Invited Talk Abstracts
Postgenomics: how to capitalize on the genome sequence
Where do we go from here? Robert L. Metzenberg, Department of Chemistry, University of California, Los Angeles.
Our challenge is to grow our imagination and our taste fast enough to match the riches we have suddenly inherited. Like most of us here, I have my own wish-list of goals I would like to see reached -- goals that are no longer the stuff of science fiction. Foremost among these is to see Neurospora more fully developed as a powerful tool for understanding the biochemical architecture and dynamics of a macroscopic, coenocytic cell. This will require a much greater degree of cytochemical sophistication than most of us now possess. Second, I think Neurospora has great potential for shedding light on the role of introns. This may be straightforward, but laborious. Finally, I think that the very complexity of Neurospora in terms of signalling mechanisms, coupled with its simplicity of genetics, offers insights that cannot be duplicated in simpler organisms, and cannot be readily understood in more complex ones.
Neurospora comes into its own. David D. Perkins. Department of Biological Sciences, Stanford University, Stanford, CA 94025-5020
As the genome sequence invigorates research, we are led to ask what will be the role of genetics in future experimental work with Neurospora, what can be done to gain wider recognition of Neurospora among those outside the community, and what is needed to make it easier for newcomers to begin using Neurospora.
Functional Analysis of a Model Filamentous Fungus. Jay Dunlap1, Hildur Colot1, Kathy Borkovich2, Gloria Turner3, Dick Weiss3, Mike Plamann4, Bruce Birren5, James Galagan5, Matt Sachs6, Louise Glass7, Mike Eisen7, Mary Anne Nelson8, Jennifer Loros1. 1Dept. Genetics, Dartmouth Medical School, Havover,NH 03755. 2Dept. Plant Pathology, UC Riverside, Riverside, CA. 3Dept. Microbiology, UCLA, Los Angeles, CA. 4Dept. Biology, Univ. Missouri,Kansas City, MO. 5MIT Center for Genome Research, Cambridge, MA. 6Oregon Health Sciences University,Portland, OR. 7Dept. Plant and Microbial Biology, UC Berkeley, Berkeley, CA. 8Dept. Biology, Univ New Mexico, Albuquerque,NM
The overall goal of the four interdependent projects in this Program Project is to carry out functional genomics, annotation, and expression analyses of Neurospora crassa, the filamentous fungus that has become a model for the assemblage of over 250,000 species of non-yeast fungi. Most Neurospora genes have no homologs in yeasts and nearly 40% have no strong homologs in any organism to date, suggesting that examination of the functions of these genes will both novel and informative. Neurospora is an important model for basic research in eukaryotes, and fungi allied to Neurospora include significant animal and plant pathogens and industrial strains yielding antibiotics, chemicals, enzymes, and pharmaceuticals. The 43 Mb Neurospora genome is completely sequenced (>16 fold archival coverage), and automated annotation using programs trained on Neurospora genes predict 10,082 proteins. The first Project will pursue the systematic disruption of genes through targeted gene replacements, preliminary phenotyping of these strains, and their distribution to the scientific community at large. Project #1 will rely on bioinformatic support from Project #2. Through a primary focus in Annotation and Genomics, Project #2 will produce a platform for electronically capturing community feedback and data about the existing annotation, while building and maintaining a database to capture and display information about phenotypes, relying on data from EST analyses in Project #4 to refine the gene structures. Oligonucleotide-based microarrays created in Project #3 will allow Transcriptional Profiling of the nearly 11,000 distinguishable transcripts in Neurospora. This effort will provide a baseline analysis of gene expression under a variety of growth conditions, and later begin to analyze the global effects of loss of novel genes in strains created by Project #1; these data will be made available through the web via structures created in Project #2. Since alternative splicing, alternative promoters, and long antisense transcripts contribute widely to the overall complexity of expressed sequences in Neurospora, in Project #4, cDNA libraries will be generated from wild type and related strains to document this complexity to aid in annotation in Project #2. Sequences from related strains will drive assembly of a SNP map. Overall this effort will help to anchor genomic exploration within the largely unexplored phylogenetic Kingdom of the Fungi.
Advances in genome defense and DNA methylation. Eric U. Selker and colleagues, Institute of Molecular Biology, University of Oregon, Eugene, OR
The availability of the Neurospora genome sequence has advanced numerous areas of study, including ours. I will give an overview of our work and highlight how we have capitalized on the genome sequence data. I will also outline our plans to further exploit the availability of this information. Genomic studies verified that most methylated regions of Neurospora are products of RIP (repeat-induced point mutation), a premeiotic homology-based genome defense system that litters duplicated sequences with C:G to T:A mutations and typically leaves them methylated at remaining cytosines. These relics of RIP consist mostly of a variety of inactivated transposon. A combination of classical genetic and genomic approaches have provided evidence that the mechanism of RIP involves modifications of both DNA and chromatin. Our efforts to elucidate the control of DNA methylation in vegetative cells have revealed clear ties between modifications of DNA and chromatin. In vegetative cells, the DIM-2 DNA methyltransferase is directed by heterochromatin protein 1 (HP1), which in turn recognizes trimethyl-lysine 9 on histone H3, placed by the DIM-5 histone H3 methyltransferase. DIM-5 is sensitive to certain modifications of histones including acetylation and phosphorylation and is presumably also directed by one or more factors that recognize DNA mutated by RIP. DNA methylation can lead to deacetylation of histones, which may aid in propagation of DNA methylation and the associated silenced chromatin state.
