mutations that occur at conserved residues within this segment. Each Taz1p mutant exhibits altered membrane association and is nonfunctional. However, the basis for Taz1p dysfunction falls into the following two categories: (1) mistargeting to the mitochondrial matrix or (2) correct localization associated with aberrant complex assembly. Thus, BTHS can be caused by mutations that alter Taz1p sorting and assembly within the mitochondrion, indicating that the lipid target of Taz1p is resident to IMS-facing leaflets.

Mitochondrial fission and fusion are linked to synaptic activity in healthy neurons and are implicated in the regulation of apoptotic cell death in many cell types. We developed fluorescence microscopy and computational strategies to directly measure mitochondrial fission and fusion frequencies and their effects on mitochondrial morphology in cultured neurons. We found that the rate of fission exceeds the rate of fusion in healthy neuronal processes, and, therefore, the fission/fusion ratio alone is insufficient to explain mitochondrial morphology at steady state. This imbalance between fission and fusion is compensated by growth of mitochondrial organelles. Bcl-[x.sub.L] increases the rates of both fusion and fission, but more important for explaining the longer organelle morphology induced by Bcl-[x.sub.L] is its ability to increase mitochondrial biomass. Deficits in these Bcl-[x.sub.L]--dependent mechanisms may be critical in neuronal dysfunction during the earliest phases of neurodegeneration, long before commitment to cell death.

[alpha]-Synuclein ([alpha]-syn), a protein of unknown function, is the most abundant protein in Lewy bodies, the histological hallmark of Parkinson's disease (PD). In yeast [alpha]-syn inhibits endoplasmic reticulum (ER)-to-Golgi (ER [right arrow] Golgi) vesicle trafficking, which is rescued by overexpression of a Rab GTPase that regulates ER [right arrow] Golgi trafficking. The homologous Rab1 rescues [alpha]-syn toxicity in dopaminergic neuronal models of PD. Here we investigate this conserved feature of [alpha]-syn pathobiology. In a cell-free system with purified transport factors [alpha]-syn inhibited ER [right arrow] Golgi trafficking in an [alpha]-syn dose-dependent manner. Vesicles budded efficiently from the ER, but their docking or fusion to Golgi membranes was inhibited. Thus, the in vivo trafficking problem is due to a direct effect of [alpha]-syn on the transport machinery. By ultrastructural analysis the earliest in vivo defect was an accumulation of morphologically undocked vesicles, starting near the plasma membrane and growing into massive intracellular vesicular clusters in a dose-dependent manner. By immunofluorescence/immunoelectron microscopy, these clusters were associated both with [alpha]-syn and with diverse vesicle markers, suggesting that [alpha]-syn can impair multiple trafficking steps. Other Rabs did not ameliorate [alpha]-syn toxicity in yeast, but RAB3A, which is highly expressed in neurons and localized to presynaptic termini, and RAB8A, which is localized to post-Golgi vesicles, suppressed toxicity in neuronal models of PD. Thus, [alpha]-syn causes general defects in vesicle trafficking, to which dopaminergic neurons are especially sensitive. endoplasmic reticulum | Rab GTPase | yeasts | vesicle trafficking | Golgi

