A basic helix-loop-helix protein with similarity to the fungal morphological regulators, Phd1p, Efg1p and StuA, controls conidiation but not dimorphic growth in Penicillium marneffei

Members of the APSES protein group are basic helix-loop-helix (bHLH) proteins that regulate processes such as mating, asexual sporulation and dimorphic growth in fungi. Penicillium marneffei is a human pathogen and is the only member of its genus to display a dimorphic growth transition. At 25 degrees C, P. marneffei grows with a filamentous morphology and produces asexual spores from multicellular con-idiophores. At 37 degrees C, the filamentous morphology is replaced by yeast cells that reproduce by fission. We have cloned and characterized an APSES protein-encoding gene from P. marneffei that has a high degree of similarity to Aspergillus nidulans stuA. Deletion of stuA in P. marneffei showed that it is required for metula and phialide formation during conidiation but is not required for dimorphic growth. This suggests that APSES proteins may control processes that require budding (formation of the metulae and phialides, pseudohyphal growth in Saccharomyces cerevisiae and dimorphic growth in Candida albicans) but not those that require fission (dimorphic growth in P. marneffei). The A. nidulans DeltastuA mutant has defects in both conidiation and mating. The P. marneffei stuA gene was capable of complementing the conidiation defect but could only inefficiently complement the sexual defects of the A. nidulans mutant. This suggests that the P. marneffei gene, which comes from an asexual species, has diverged significantly from the A. nidulans gene with respect to sexual but not asexual development.     

Interactions between the developmental program and cell cycle regulation of Aspergillus nidulans

Aspergillus nidulans is a multicellular fungus being used to study developmental regulation and cell cycle regulation. Genetic and molecular mechanisms underlying both processes have been characterized. Two types of observations suggest that there is significant interaction between cell cycle and developmental regulatory mechanisms. First, A. nidulans development involves the formation of specialized cell types that contain different, but specific, numbers of nuclei that are differentially regulated for cell cycle progression. Second, mutations directly affecting nuclear division can have major affects on cell differentiation during development. In this essay we describe these interactions and point out potential mechanisms for the cross talk between morphogenesis and the cell cycle that are tractable for future experimental investigation.     

Novel response to microtubule perturbation in meiosis

During the mitotic cell cycle, microtubule depolymerization leads to a cell cycle arrest in metaphase, due to activation of the spindle checkpoint. Here, we show that under microtubule-destabilizing conditions, such as low temperature or the presence of the spindle-depolymerizing drug benomyl, meiotic budding yeast cells arrest in G(1) or G(2), instead of metaphase. Cells arrest in G(1) if microtubule perturbation occurs as they enter the meiotic cell cycle and in G(2) if cells are already undergoing premeiotic S phase. Concomitantly, cells down-regulate genes required for cell cycle progression, meiotic differentiation, and spore formation in a highly coordinated manner. Decreased expression of these genes is likely to be responsible for halting both cell cycle progression and meiotic development. Our results point towards the existence of a novel surveillance mechanism of microtubule integrity that may be particularly important during specialized cell cycles when coordination of cell cycle progression with a developmental program is necessary.     

Silence is golden: combining RNAi and live cell imaging to study cell cycle regulatory genes during Caenorhabditis elegans development

Much of the pioneering work on the genetics of cell cycle regulation was accomplished using budding and fission yeast. The relative simplicity of these single-celled organisms allowed investigators to readily identify and assign roles to individual genes. While the molecular mechanisms worked out in yeast are more or less identical to those operating in higher organisms, additional layers of control must exist in multicellular organisms to coordinate the timing of developmental events occurring in different cells and tissues. Here we discuss experimental approaches for studying cell cycle processes in the nematode Caenorhabditis elegans.     

The COP9 signalosome counteracts the accumulation of cullin SCF ubiquitin E3 RING ligases during fungal development

Defects in the COP9 signalosome (CSN) impair multicellular development, including embryonic plant or animal death or a block in sexual development of the fungus Aspergillus nidulans. CSN deneddylates cullin-RING ligases (CRLs), which are activated by covalent linkage to ubiquitin-like NEDD8. Deneddylation allows CRL disassembly for subsequent reassembly. An attractive hypothesis is a consecutive order of CRLs for development, which demands repeated cycles of neddylation and deneddylation for reassembling CRLs. Interruption of these cycles could explain developmental blocks caused by csn mutations. This predicts an accumulation of neddylated CRLs exhibiting developmental functions when CSN is dysfunctional. We tested this hypothesis in A. nidulans, which tolerates reduced levels of neddylation for growth. We show that only genes for CRL subunits or neddylation are essential, whereas CSN is primarily required for development. We used functional tagged NEDD8, recruiting all three fungal cullins. Cullins are associated with the CSN1/CsnA subunit when deneddylation is defective. Two CRLs were identified which are specifically involved in differentiation and accumulate during the developmental block. This suggests that an active CSN complex is required to counteract the accumulation of specific CRLs during development.     

