Many muscular and neurological disorders are associated with mitochondrial dysfunction and are often accompanied by changes in mitochondrial morphology. Mutations in the gene encoding OPA1, a protein required for fusion of mitochondria, are associated with hereditary autosomal dominant optic atrophy type I. Here we show that mitochondrial fragmentation correlates with processing of large isoforms of OPA1 in cybrid cells from a patient with myoclonus epilepsy and ragged-red fibers syndrome and in mouse embryonic fibroblasts harboring an error-prone mitochondrial mtDNA polymerase ␥. Furthermore, processed OPA1 was observed in heart tissue derived from heart-specific TFAM knock-out mice suffering from mitochondrial cardiomyopathy and in skeletal muscles from patients suffering from mitochondrial myopathies such as myopathy encephalopathy lactic acidosis and stroke-like episodes. Dissipation of the mitochondrial membrane potential leads to fast induction of proteolytic processing of OPA1 and concomitant fragmentation of mitochondria. Recovery of mitochondrial fusion depended on protein synthesis and was accompanied by resynthesis of large isoforms of OPA1. Fragmentation of mitochondria was prevented by overexpressing OPA1. Taken together, our data indicate that proteolytic processing of OPA1 has a key role in inducing fragmentation of energetically compromised mitochondria. We present the hypothesis that this pathway regulates mitochondrial morphology and serves as an early response to prevent fusion of dysfunctional mitochondria with the functional mitochondrial network.

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SummaryThe fungal cell wall, a conserved and highly dynamic structure, is essential for virulence and viability of fungal pathogens and is an important antifungal drug target. The cell wall integrity (CWI) signalling pathway regulates shape and biosynthesis of the cell wall. In this work we identified, localized and functionally characterized four putative CWI stress sensors of Aspergillus fumigatus, an airborne opportunistic human pathogen and the cause of invasive aspergillosis. We show that Wsc1 is specifically required for resistance to echinocandin antifungals. MidA is specifically required for elevated temperature tolerance and resistance to the cell wall perturbing agents congo red and calcofluor white. Wsc1, Wsc3 and MidA additionally have overlapping functions and are redundantly required for radial growth and conidiation. We have also analysed the roles of three Rho GTPases that have been implicated in CWI signalling in other fungi. We show that Rho1 is essential and that conditional downregulation of rho1 or deletion of rho2 or rho4 results in severely impaired CWI. Our data indicate significant functional differences between the CWI stress sensors of yeasts and moulds.

The morphology of mitochondria in mammalian cells is regulated by proteolytic cleavage of OPA1, a dynamin-like GTPase of the mitochondrial inner membrane. The mitochondrial rhomboid protease PARL, and paraplegin, a subunit of the ATP-dependent m-AAA protease, were proposed to be involved in this process. Here, we characterized individual OPA1 isoforms by mass spectrometry, and we reconstituted their processing in yeast to identify proteases involved in OPA1 cleavage. The yeast homologue of OPA1, Mgm1, was processed both by PARL and its yeast homologue Pcp1. Neither of these rhomboid proteases cleaved OPA1. The formation of small OPA1 isoforms was impaired in yeast cells lacking the m-AAA protease subunits Yta10 and Yta12 and was restored upon expression of murine or human m-AAA proteases. OPA1 processing depended on the subunit composition of mammalian m-AAA proteases. Homo-oligomeric m-AAA protease complexes composed of murine Afg3l1, Afg3l2, or human AFG3L2 subunits cleaved OPA1 with higher efficiency than paraplegin-containing m-AAA proteases. OPA1 processing proceeded normally in murine cell lines lacking paraplegin or PARL. Our results provide evidence for different substrate specificities of m-AAA proteases composed of different subunits and reveal a striking evolutionary switch of proteases involved in the proteolytic processing of dynamin-like GTPases in mitochondria. INTRODUCTIONMitochondria form large networks of interconnected tubules that are maintained by balanced fission and fusion events (Nunnari et al., 1997;Okamoto and Shaw, 2005). The morphology and ultrastructure of mitochondria depend on the tissue, on the physiological condition of the cell, and, in particular, on the functional status of mitochondria. Dynamic processes associated with mitochondria are apparently crucial for the cell, e.g., in apoptosis (Frank et al., 2001;Karbowski et al., 2002;Lee et al., 2004;Jagasia et al., 2005). Likewise, formation of dendritic spines and synapses (Li et al., 2004;Verstreken et al., 2005) and functional complementation of mitochondrial DNA (mtDNA) mutations by content mixing Ono et al., 2001) depend on dynamics of mitochondria. In contrast, vast morphological alterations of mitochondria have been reported to occur in human disorders. Impairment of mitochondrial fusion is causative of neurodegenerative diseases such as CharcotMarie-Tooth disease type 2A and 4A, and optic atrophy type 1 (Alexander et al., 2000;Delettre et al., 2000;Zuchner et al., 2004;Niemann et al., 2005).A key player in regulating mitochondrial fusion is the dynamin-like GTPase OPA1 (Olichon et al., 2003;Cipolat et al., 2004). Mutations in the OPA1 gene cause autosomal dominant optic atrophy type I, a prevalent hereditary neuropathy of the optic nerve (Alexander et al., 2000;Delettre et al., 2000). Down-regulation of OPA1 leads to fragmentation of mitochondria, mitochondrial dysfunction, altered maintenance of mtDNA, altered mitochondrial inner membrane morphology, and increased propensity for apoptosis (Olichon et al., 2003;Griparic ...