Spores of the anaerobic bacterium Clostridium novyi NT are able to germinate in and destroy hypoxic regions of tumors in experimental animals. Future progress in this area will benefit from a better understanding of the germination and outgrowth processes that are essential for the tumorilytic properties of these spores. Toward this end, we have used both transmission electron microscopy and atomic force microscopy to determine the structure of both dormant and germinating spores. We found that the spores are surrounded by an amorphous layer intertwined with honeycomb parasporal layers. Moreover, the spore coat layers had apparently self-assembled, and this assembly was likely to be governed by crystal growth principles. During germination and outgrowth, the honeycomb layers, as well as the underlying spore coat and undercoat layers, sequentially dissolved until the vegetative cell was released. In addition to their implications for understanding the biology of C. novyi NT, these studies document the presence of proteinaceous growth spirals in a biological organism.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2007.06.026 Byline: Hsiuchen Chen (1), J. Michael McCaffery (2), David C. Chan (1) Keywords: CELLBIO; HUMDISEASE; MOLNEURO Abstract: Mutations in the mitochondrial fusion gene Mfn2 cause the human neurodegenerative disease Charcot-Marie-Tooth type 2A. However, the cellular basis underlying this relationship is poorly understood. By removing Mfn2 from the cerebellum, we established a model for neurodegeneration caused by loss of mitochondrial fusion. During development and after maturity, Purkinje cells require Mfn2 but not Mfn1 for dendritic outgrowth, spine formation, and cell survival. In vivo, cell culture, and electron microscopy studies indicate that mutant Purkinje cells have aberrant mitochondrial distribution, ultrastructure, and electron transport chain activity. In fibroblasts lacking mitochondrial fusion, the majority of mitochondria lack mitochondrial DNA nucleoids. This deficiency provides a molecular mechanism for the dependence of respiratory activity on mitochondrial fusion. Our results show that exchange of mitochondrial contents is important for mitochondrial function as well as organelle distribution in neurons and have important implications for understanding the mechanisms of neurodegeneration due to perturbations in mitochondrial fusion. Author Affiliation: (1) Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA (2) Integrated Imaging Center, Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA Article History: Received 16 February 2007; Revised 11 May 2007; Accepted 12 June 2007 Article Note: (miscellaneous) Published: August 9, 2007

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.09.021 Byline: Shelly Meeusen (1), Rachel DeVay (1), Jennifer Block (1), Ann Cassidy-Stone (1), Sarah Wayson (1), J. Michael McCaffery (2), Jodi Nunnari (1) Abstract: Mitochondrial outer- and inner-membrane fusion events are coupled in vivo but separable and mechanistically distinct in vitro, indicating that separate fusion machines exist in each membrane. Outer-membrane fusion requires trans interactions of the dynamin-related GTPase Fzo1, GTP hydrolysis, and an intact inner-membrane proton gradient. Inner-membrane fusion also requires GTP hydrolysis but distinctly requires an inner-membrane electrical potential. The protein machinery responsible for inner-membrane fusion is unknown. Here, we show that the conserved intermembrane-space dynamin-related GTPase Mgm1 is required to tether and fuse mitochondrial inner membranes. We observe an additional role of Mgm1 in inner-membrane dynamics, specifically in the maintenance of crista structures. We present evidence that trans Mgm1 interactions on opposing inner membranes function similarly to tether and fuse inner membranes as well as maintain crista structures and propose a model for how the mitochondrial dynamins function to facilitate fusion. Author Affiliation: (1) Section of Molecular and Cellular Biology, Center of Genetics and Development, University of California, Davis, Davis, CA 95616, USA (2) Department of Biology and Integrated Imaging Center, Johns Hopkins University, Baltimore, MD 21218, USA Article History: Received 4 May 2006; Revised 19 June 2006; Accepted 4 September 2006 Article Note: (miscellaneous) Published: October 19, 2006

This study was undertaken to investigate the molecular constituents mediating LS174T colon adenocarcinoma cell adhesion to 4-h TNF-[alpha]-stimulated human umbilical vein endothelial cells (HUVECs) under flow. At 1 dyn/[cm.sup.2], ~57% of cells rolled and then became firmly adherent, whereas others continuously rolled on endothelium. Initial cell binding was primarily mediated by endothelial E-selectin. By using neuraminidase, glycolipid biosynthesis inhibitor d,l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-l-propanol*HCl, trypsin, and flow cytometry, LS174T cells were shown to express sialyl [Lewis.sup.x] ([sLe.sup.x])- and di-[sLe.sup.x]-decorated, but not [sLe.sup.a]-decorated, glycolipid and glycoprotein ligands for E-selectin. The cells preferentially employed sialylated glycoproteins over glycolipids in adhesion as measured by conversion of rolling to firm adhesion, resistance to detachment by increased shear stress, and rolling velocity. However, a nonsialylated E-selectin counterreceptor also exists. Furthermore, LS174T [[alpha].sub.2], [[alpha].sub.6], and [[beta].sub.1] integrins support a minor pathway in adhesion to HUVECs. Finally, tumor cell attachment specifically increases HUVEC endocytosis of E-selectin. Altogether, the data indicate the complexity of carcinoma cell-endothelium adhesion via sialylated glycoconjugates, integrins, and their respective counterreceptors. E-selectin; sialyl [Lewis.sup.x]; glycolipid; shear stress