Impact of the genome sequence on analysis of signal transduction pathways in Neurospora. Katherine A. Borkovich, Department of Plant Pathology, University of California, Riverside, CA
The availability of the complete genome sequence of Neurospora crassa has greatly impacted study of gene products involved in signal transduction pathways. Genes encoding signaling components have been identified and tentatively assigned to pathways, allowing the design of experiments to test both cellular function and epistatic relationships between gene products. In many cases, identification of these gene products was not feasible prior to the completion of the genome sequence. For example, cloning of seven transmembrane helix, G protein coupled receptors (GPCRs) is very difficult using degenerate PCR or low-stringency hybridization, as the regions of homology are usually confined to the transmembrane helices, leading to relatively low similarity. However, using the genome sequence, our laboratory has identified 10 putative GPCRs and has utilized a gene replacement strategy to create null mutations in the respective genes. Results will be presented for the analysis of two pheromone receptors, as well as a class of GPCRs not found in budding or fission yeasts.
Organismal Biology
Non-self recognition and programmed cell death in Neurospora crassa. N. Louise Glass, Gopal Iyer, Qijun Xiang, Isao Kaneko, Amita Pandey and Karine Dementhon. Department of Plant and Microbial Biology, The University of California, Berkeley, CA 94720-3102
Nonself recognition during vegetative growth in filamentous fungi is mediated by heterokaryon incompatibility (het) loci. In Neurospora crassa, het-c is one of eleven het loci. Three allelic specificity groups, termed het-cOR, het-cPA and het-cGR, exist in natural populations. Heterokaryons, partial diploids or transformants that contain het-c alleles of alternative specificity show severe growth inhibition, repression of conidiation and hyphal compartmentation and death (HCD). Nonself recognition is associated with presence of a plasma membrane associated heterocomplex composed of polypeptides encoded by het-c alleles of alternative specificity. Mutations in the vib-1 locus suppress growth inhibition and conidiation repression by genetic differences at het-c and mat and reduce HCD. The vib-1 locus encodes a nuclear localized putative transcriptional regulator. HET-C heterocomplex formation occurs in the vib-1 mutant, indicating that VIB-1 functions downstream of HET-C heterocomplex formation and may mediate some of the phenotypic responses to heterokaryon incompatibility at the transcriptional level. Mutations at a second locus, vib-2, confer temperature-dependent het-c heterokarkyon incompatibility. Initial data suggests that vib-2 may encode a protein related to TOL, which is required for mat incompatibility. Our finding is a step toward understanding nonself recognition mechanisms that operate during vegetative growth in filamentous fungi and provide a model for investigating relationships between recognition mechanisms and cell death.
An incompatibility supergene at het-6 in Neurospora crassa. Myron Smith. Department of Biology, Carleton University, Ottawa, Canada.
We constructed a lambda DNA library of the "Panama" (PA) background strain FGSC 1131 to clone and characterize PA alleles of het-6 and un-24, two linked genes that both have heterokaryon incompatibility function. In the process, we identified a ~19 kbp paracentric inversion, In(het-6), near het-6 on linkage group II that differentiates het-6OR from het-6PA strains. The structural features of this inversion explain our earlier observations that het-6 and un-24 are in severe linkage disequilibrium. For example, the PA and OR haplotypes are completely dissimilar in the region around breakpoint1 (brk-1) that is situated between un-24 and het-6 in thePA haplotype. This arrangement guarantees that the PA-OR and OR-PA combinations of het-6-un-24 cannot arise through homologous recombination between OR-OR and PA-PA forms. We hypothesize this inversion created an incompatibility supergene (comprising un-24OR het-6OR or un-24PA het-6PA) that underwent a selective sweep because it has a "strong" incompatibility function. This hypothesis is based partly on the elegant structure of the inversion but also on our observations of the characteristics of the PA alleles. Whereas the OR alleles at both loci cause cell death of PA strains in transformation-based assays, transforming the PA forms of either gene into OR background cells results in the recovery of inhibited, but viable colonies. From these self-incompatible transformed strains we recovered escapes that are functionally PA-OR or OR-PA at het-6-un-24. These escape strains will be used to test predictions of the incompatibility supergene hypothesis.
The genetic basis of adaptation: How good it's gonna be. John W. Taylor1, Takao Kasuga1, Luz B. Gilbert1, Jeff Townsend1, Jeremy Dettman1, David Jacobson1, 2, Anne Pringle1, Elizabeth Turner1, Don Natvig3, and Louise Glass1. 1Department of Plant and Microbial Biology, University of California, Berkeley, CA, 94720-3102, USA. 2Department of Biological Sciences, Stanford University, Stanford, CA, 94305, USA. 3Department of Biology, University of New Mexico, Albuquerque, NM, 87131, USA.
