Paul R. Fisher

A SLOW SUSTAINED INCREASE IN CYTOSOLIC CA2+ LEVELS MEDIATES STALK GENE INDUCTION BY DIFFERENTIATION INDUCING FACTOR IN DICTYOSTELIUM

During Dictyostelium stalk cell differentiation, cells vacuolate, synthesize a cellulose cell wall and die. This process of programmed cell death is accompanied by expression of the prestalk gene ecmB and induced by the differentiation inducing factor DIF. Using cell lines expressing the recombinant Ca2+‐sensitive photoprotein apoaequorin, we found that 100 nM DIF increases cytosolic Ca2+ ([Ca2+]i) levels from approximately 50 to 150 nM over a period of 8 h. The Ca2+‐ATPase inhibitor 2,5‐di(tert‐butyl)‐1,4‐hydroquinone (BHQ) induced a similar increase in [Ca2+]i levels and induced expression of the prestalk gene ecmB to the same level as DIF. The [Ca2+]i increases induced by DIF and BHQ showed similar kinetics and preceded ecmB gene expression by approximately 1–2 h. The Ca2+ chelator 1,2‐bis(o‐aminophenoxy)‐ethane‐N,N,N′N′‐tetra‐acetic acid (BAPTA) efficiently inhibited the BHQ‐induced [Ca2+]i increase and blocked DIF‐induced expression of the ecmB gene. These data indicate that the effects of DIF on stalk gene expression are mediated by a sustained increase in [Ca2‐]i. Sustained [Ca2+]i elevation mediates many forms of programmed cell death in vertebrates. The Dictyostelium system may be the earliest example of how this mechanism developed during early eukaryote evolution.

The heat shock cognate protein from Dictyostelium affects actin polymerization through interaction with the actin-binding protein cap32/34.

During isolation of the F‐actin capping protein cap32/34 from Dictyostelium discoideum, a 70 kDa protein was copurified which by cloning and sequencing was identified as a heat shock cognate protein (hsc70). This protein exhibited a specific and MgATP‐dependent interaction with the heterodimeric capping protein. To investigate the protein‐protein interaction in vitro, we expressed all three polypeptides separately in Escherichia coli and performed reconstitution experiments of complete or truncated hsc70 with the 32 and 34 kDa subunits of the capping protein. Viscosity measurements and studies on the polymerization kinetics of pyrene‐labeled actin showed that hsc70 increased the capping activity of cap32/34 up to 10‐fold, whereas hsc70 alone had no effect on actin polymerization. In addition, hsc70 acted as a molecular chaperone by stimulating the refolding of the denatured 32 and 34 kDa subunits of the capping protein. To study the interaction of the two domains of hsc70 with cap32/34, the N‐terminal 42 kDa ATPase region and the C‐terminal 30 kDa tail of hsc70 were expressed separately in E. coli. The 32 and 34 kDa subunits were capable of associating with both domains of hsc70. The ATPase domain of hsc70, which is structurally related to actin, proved to be responsible for the increased capping activity of cap32/34, whereas the C‐terminal tail of hsc70 was involved in folding of the subunits of cap32/34. Our data indicate a novel linkage between 70 kDa heat shock proteins and the actin cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)

Two Dictyostelium Orthologs of the Prokaryotic Cell Division Protein FtsZ Localize to Mitochondria and Are Required for the Maintenance of Normal Mitochondrial Morphology

ABSTRACT In bacteria, the protein FtsZ is the principal component of a ring that constricts the cell at division. Though all mitochondria probably arose through a single, ancient bacterial endosymbiosis, the mitochondria of only certain protists appear to have retained FtsZ, and the protein is absent from the mitochondria of fungi, animals, and higher plants. We have investigated the role that FtsZ plays in mitochondrial division in the genetically tractable protist Dictyostelium discoideum, which has two nuclearly encoded FtsZs, FszA and FszB, that are targeted to the inside of mitochondria. In most wild-type amoebae, the mitochondria are spherical or rod-shaped, but in fsz-null mutants they become elongated into tubules, indicating that a decrease in mitochondrial division has occurred. In support of this role in organelle division, antibodies to FszA and FszA-green fluorescent protein (GFP) show belts and puncta at multiple places along the mitochondria, which may define future or recent sites of division. FszB-GFP, in contrast, locates to an electron-dense, submitochondrial body usually located at one end of the organelle, but how it functions during division is unclear. This is the first demonstration of two differentially localized FtsZs within the one organelle, and it points to a divergence in the roles of these two proteins.

