Talks: Fungal Cell Biology and Morphogenesis

The ultimate purpose of mitosis is to separate replicated DNA precisely into identical, and viable daughter nuclei. A subset of temperature sensitive cell cycle mutants are unable to complete the later stages of mitosis. These include alleles of bimG, various hfa genes and several nim genes. Mutations in all of these genes lead to the inability, at restrictive temperature, to separate daughter nuclei. Molecular analysis revealed that bimG encodes a regulatory protein phosphatase very similar both structurally and functionally to mammalian type 1 phosphatase. A single nucleotide change, responsible for the mutant phenotype, alters the 5' splice site of the second intron and leads to a temperature sensitive defect in splicing. Overexpression of the mutant gene from palcA produces a dominant lethal phenotype supporting the idea that aberrant splicing leads to the production of a toxic peptide which interferes with normal phosphatase function. To identify gene products which might interact with the phosphatase, extra-genic suppressors have been identified in three different genes, sugA, B & C. Mutations in these genes cause defective nuclear separation when grown at low temperature. Other genes involved in late mitosis include the nimU gene. nimU encodes a very basic novel protein, whose amino acid sequence suggests the potential for direct binding to DNA. The nimU24 mutation is partly epistatic to the bimE7 mutation (which causes DNA to become highly condensed) suggesting that NIMU may be required for normal chromosome condensation.

Nuclear positioning in Aspergillus nidulans

Organellar movement is an important feature of all eukaryotic cells. Vesicles, chloroplasts, mitochondria and nuclei migrate within the cytoplasm. Rapid, long distance nuclear migration has been observed in filamentous fungi for more than 60 years. However, the understanding of the enzymatic and molecular mechanisms driving nuclei are still at the beginning. In Aspergillus nidulans nuclei migrate along microtubules, probably driven by the microtubule dependent motor protein dynein. Besides a "basic translocation machinery" auxiliary components are required for triggering the movement and its coordination with other cellular functions. As one of the latter components apsA (anucleate primary sterigmata) was characterized. ApsA is a coiled coil protein required for nuclear positioning in hyphae and also in conidiophores, where it is needed for conidia production. The 180 kD protein was detected in crude protein extracts of hyphae with polyclonal antibodies rose against a part of ApsA expressed in E. coli. In a mutational screening of the apsA- strain (no conidia) a partially suppressed, conidiating strain was discovered. Formal genetics suggested that this suppression was due to a single mutated gene, extragenic to apsA. In a wild type (apsA+) background, the mutated suppressor gene had no obvious effect on conidiation, but a slight inhibitory effect on hyphal growth rate, and sexual spore formation was completely prohibited. Molecular characterization of the suppressor gene might shed some light on the function of apsA and the suppressor gene in the nuclear migration and positioning process.

Breaking the mold: cytokinesis in Aspergillus nidulans

Fungal cells are divided by crosswalls termed septa. We are determining the order of events in septum formation. These events include mechanisms that coordinate septum formation with cell growth and nuclear division; positioning a site in the cortex for septum formation; constricting of the protoplasm; recruitment of cell wall biosynthetic capacity to a site distal from the growing point (the hyphal tip), and assembly of the septal pore complex. Thus, septum formation represents a complex process of cellular morphogenesis. Physiological and cytological studies have demonstrate that nuclear positioning plays a critical role in determining the placement of the fungal septum site. The capacity to initiate septum formation requires nuclear division. Once the fungal septation site is established, septation can occur independently of nuclear division. Actin is recruited to the site of the incipient septum. Confocal microscopy demonstrates that actin structures at this site closely resemble the animal cell contractile ring. Genetic analysis has defined a pathway for septation; four genes act before site establishment, and four genes act after site establishment. The failure of the A. nidulans cells to establish a site for septum formation results in a cessation of growth, nuclear division and eventual cell death.