Adaptation to novel environments is easily inferred by comparisons between species, for example, Darwin's finches. Characterizing adaptation within a species and its genetic basis, perhaps as a prelude to or simultaneous with speciation, is more difficult. The keys will be to study a confirmed single species that ranges across different environments and to concentrate on traits that contribute to fitness and for which genetic control can be assigned. Neurospora discreta will be our model species because it thrives in different environments over a tremendous latitudinal gradient in western North America (Jacobson et al. 2004 Mycologia 96:66-74), a range that offers variation in abiotic factors (e.g., day length and temperature) and biotic factors (e.g., host tree and coexisting microbes). To ensure conspecificity, we are applying phylogenetic species recognition to the N. discreta clade as we have done for the other outbreeding Neurospora species (Dettman et al. Evolution 57:2703-2741). We will start with comparing a simple trait, mitospore germination, between N. discreta individuals in a common garden experiment, using strains collected at the extreme ends of the range and using temperature as the environmental variable. Eventually, we will measure other fitness components, such as growth and sporulation. To assay genome activity, we are profiling transcription using microarrays designed from the N. crassa genomic sequence. Hypotheses about the adaptive role of differentially expressed genes identified by transcriptional profiling (e.g., as compared to drift)combined with existing knowledge of Neurospora biology can be challenged by genetic investigation ranging from QTL analysis to molecular manipulation.
What we know about regional diversity in Neurospora. David Jacobson1,5, Cornelia Boesl2, Shahana Sultana2, Till Roenneberg2, Martha Merrow2, Margarida Duarte3, Isabel Marques3, Alexandra Ushakova3, Patricia Carneiro3, ArnaldoVideira3, Donald O. Natvig4, and John Taylor5. 1Dept. of Biological Sciences, Stanford University, California. 2Institute for Medical Psychology, University of Munich, Germany 3Instituto deBiologia Molecular e Celular, Porto, Portugal. 4Dept. of Biology,University of New Mexico, Albuquerque. 5Dept. of Plant and Microbial Biology, University of California, Berkeley.
Neurospora was previously considered primarily a tropical or subtropical genus. However, recent field surveys found Neurospora occupying an entirely new ecological niche under the bark of fire-damaged trees in dry and cold habitats within a new geographic range, western North America, from New Mexico (34°N) to Alaska (64°N) (Jacobson et al. 2004 Mycologia 96:55-74). Isolates from these sites were comprised predominantly (95%) of a single species, N. discreta, heretofore the least common species of Neurospora collected. In autumn 2003, a multinational effort surveyed southern Europe for Neurospora after unusually devastating wildfires. Neurospora was found from southern Portugal (37°N) to Switzerland (46°N). Species collected included N. crassa, N. discreta, N. sitophila, and N. tetrasperma. Although the latitude, climate and vegetation are similar to western North America, species distribution and spatial dynamics were quite different. Rather, these characteristics are more similar between southern Europe and semitropical Florida, where four different species are also present over very small spatial scales (Powell et al. 2003 Mycologia 95:809-819). These differences in regional diversity will form the basis of testable hypotheses, furthering the value of this model organism as a subject for studying fungal ecology.
What we know about local diversity in Neurospora. D.O. Natvig1, D.J. Jacobson2, G.S. Saenz1, A.J. Powell1, W.H. Dvorachek, Jr.1 and C. Kitchen1.1University of New Mexcio and 2Stanford University.
Studies with natural isolates from diverse locations, pioneered by David Perkins, set the stage to make Neurospora a model for evolutionary, systematic and population biology. This foundation is strengthened by the recent discovery that species are observed predictably after fires in western US forests. In addressing questions in realms such as population structure and gene flow, it is important to know whether isolates collected from a given location exhibit diversity. Typically, Neurospora species are observed in local "blooms" with copious conidia. Perithecia have been observed in nature rarely. It could be assumed that a bloom represents dispersal of conidia from a few early colonizers, or even a single colony. This is far from the truth. The Perkins collections revealed that multiple mating types of a given species, and at least occasionally, different species can be collected from a single "colony." Recently, we have shown that many distinct individuals can be obtained over small scales. In surveys of N. discreta, we found 9 different genotypes among 24 isolates from a single tree in New Mexico and 4 genotypes among 9 isolates from a single site in Alaska. Similarly, a survey of 16 isolates from adjacent cane stems in the Everglades revealed three species and 8 genotypes; and among 4 isolates from a single shrub, we observed three species and four genotypes. These results confirm the value of Neurospora for population studies. In addition, they highlight a poor understanding of modes of colonization and dispersal that result in blooms that appear within a few days after fire.