Aspergillus SteA (sterile12-like) is a homeodomain-C2/H2-Zn+2 finger transcription factor required for sexual reproduction

Saccharomyces cerevisiae Ste12p plays a key role in coupling signal transduction through MAP kinase modules to cell-specific or morphogenesis-specific gene expression required for mating and pseudohyphal (PH)/filamentous growth (FG). Ste12p homologues in the pathogenic yeasts Candida albicans and Filobasidiela neoformans apparently play similar roles during dimorphic transitions. Here we report the isolation and characterization of the first Ste12 protein from a true filamentous fungus. Aspergillus nidulans steA encodes a protein with a homeodomain 63-75% identical to those of other Ste12 proteins, with greatest similarity to FnSte12alphap. SteAp and Ste12alphap lack the pheromone induction domain found in budding yeast Ste12p, but have C-terminal C2/H2-Zn+2 finger domains not present in the other Ste12 proteins. A DeltasteA strain is sterile and differentiates neither ascogenous tissue nor fruiting bodies (cleistothecia). However, the development of sexual cycle-specific Hülle cells is unaffected. Filamentous growth, conidiation and the differentiation of PH-like asexual reproductive cells (metulae and phialides) are normal in the deletion strain. Northern analysis of key regulators of the asexual and sexual reproductive cycles support the observation that although SteAp function is restricted to the sexual cycle, cross regulation between the two developmental pathways exists. Our results further suggest that while several classes of related proteins control similar morphogenetic events in A. nidulans and the dimorphic yeasts, significant differences must exist in the regulatory circuitry.     

A novel human protein of the maternal centriole is required for the final stages of cytokinesis and entry into S phase

Centrosomes nucleate microtubules and contribute to mitotic spindle organization and function. They also participate in cytokinesis and cell cycle progression in ways that are poorly understood. Here we describe a novel human protein called centriolin that localizes to the maternal centriole and functions in both cytokinesis and cell cycle progression. Centriolin silencing induces cytokinesis failure by a novel mechanism whereby cells remain interconnected by long intercellular bridges. Most cells continue to cycle, reenter mitosis, and form multicellular syncytia. Some ultimately divide or undergo apoptosis specifically during the protracted period of cytokinesis. At later times, viable cells arrest in G1/G0. The cytokinesis activity is localized to a centriolin domain that shares homology with Nud1p and Cdc11p, budding and fission yeast proteins that anchor regulatory pathways involved in progression through the late stages of mitosis. The Nud1p-like domain of centriolin binds Bub2p, another component of the budding yeast pathway. We conclude that centriolin is required for a late stage of vertebrate cytokinesis, perhaps the final cell cleavage event, and plays a role in progression into S phase.     

Cell cycling through development

Regardless of the species, the development of a multicellular organism requires the precise execution of essential developmental processes including patterning, growth, proliferation and differentiation. The cell cycle, in addition to its role as coordinator of DNA replication and mitosis, is also a coordinator of developmental processes, and is a target of developmental signaling pathways. Perhaps because of its central role during development, the cell cycle mechanism, its regulation and its effects on developing tissues is remarkably complex. It was in this light that the Keystone meeting on the cell cycle and development at Snowbird, Utah in January 2004 was held.     

Microtubule function and its relation to cellular development and the yeast cell cycle in Wangiella dermatitidis

The microtubule inhibitor nocodazole {methyl-5-[2-(thienylcarbonyl)-1H-benzimidazol-2-yl]-carbamate} prevented nuclear migration and nuclear division in yeasts and developing multicellular forms of the polymorphic fungus Wangiella dermatitidis. It did not prevent yeast bud formation during at least two or three budding cycles, and caused yeasts to accumulate as premitotic forms with one to three buds. The effects of the drug suggested that at least three control pathways were involved in the yeast cell cycle; that the nocodazole block point was separate from the execution points of two temperature-sensitive mutations which lead to multicellularity; and that microtubules were controlling neither the yeast budding process nor the development of multicellular forms.Non-standard Abbreviations DMSO dimethylsulfoxide; nocodazole, methyl-5-[2-(thienylcarbonyl)-1H-benzimidazol-2-yl]-carbamate