In two studies, recall was best when subjects' manipulated facial expressions were consistent with the emotional content of material recalled. In both studies, subjects' mood response to manipulated expressions was first assessed in a separate procedure. Subjects whose mood was affected were designated the selfproduced cue group. In Study 1, 60 undergraduate volunteers then read two passages of the same emotional content, either anger-provoking editorials or humorous selections by Woody Allen. Later, one selection was recalled while frowning and one while smiling. In the self-produced cue group, recall was significantly better for the editorials when frowning and for the Woody Allen stories when smiling. In the nonself cue group, expressions did not affect recall. In Study 2, 20 subjects heard and recalled three kinds of sentences, angry, sad, or fearful, in the same three expressions. Again, only in the self-produced cue group was recall significantly better when sentence and expression were consistent. The combination of studies indicates that these effects are due to mood rather than expression and to the match between expression and content at the time of recall.

Summary Fungi are surrounded by a rigid structure, the fungal cell wall. Its plasticity and composition depend on active regulation of the underlying biosynthesis and restructuring processes. This involves specialised signalling pathways that control gene expression and activities of biosynthetic enzymes. The cell wall integrity (CWI) pathway is the central signalling cascade required for the adaptation to a wide spectrum of cell wall perturbing conditions, including heat, oxidative stress and antifungals. In the recent years, great efforts were made to analyse the CWI pathway of diverse fungi. It turned out that the CWI signalling cascade is mostly conserved in the fungal kingdom. In this review, we summarise as well as compare the current knowledge on the canonical CWI pathway in the human pathogenic fungi Candida albicans, Candida glabrata, Aspergillus fumigatus and Cryptococcus neoformans. Understanding the differences and similarities in the stress responses of these organisms could become a key to improving existing or developing new antifungal therapies.

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Proteins entering the eukaryotic secretory pathway commonly are glycosylated. Important steps in this posttranslational modification are carried out by mannosyltransferases. In this study, we investigated the putative ␣-1,2-mannosyltransferase AfMnt1 of the human pathogenic mold Aspergillus fumigatus. AfMnt1 belongs to a family of enzymes that comprises nine members in Saccharomyces cerevisiae but only three in A. fumigatus. A ⌬afmnt1 mutant is viable and grows normally at 37°C, but its hyphal cell wall appears to be thinner than that of the parental strain. The lack of AfMnt1 leads to a higher sensitivity to calcofluor white and Congo red but not to sodium dodecyl sulfate. The growth of the mutant is abrogated at 48°C but can be restored by osmotic stabilization. The resulting colonies remain white due to a defect in the formation of conidia. Electron and immunofluorescence microscopy further revealed that the observed growth defect of the mutant at 48°C can be attributed to cell wall instability resulting in leakage at the hyphal tips. Using a red fluorescence fusion protein, we localized AfMnt1 in compact, brefeldin A-sensitive organelles that most likely represent fungal Golgi equivalents. The tumor necrosis factor alpha response of murine macrophages to hyphae was not affected by the lack of the afmnt1 gene, but the corresponding mutant was attenuated in a mouse model of infection. This and the increased sensitivity of the ⌬afmnt1 mutant to azoles, antifungal agents that currently are used to treat Aspergillus infections, suggest that ␣-1,2-mannosyltransferases are interesting targets for novel antifungal drugs.