Photosensory and thermosensory responses in Dictyostelium slugs are specifically impaired by absence of the F-actin cross-linking gelation factor (ABP-120)

Chemotactic aggregation of starving amoebae of Dictyostelium discoideum leads to formation of a motile, multicellular organism - the slug - whose anterior tip controls its phototactic and thermotactic behaviour. To determine whether proteins that regulate the in vitro assembly of actin are involved in these responses, we tested phototaxis and thermotaxis in mutant slugs in which the gene encoding one of five actin-binding proteins had been disrupted. Of the proteins tested - severin, alpha-actinin, fimbrin, the 34 kD actin-bundling protein and the F-actin cross-linking gelation factor (ABP-120) - only ABP-120 proved essential for normal phototaxis and thermotaxis in the multicellular slugs. The related human protein ABP-280 is required for protein phosphorylation cascades initiated by lysophosphatidic acid and tumor necrosis factor alpha. The repeating segments constituting the rod domains of ABP-120 and ABP-280 may be crucial for the function of both proteins in specific signal transduction pathways by mediating interactions with regulatory proteins.

MidA is a putative methyltransferase that is required for mitochondrial complex I function

Dictyostelium and human MidA are homologous proteins that belong to a family of proteins of unknown function called DUF185. Using yeast two-hybrid screening and pull-down experiments, we showed that both proteins interact with the mitochondrial complex I subunit NDUFS2. Consistent with this, Dictyostelium cells lacking MidA showed a specific defect in complex I activity, and knockdown of human MidA in HEK293T cells resulted in reduced levels of assembled complex I. These results indicate a role for MidA in complex I assembly or stability. A structural bioinformatics analysis suggested the presence of a methyltransferase domain; this was further supported by site-directed mutagenesis of specific residues from the putative catalytic site. Interestingly, this complex I deficiency in a Dictyostelium midA− mutant causes a complex phenotypic outcome, which includes phototaxis and thermotaxis defects. We found that these aspects of the phenotype are mediated by a chronic activation of AMPK, revealing a possible role of AMPK signaling in complex I cytopathology.

Legionella pneumophila multiplication is enhanced by chronic AMPK signalling in mitochondrially diseased Dictyostelium cells.

SUMMARY Human patients with mitochondrial diseases are more susceptible to bacterial infections, particularly of the respiratory tract. To investigate the susceptibility of mitochondrially diseased cells to an intracellular bacterial respiratory pathogen, we exploited the advantages of Dictyostelium discoideum as an established model for mitochondrial disease and for Legionella pneumophila pathogenesis. Legionella infection of macrophages involves recruitment of mitochondria to the Legionella-containing phagosome. We confirm here that this also occurs in Dictyostelium and investigate the effect of mitochondrial dysfunction on host cell susceptibility to Legionella. In mitochondrially diseased Dictyostelium strains, the pathogen was taken up at normal rates, but it grew faster and reached counts that were twofold higher than in the wild-type host. We reported previously that other mitochondrial disease phenotypes for Dictyostelium are the result of the activity of an energy-sensing cellular alarm protein, AMP-activated protein kinase (AMPK). Here, we show that the increased ability of mitochondrially diseased cells to support Legionella proliferation is suppressed by antisense-inhibiting expression of the catalytic AMPKα subunit. Conversely, mitochondrial dysfunction is phenocopied, and intracellular Legionella growth is enhanced, by overexpressing an active form of AMPKα in otherwise normal cells. These results indicate that AMPK signalling in response to mitochondrial dysfunction enhances Legionella proliferation in host cells.

Import of nuclear-encoded mitochondrial proteins: a cotranslational perspective.

A growing amount of evidence suggests that the cytosolic translation of nuclear-encoded mitochondrial proteins and their subsequent import into mitochondria are tightly coupled in a process termed cotranslational import. In addition to the original posttranslational view of mitochondrial protein import, early literature also provides both in vitro and in vivo experimental evidence supporting the simultaneous existence of a cotranslational protein-import mechanism in mitochondria. Recent investigations have started to reveal the cotranslational import mechanism which is initiated by transporting either a translation complex or a translationally competent mRNA encoding a mitochondrial protein to the mitochondrial surface. The intracellular localization of mRNA to the mitochondrial surface has emerged as the latest addition to our understanding of mitochondrial biogenesis. It is mediated by targeting elements within the mRNA molecule in association with potential mRNA-binding proteins.