Photoperiodism in Neurospora crassa. Ying Tan1, Zdravko Dragovic1, Cornelia Boesl1, Shahana Sultana1, David Jacobson2, Till Roenneberg1 and Martha Merrow1. 1Institute for Medical Psychology, Munich, Germany. 2Stanford University, Palo Alto, CA, USA
Seasonality is common in nature. It is manifest in migration, hibernation, reproduction and pelage in animals, in flowering time and seed production in plants, and even in human psychology and fertility. Clever experiments and mutant plants and animals demonstrate that this regulation is part of the circadian system. Yet, circadian model organisms in which photoperiodism has been defined are inherently complex. We therefore investigated regulation of propagation, reproduction and light-regulated physiology in the fungal clock model system, Neurospora crassa. Regulation of all three of these functions can be demonstrated as sensitive to day or night length. Enhanced responses in 12, 14 or 14 to 20 h photoperiods, respectively, are specific for light duration and do not represent simple irradiance responses, as cultures grown in constant light resemble those grown in constant darkness. Night break experiments suggest that at least part of the timing mechanism measures night length. Clock mutant strains confirm that, like in other model organisms, photoperiodism in Neurospora depends on an intact circadian system. Recent collections over a wide range of latitudes suggest an adaptive advantage to using night or day length to program reproductive or metabolic function to certain seasons. Preliminary experiments with these strains demonstrate phenotypic diversity – perhaps comparable to that in the human population – in Neurospora chronotypes.
Morphogenesis
The role of the Spitzenkörper in hyphal morphogenesis. S. Bartnicki-Garcia1, Meritxell Riquelme1 and Robert W. Roberson2. 1División de Biología Experimental y Aplicada, CICESE, Ensenada, Baja California, México. 2Department of Plant Biology, Arizona State University, Tempe, AZ.
Mathematical modeling of the secretory process led to the concept that the polarized growth and shape of a fungal hypha is generated by the movement of the Spitzenkörper (Spk) acting as a vesicle supply center (VSC). The VSC model provides the basis for explaining how a fungal cell generates and changes its morphology. Tested mainly by high resolution video microscopy and image analysis, the model provided an explanation of how the Spk controls growth direction in hyphae of Neurospora crassa. Experiments with cytoskeleton inhibitors and light- and electron- microscopic examination of two ropy mutants of N. crassa led to the conclusion that a fully functional microtubular cytoskeleton is essential to maintain a steady, well developed Spk, and thus regular (hyphoid) morphology and a vigorous elongation rate. Immunofluorescence microscopy of ro-1 hyphae confirmed that the microtubule cytoskeleton was severely disrupted. A comparison of lateral vs. apical branching in hyphae of N. crassa disclosed a major difference in Spk ontogeny. Lateral branching occurs without affecting the behavior of the primary Spk or the shape and elongation rate of the primary hyphae. Lateral branching is probably triggered by the accumulation of a critical excess of wall precursors (mainly wall-building vesicles) in the subapical region. The trigger of apical branching may be traced to a sudden discrete disruption in cytoplasmic organization (cytoplasmic contraction) that causes the disappearance of the primary Spk, followed by the development of two new Spk.
Suppression of N. crassa cot-1 morphology by environmental stresses and Farnesol suggest that COT1 is involved in environmental stress response and Quorum sensing. Oded Yarden, Nourit Cohen, Carmit Ziv, Haya Sandori, Leonid Chernin, Zohar Kerem and Rena Gorovits. Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot Israel.
cot-1 is a Neurospora crassa colonial temperature-sensitive mutant that ceases to elongate and produces multiple branches at the restrictive temperature. Various environmental stresses (e.g., NaCl, Sorbitol, H2O2, ethanol or reduced availability of fermentable carbon sources) significantly suppressed the cot-1 phenotype. These effects are not dependent on os-2 or on mak-3 (inactivated by RIP) MAPK function, but are accompanied by alterations in PKA activity. When grown at 10-fold cell density (106 conidia/ml), cot-1exhibited near-wild type morphology (at restrictive temperatures), indicating that a biotic-derived environmental signal can phenocopy the effect of abiotic stresses. Replacing low-density cell medium with spent medium obtained from high-density cultures of cot-1 or wild type abrogated the cot-1 hyperbranching phenotype. Similarly, culturing cot-1 in the presence of 40-70 micromolar farnesol (a compound shown to be involved in Candida albicans Quorum sensing) suppressed the cot-1 phenotype. Chemical analysis confirmed that N. crassa produces and secretes farnesol in a cell density-dependent manner. Based on our results, we propose that COT1 is involved in environmental stress response and Quorum sensing. Use of transcription profiling has facilitated the identification of several genes differentially expressed in cot-1 cultures shortly after being shifted to the restrictive temperature. The possible involvement of these genes in the cellular response to environmental changes is being analyzed.
Identifying genes required for morphogenesis by a map and sequence strategy. Stephen J.Free, Amy Piwowar, and Shaun Bowman. Dept. of Biological Sciences, University at Buffalo, Buffalo, NY 14260.
We have used a map and sequence approach to identify genes involved in regulating the morphogenesis of Neurospora crassa. We initially isolated a number of morphological mutants using UV mutagenesis and then mapped the mutations using standard mapping procedures. We then identified candidate genes at the mapped loci and used PCR to amplify and sequence the candidate genes from mutant genomes. Using this approach we have identified new genes that are required for normal morphogenesis. These include an O-mannosyltransferase involved in cell wall biosynthesis and a gene thought to be involved in generating GPI-anchored proteins.
Genetic analysis of cytoplasmic dynein heavy chain. D. Madole, D. Gandhe and M. Plamann, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110-2499.