Genetic dissection of cytoplasmic dynein in Neurospora

Cytoplasmic dynein is a multisubunit, microtubule-dependent mechanochemical enzyme that has been proposed to function in a variety of intracellular movements, including minus-end-directed transport of organelles. A second protein complex referred to as the Glued or dynactin complex is required for cytoplasmic dynein to mediate efficient microtubule-dependent transport of organelles in vitro. We have determined that mutants defective in cytoplasmic dynein or the associated Glued/dynactin complex can act as partial suppressors of a Neurospora crassa protein kinase mutant (cot-1). These mutants, defined as ropy, display curled hyphal growth and abnormal nuclear distribution. We have shown that ro-1 encodes the heavy chain of cytoplasmic dynein and ro-4 encodes the actin-related protein Arp1, which is the most abundant subunit of the Glued/dynactin complex. In addition, we have isolated and characterized additional ro genes and determined that ro- 3 encodes the largest subunit of the Glued/dynactin complex. We have now isolated >2000 ro mutants in an effort to identify all genes encoding subunits or specific regulators of cytoplasmic dynein. Complementation analysis of ro mutants has identified approximately 20 complementation groups. Many of the ro mutants show complex genetic interactions with examples of unlinked noncomplementation and allelic complementation being common.

asm-1: a gene involved in formation of protoperithecia and ascospore maturation in Neurospora crassa

To identify novel, key regulatory genes of sexual development in Neurospora crassa, we explored the possibility that regulatory genes known to affect sexual development in Aspergillus nidulans may be evolutionary conserved and play similar roles in N. crassa. Among the classical developmental mutants of conidiophore development in A. nidulans, two genes, stuA (stunted A) and medA (Medusa A), are the only ones whose loss of function mutations completely block sexual development in this organism. Using stuA as heterologous probe in low stringency hybridizations we have cloned asm-1+ (ascus maturation 1) from Neurospora crassa. asm-1 was mapped to linkage group VR, near al-3, using RFLP analysis. We determined by Northern and Western blot analysis that both its message and its protein product are very abundant and constitutively expressed in both low and high nitrogen media. Loss of function asm-1 mutants (obtained by gene replacement) present multiple phenotypes that affect both the asexual and sexual phases of the life cycle. During asexual development asm-1 loss of function mutants lack aerial mycelia and conidiate rather close to the agar surface. During sexual development the mutant strain is unable to form protoperithecia and is therefore female sterile. As a male, in heterozygous crosses, the mutant presents a dominant phenotype. This dominant phenotype is characterized by the production of 99.9% of very small, white, and inviable ascospores. The rare, viable black ascospores that have been tested have always proven to be asm-1+, except for a single heterokaryotic segregant which must have come through meiosis as a heterozygous disomic. The loss of function mutation can be complemented when the asm- 1 chromosomal region is integrated at the his-3 locus. The resulting strain presents a normal vegetative phenotype as well as protoperithecia formation. The asm-1 loss of function phenotype presents formally similar characteristics to those of other ascus dominant (zygote dominant) mutations like Banana (Ban), Perforated ascus (Pfr), Indurated ascus (Iasc), and Round ascospores (R). Possible modes of action and regulation of asm-1 during sexual development were discussed.

The nudF and nudC genes are required for nuclear migration in Aspergillus nidulans

The nudF gene was identified as an extra copy suppressor of the temperature sensitive (ts) nudC3 mutation that blocks mycelial and conidial nuclear migration in Aspergillus nidulans. NudF encodes a protein similar to the beta-transducin (WD-40) family of heteromeric G-protein subunits and has a molecular weight of 49 kDa. In sucrose gradients the NUDF protein has an S value greater than that of albumin (68 kDa), suggesting that it is sedimenting as a subunit of a protein complex. Ts mutations in nudF lead to failure of nuclear migration and an almost complete disappearance of the protein. The ts nudC3 mutation also causes a disappearance of NUDF protein at restrictive temperature. Nine different extragenic suppressors of nudC3 restore the depleted NUDF protein toward normal levels and allow growth at restrictive temperature. We conclude that maintainance of a normal intracellular level of NUDF protein is required for nuclear migration to occur.

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