The Dictyostelium genome encodes numerous RasGEFs with multiple biological roles

BackgroundDictyostelium discoideum is a eukaryote with a simple lifestyle and a relatively small genome whose sequence has been fully determined. It is widely used for studies on cell signaling, movement and multicellular development. Ras guanine-nucleotide exchange factors (RasGEFs) are the proteins that activate Ras and thus lie near the top of many signaling pathways. They are particularly important for signaling in development and chemotaxis in many organisms, including Dictyostelium.ResultsWe have searched the genome for sequences encoding RasGEFs. Despite its relative simplicity, we find that the Dictyostelium genome encodes at least 25 RasGEFs, with a few other genes encoding only parts of the RasGEF consensus domains. All appear to be expressed at some point in development. The 25 genes include a wide variety of domain structures, most of which have not been seen in other organisms. The LisH domain, which is associated with microtubule binding, is seen particularly frequently; other domains that confer interactions with the cytoskeleton are also common. Disruption of a sample of the novel genes reveals that many have clear phenotypes, including altered morphology and defects in chemotaxis, slug phototaxis and thermotaxis.ConclusionThese results suggest that the unexpectedly large number of RasGEF genes reflects an evolutionary expansion of the range of Ras signaling rather than functional redundancy or the presence of multiple pseudogenes.

Chaperonin 60 and mitochondrial disease in Dictyostelium

The single Dictyostelium chaperonin 60 gene, hspA, was cloned, sequenced and characterized. Sequence comparisons and a three-dimensional model for the structure of the encoded protein showed that it exhibits the conserved sequence and structural features expected for its role as the Dictyostelium mitochondrial chaperonin 60. Dictyostelium hspA contains two introns and, unusually for a member of this major heat shock gene family, is not stress-inducible in response to heat, cold or cadmium ions. Although transcription of hspA is down regulated during early Dictyostelium development in response to starvation, the levels of the chaperonin 60 protein remain constant throughout the life cycle. Consistent with the essential role of chaperonin 60 in mitochondrial biogenesis, we were unable to isolate mutants in which the hspA gene had been disrupted. However, transformants were isolated that exhibited differing levels of antisense inhibition of chaperonin 60 expression, depending upon the number of copies of the antisense-expressing plasmid in the genome. Orientation in phototaxis (and thermotaxis) was severely impaired in all antisense transformants, while growth and morphogenesis were markedly defective only in transformants with higher levels of antisense inhibition. This pattern of phenotypes is similar to that reported previously to result from targeted disruption of the mitochondrial large subunit rRNA gene in a subpopulation of mitochondria. This suggests that, regardless of the nature of the underlying genetic defect, mitochondrial deficiency impairs signal transduction more sensitively than other cellular activities.

Mitochondrial biology and disease in Dictyostelium.

The cellular slime mold Dictyostelium discoideum has become an increasingly useful model for the study of mitochondrial biology and disease. Dictyostelium is an amoebazoan, a sister clade to the animal and fungal lineages. The mitochondrial biology of Dictyostelium exhibits some features which are unique, others which are common to all eukaryotes, and still others that are otherwise found only in the plant or the animal lineages. The AT-rich mitochondrial genome of Dictyostelium is larger than its mammalian counterpart and contains 56kb (compared to 17kb in mammals) encoding tRNAs, rRNAs, and 33 polypeptides (compared to 13 in mammals). It produces a single primary transcript that is cotranscriptionally processed into multiple monocistronic, dicistronic, and tricistronic mRNAs, tRNAs, and rRNAs. The mitochondrial fission mechanism employed by Dictyostelium involves both the extramitochondrial dynamin-based system used by plant, animal, and fungal mitochondria and the ancient FtsZ-based intramitochondrial fission process inherited from the bacterial ancestor. The mitochondrial protein-import apparatus is homologous to that of other eukaryote, and mitochondria in Dictyostelium play an important role in the programmed cell death pathways. Mitochondrial disease in Dictyostelium has been created both by targeted gene disruptions and by antisense RNA and RNAi inhibition of expression of essential nucleus-encoded mitochondrial proteins. This has revealed a regular pattern of aberrant mitochondrial disease phenotypes caused not by ATP insufficiency per se, but by chronic activation of the universal eukaryotic energy-sensing protein kinase AMPK. This novel insight into the cytopathological mechanisms of mitochondrial dysfunction suggests new possibilities for therapeutic intervention in mitochondrial and neurodegenerative diseases.