The three major families of motor proteins operating in eukaryotic organisms are the actin-associated myosin motors and the microtubule-associated kinesin and dynein motors. The mechanisms controlling myosin and kinesin motor function are relatively well understood; however, the molecular details of dynein motor function are largely unknown. Difficulties in understanding the dynein motor are due in part to the large size of its motor domain (~350 kDa vs. ~35 kDa for kinesins) and the involvement of >15 additional subunits. In Neurospora crassa, cytoplasmic dynein is highly conserved relative to vertebrates and is required for many of the same cellular activities. However, Neurospora dynein is not essential for viability and this allows the isolation of mutants defective in dynein function including many dynein heavy chain mutants. From a collection of >300 dynein heavy chain mutants, we have identified >80 mutants that produce full-length, but defective, heavy chain. We have defined the mutations in many of these mutants and have identified specific regions of the heavy chain that are essential for function of the dynein motor. Interestingly, the dynein motor mutants show significant variation in their cellular phenotypes. These results represent the first large-scale genetic analysis of the dynein motor domain.
Cell Biology
Does transport of calcium into the vacuole affect hyphal morphology and tolerance of high external calcium? Barry Bowman and Stephen Abreu, Department of Molecular Cell and Developmental Biology, University of California, Santa Cruz CA 95064
The vacuole of Neurospora crassa contains a relatively high concentration of calcium, which has been implicated as an important effector of hyphal morphology. Null mutations in the vacuolar ATPase in S. cerevisiae disrupt the ability of the vacuole to sequester calcium. In N. crassa loss of the vacuolar ATPase causes severe changes in hyphal morphology. We have characterized strains with null mutations in nca-2, nca-3, and cax, genes predicted to encode proteins that transport calcium into the vacuole. All three genes are expressed in standard minimal medium and expression is quickly elevated if 50-200 mM calcium is added to the external medium. Growth in medium with high calcium is partially affected by inactivation of the nca-2 gene, and severely affected if both nca-2 and cax are mutated. Inactivation of nca-3 causes no change in phenotype. The data suggest that nca-2,cax double mutants have lost the ability to sequester calcium in the vacuole, but surprisingly, these mutant strains have normal hyphal morphology. We also examined strains lacking pmr, the gene encoding a manganese or calcium transporter in the ER/golgi. These strains exhibit mild changes in hyphal morphology but growth is inhibited very little by high calcium in the medium.
Hyphal Ion Currents, Calcium and Tip Growth. Roger R. Lew. Department of Biology, York University, Toronto, Ontario Canada
Fungi (and specialized cells in other organisms) use polarized cellular extensions to explore new territory. At the growing tip, there can be assymetric distributions of ion fluxes, oxygen fluxes, and ionic gradients which may or may not be correlated spatially with cytological features, depending upon the organism and cell type. The highly coordinated process of tip extension always involves the presence of an internal tip-high gradient of calcium ions. By mapping channel distributions, extracellular ionic currents and cytosolic ion gradients along growing hyphal tips, we are characterizing the mechanisms responsible for generation and maintenance of the calcium gradient during hyphal growth in Neurospora crassa. The gradient is generated from internal stores by the activity of IP3-activated calcium channel. We envisage a mechanism that begins with tip-localized IP3 production by a stretch-activated phospholipase C, followed by IP3activation of calcium channels on calcium-containing vesicles. Elevated calcium then mediates the vesicle fusion required for continued hyphal tip expansion. Calcium sequestration occurs behind the growing apex into endoplasmic reticulum.
The research was funded by NSERC.
Integrative Analysis of the Mitochondrial Proteome in Yeast. Holger Prokisch, Lars Steinmetz, Curt Scharfe, David Camp, Wenzhong Xiao, Peter Oefner, Richard Smith, Ronald Davis, Thomas Meitinger
We use yeast and Neurospora mitochondria as a system to apply, integrate and compare different functional genomic approaches to define an organellar proteome.We first applied liquid chromatography mass spectrometry on purified organelles of yeast and Neurospora to identify 546 and 200 mitochondrial proteins, respectively. We then integrated proteomic with genomic approaches including deletion phenotype screening, expression profiling, subcellular localization, protein interaction analysis and computational predictions to derive a more comprehensive identification of yeast mitochondrial proteins. The integrative analysis achieves an accuracy higher than that of any single approach, predicts 650 yeast mitochondria localized proteins, and allows an evaluation of the success of individual methods. We show that two complementary systematic studies in combination, like deletion phenotype screening and mass spectrometry, identify 76% of the known mitochondrial proteome. These findings have implications for approaching other cellular systems, such as organelles and pathways in various species, using a minimal number of optimal genome-wide approaches. A comprehensive view of mitochondrial function and biogenesis will accelerate the understanding of Mendelian and complex mitochondrial disorders.
Assembly of the mitochondrial TOM complex. Frank E. Nargang, Rebecca D. Taylor, and Suzanne C. Hoppins. Dept. of Biological Sciences, University of Alberta, Edmonton, Alberta.
Over 95% of proteins found in mitochondria are encoded by genes in the nucleus, synthesized as precursor proteins on cytosolic ribosomes, and imported into the organelle. Mitochondrial precursor proteins contain targeting information which is recognized by multicomponent translocases in the mitochondrial membranes. The TOM complex (translocase of the outer membrane) is responsible for recognition of all mitochondrial precursor proteins and for their transfer into or across the mitochondrial outer membrane. In recent years, my laboratory has been interested in the assembly of the TOM complex. The major component of the complex is the beta-barrel protein Tom40, which forms the actual translocation pore. Tom40 requires pre-existing TOM complex for its own import into mitochondria. The Tom40 preprotein is incorporated into the complex via a well-defined pathway of intermediates that can be detected by blue native gel electrophoresis following import of radiolabeled Tom40 precursor intoisolated mitochondria. The Tim8-Tim13 complex exists in the intermembrane space and is traditionally considered a factor required for the import of the Tim23 protein. We have shown that mitochondria lacking the Tim8-Tim13 complex are deficient in the import of Tom40 and porin, another beta-barrel protein of the outer membrane. Crosslinking studies demonstrate that the Tom40 precursor contacts the Tim8-Tim13 complex. The complex is involved at an early point in the Tom40 assembly pathway since crosslinks can only be detected during theinitial stages of Tom40 import.
Analysing the cell biology of macroconidial germination and early colony development in Neurospora crassa. Nick D. Read, Fungal Cell Biology Group, Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 3JH, Scotland
In recent years there have been important developments in live-cell analytical techniques (e.g. confocal microscopy, vital and GFP probes, and laser tweezer micromanipulation) applied to studies on filamentous fungi. Much of the work of my lab has focused on developing and optimising these powerful technologies in applications to provide novel insights into the biology and dynamics of living fungal cells. We are currently using these approaches to investigate different aspects of the cell biology of macroconidial germination and early colony development in Neurospora. The process of hyphal homing and fusion (anastomosis) between conidial germlings of labelled has been imaged using different vital dyes and GFP probes. Both germ tubes and another type of specialised, morphologically distinct hypha (called a conidial anastomosis tube [CAT]) are produced by macroconidia. In wild type strains, CATs are thinner than germ tubes and do not undergo branching. In contrast to germ tubes, conidial anastomosis tubes grow towards each other. We have developed a simple laser tweezer technique to optically manipulate whole spores and germlings. When homing germlings are moved relative to each other the CATs subsequently reorientate themselves and grow back towards each other. This provides clear evidence for the existence of, as yet unknown, diffusible chemotropic signals being involved in the homing response of CATs. This experimental manipulation of macroconidia is being used in assays to determine whether strains of different genetic backgrounds can home towards or fuse with each other. In this way we have shown that fusion between CATs is independent of mating type. Nuclear movement, and continuity of the microtubular cytoskeleton between fused germlings, have been imaged. Endocytosis, as indicated by the internalisation of the membrane-selective probe FM4-64, is initiated after spore hydration before the emergence of germ tubes or CATs. Interestingly, the Spitzenkörper in germ tubes is not stained by the dye as it is in vegetative hyphae suggesting differences in the pathways of vesicle trafficking in these different cell types.
Exceptionally fast RNA cleavage and ligation catalyzed by a Neurospora Varkud Satellite ribozyme. Rick Collins.
Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada
Small ribozymes that use an internal phosphoester transfer mechanism to catalyze site-specific RNA cleavage have been identified in a several organisms and in in vitro selection experiments. All of these have been thought to be rather poor catalysts, exhibiting apparent cleavage rate constants typically less than 2 min-1. We have identified variants of one of these, the Neurospora VS ribozyme, that self-cleave with experimentally-measured apparent rate constants of up to 10 s-1 (600 min-1), about two orders of magnitude faster than any previously-characterized self-cleaving RNA. We describe structural features of the cleavage-site loop and an adjacent helix that affect the apparent rate constants for cleavage and ligation, and the equilibrium between them. These data show that the phosphoester transfer ribozymes can catalyze reactions with rate constants much larger than previously appreciated, and in the range of those of protein enzymes that perform similar reactions. These observations provide support for the feasibility of the ”RNA World” and for improving the performance of ribozyme-based therapeutic agents.
Regulation
Molecular Biology of the Neurospora Circadian Clock. Jennifer J. Loros, Hildur V. Colot, Jay C. Dunlap Department of Genetics, Dartmouth Medical School, Hanover, NH 03755
Circadian rhythms are a subset of biological rhythms having specific characteristics: they display period lengths close to 24 hrs, are readily entrainable to environmental cues including light and temperature, and their period lengths are compensated such they are close to the same under different ambient temperatures and nutritional conditions. Although noncircadian rhythms, rhythms lacking some or all of these characteristics, have been described in Neurospora, the circadian clock is one of the major regulators of conidiation in this organism. In all genetically studied model organisms, a negative feedback loop of gene expression makes a major contribution to the circadian rhythm mechanism. In animals and fungi this core loop involves a transcription factor (a heterodimer of proteins that interact via PAS domains) that activates expression of genes encoding proteins that feed back to depress the activity of the heterodimer. Additional feedback loops link to and close around this central loop. In Neurospora, a heterodimer of WC-1 and WC-2 makes up the PAS/PAS transcriptional activator and it acts to regulate the daily cyclical expression of the frq gene whose rhythmic expression is essential for all true circadian rhythms in this organism. FRQ is seen in the cell in two forms, a long form of 989 amino acids and a shorter form of 890 amino acids; the total amount of protein and the ratio between the two forms are regulated by ambient temperature. Based on limited frq cDNA analyses that had identified no introns, we believed that the transcription unit was simple and that the origin of the forms therefore lay in translational control. To examine this regulation we looked at transcripts, and when strand-specific primers were used to examine specifically the sense strand (with anti-sense strand primers as controls) a number of surprises emerged: (1) primary transcripts of frq are multiply spliced in a complex manner; (2) subsequent 5'RACE has revealed use of multiple promoters; (3) choice of alternative splice site within the 5'UTR is regulated by temperature as is use of promoters. The existence of an antisense transcript was also confirmed and has since been studied extensively by Crosthwaite and colleagues (Kramer et al, Nature 421:848-952, 2003). This complex environmentally regulated use of alternative splicing and multiple promoters to regulate the expression of frq provides a venue for examining the molecular biology of these phenomena in a genetically and molecularly tractable system.
Meiotic Silencing by Unpaired DNA. Patrick Shiu, Namboori Raju, Denise Zickler, and Robert Metzenberg. Department of Chemistry and Biochemistry, University of California, Los Angeles, CA; Department of Biological Sciences, Stanford University, CA. Institut de Genetique et Microbiologie, Universite Paris-Sud, Orsay, France.
Recently we have reported a novel system of targeted gene silencing that operates after karyogamy, namely Meiotic Silencing by Unpaired DNA (MSUD). In the MSUD system, the diploid cell scans the genome and compares a gene with its homolog during pachytene pairing. A gene which fails to be paired with a homolog generates a signal that transiently silences all sequences homologous to it. We have isolated a class of mutants called Sad (Suppressor of ascus dominance) that fail to perform MSUD. Sad mutants also suppress several classical "ascus-dominant" mutants, suggesting that these, too, owe their ascus dominance to the MSUD mechanism. MSUD is not restricted to a few ascus-dominant genes, but is applicable to virtually the entire genome. This can be seen from the fact that a variety of genes are meiotically silenced if they are manipulated so as to be unpaired during meiotic prophase. The sad-1 gene encodes an RNA-directed RNA polymerase (RdRP). RdRP has been implicated in many post-transcriptional gene silencing systems, such as co-suppression in plants, RNA interference in animals, and quelling in fungi. Owing to its ability to compare the genomes of two mating partners, MSUD has implications not only for surveillance against invading sequences but also for reproductive behavior.
The RNA-dependent RNA Polymerase, QDE-1 is a Rate-Limiting Factor in Post-Transcriptional Gene Silencing in Neurospora crassa. Emma C Forrest, Carlo Cogoni, and Giuseppe Macino. Dipartimento di Biotecnologie Cellulari ed Ematologia, Sezione di Genetica Molecolare, di Roma La Sapienza, Viale Regina Elena, 324, 00161 Roma, Italy
The RNA-dependent RNA polymerase qde-1 is an essential component of post-transcriptional gene silencing, termed “quelling” in the fungus Neurospora crassa. Here we show that over-expression of qde-1 results in a dramatic both a substantial increase in the efficiency of quelling, with a concomitant net increase in the quantity of al-1 siRNAs. Moreover, in over-expressed strains there is a significant reduction in the threshold number of transgenes required to induce quelling, and an increase in the phenotypic stability of quelling despite progressive loss of tandemly repeated transgenes which normally determines reversion of a silenced phenotype to wild-type. These data demonstrate that QDE-1 is the a rate-limiting factor of the silencing mechanism pathway, and suggests the existence of a mechanism able to detect and count transgenes, prompting a silencing response above a certain threshold. Moreover, the stability of quelling at the phenotypic level is increased in strains over-expressing qde-1, despite progressive loss of tandem arrays of transgenes during vegetative growth which normally determines reversion of a silenced phenotype to wild-type.
A Neurospora heterochromatin protein is essential for DNA methylation. Michael Freitag, Tamir K. Khlafallah, and Eric U. Selker, Institute of Molecular Biology, University of Oregon, Eugene, OR 97403
Methylation of cytosines silences transposable elements and certain cellular genes in mammals, plants and some fungi. Recent studies have advanced the understanding of what controls eukaryotic DNA methylation. All DNA methylation in Neurospora, and some in plants and animals, depends on histone H3 Lys9 methylation by DIM-5. Biochemical studies showed that methylated Lys9 is bound by the chromo domain of HP1, a heterochromatin protein originally identified in Drosophila and implicated in silencing in Drosophila, fission yeast and mammals. We therefore investigated the possibility that a Neurospora HP1 homologue reads the methyl-Lys9 mark to signal DNA methylation. We identified an HP1 homologue and showed that it is essential for DNA methylation, is localized to heterochromatic foci, and that this localizationis dependent on the catalytic activity of DIM-5. We conclude that HP1 serves as an adapter between methylated histone H3 Lys9 and the DNA methylation machinery. Unlike DNA methyltransferase mutants, HP1 mutants exhibit severe growth defects. This suggests that HP1 is required for processes besides DNA methylation. We identified eight additional Neurospora chromo domain proteins in the genome sequence. At least one of these also binds heterochromatic regions, partially co-localizing with HP1.
A Neurospora nascent polypeptide domain that regulates translation elongation. Peng Fang, Christina C. Spevak, Cheng Wu and Matthew S. Sachs. Oregon Health & Science University, Beaverton, OR 97006
The Neurospora crassa arginine attenuator peptide (AAP) specified in the 5'-leader of the arg 2 transcript acts as a nascent peptide to stall the translating ribosome in response to the presence of a high concentration of the amino acid arginine. We examined whether the AAP maintains regulatory function in N. crassa, wheat germ and reticulocyte cell-free translation systems when placed as a domain near the N-terminus or internally within a large polypeptide. Pulse-chase analyses of radiolabeled polypeptides indicated that the wild-type AAP functions at either position to stall polypeptide synthesis in response to arginine. Toeprint analyses performed to map the positions of stalled ribosomes on transcripts in the N. crassa system showed that ribosome stalling required translation of the AAP coding sequence. The positions of the stalled ribosomes were consistent with the sizes of the radiolabeled polypeptide intermediates. These findings demonstrate that an internal polypeptide domain in a nascent chain can regulate eukaryotic translational elongation in response to a small molecule. Apparently the peptide sensing features are conserved in fungal, plant and animal ribosomes. These data provide precedence for translational strategies that would allow domains within nascent polypeptide chains to modulate gene expression.
Posttranslational and light regulation of the Neurospora circadian clock. Yi Liu, Ping Cheng, Yuhong Yang, Qiyang He, & Qun He. Department of Physiology, UT Southwestern Medical Center, Dallas, TX 75390
FREQUENCY (FRQ), WHITE COLLAR-1 (WC-1) and WC-2 proteins are three critical components forming the circadian negative feedback loop in Neurospora. FRQ is progressively phosphorylated over time, and its level decreases when it is extensively phosphorylated. To identify the kinase phosphorylating FRQ and to understand the function of FRQ phosphorylation, two FRQ phosphorylating kinase was purified and identified as casein kinase II (CKII) and a calcium/calmodulin-dependent kinase. Our data showed that CKII is an essential clock components, and that the phosphorylation of FRQ by CKII promotes FRQ degradation and is important for the closing of the circadian negative feedback loop. On the hand, two protein phosphatases, PP1 and PP2A, play distinct roles in the Neurospora clock: PP1 regulating FRQ stability while PP2A is important for the function of the circadian feedback loop. After FRQ is phosphorylated, it is degraded through the ubiquitin-proteasome pathway. Such degradation is mediated by a E3 ligase, FWD-1 (an F-box/WD-40 repeat-containing protein), which is the Neurospora homolog of the Drosophila Slimb protein. The conservation of the posttranslational regulators in the Neurospora and animal circadian systems suggests that the molecules mediating the posttranslational regulation of clock proteins may be the common evolutionary link among distinct eukaryotic circadian systems.
In addition to the critical role of WC-1 and WC-2 in the circadian feedback loop, both proteins are essential components for the light input of various blue light responses, including the light entrainment of the circadian clock. We showed that the putative flavin-binding domain of WC-1, its LOV domain, is required for light responses. By purifying the endogenous WC complex from Neurospora, we showed that it is associated with FAD, suggesting that WC-1 is the blue light photoreceptor mediating light responses in Neurospora. VVD is a LOV domain protein regulating photoadaptation in Neurospora. By creating a Neurospora strain in which the LOV domain of WC-1 is swapped with that of VVD, we showed that the LOV domain of VVD partially replaces the function of the WC-1 LOV domain, suggesting that VVD is another blue light photoreceptor in Neurospora.
Genetics and Kinetics, Stuart Brody Molecular Biology Section, Division of Biological Sciences, University of California, San Diego
February of 1964 was the beginning of my research on Neurospora. In those forty years, my studies have ranged from morphological mutants to conidial germination to membrane lipid composition to mitochondria, and of course, to circadian rhythms. Through all of these topics, a common theme emerged: the effect of mutations on rates. After reviewing some of these subjects briefly, I will make the following predictions / "wish list" for Neurospora research.
1) More studies are needed as to how Neurospora interacts with / modifies its environment. On the biological side, the study of volatile signaling molecules has been neglected, as has "quorum sensing".
2) We need more and better ways of analyzing physical effects, such as pressure, charge fields, etc., on and in this organism, not just its biology and chemistry.
3) We should apply the techniques of "systems biology" to Neurospora studies so that we can progress from individual components to pathways to groups of pathways (nodes) to interactions between nodes.
4) We should start to look past the Y2H studies to describe the other ways that proteins communicate with each other and to develop methodology that allows a readout of large numbers of intermediates in a cell at onetime (metabolosome?) and under different conditions, etc.
5) For circadian rhythms, this area could use a big push of quantitation in terms of reaction rates, affinity constants, etc. We should also figure out how to measure quantitatively another important kinetic property of oscillators, i.e. their amplitude.
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These are published as an online supplement to the Fungal Genetics Newsletter #51. You may cite them as Fungal Genet. Newsl. 51S
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3/8/04 KMC