Potential Link between the NIMA Mitotic Kinase and Nuclear Membrane Fission during Mitotic Exit inAspergillus nidulans

Abstract

We have isolated TINC as a NIMA-interacting protein by using the yeast two-hybrid system and have confirmed that TINC interacts with NIMA in Aspergillus nidulans. The TINC-NIMA interaction is stabilized in the absence of phosphatase inhibitors and in the presence of kinase-inactive NIMA, suggesting that the interaction is enhanced when NIMA is not fully activated. TINC is a cytoplasmic protein. TINC homologues and a TINC-like protein (A. nidulans HETC) are conserved in other filamentous fungi. Neither deletion of tinC nor deletion of both tinC and A. nidulans hetC is lethal, but deletion of tinC does produce cold sensitivity as well as osmotic sensitivity. Expression of an amino-terminal-truncated form of TINC (⌬N-TINC) inhibits colony growth in Aspergillus and localizes to membrane-like structures within the cell. Examination of cell cycle progression in these cells reveals that they progress through multiple defective mitoses. Many cells contain large polyploid single nuclei, while some appear to have separated masses of DNA. Examination of the nuclear envelopes of cells containing more than one DNA mass reveals that both DNA masses are contained within a single nuclear envelope, indicating that nuclear membrane fission is defective. The ability of these cells to separate DNA segregation from nuclear membrane fission suggests that this coordination is normally a regulated process in A. nidulans. Additional experiments demonstrate that expression of ⌬N-TINC results in premature NIMA disappearance in mitotic samples. We propose that TINC's interaction with NIMA and the cell cycle defects produced by ⌬N-TINC expression suggest possible roles for TINC and NIMA during nuclear membrane fission.Entry into mitosis represents the irreversible commitment by a cell to segregate duplicated DNA equally to form two independent nuclei. Entry into mitosis with damaged DNA or incompletely replicated DNA, failure to establish a proper bipolar spindle, and premature exit from mitosis can all have disastrous consequences for the cell. As such, entry into, progression through, and exit from mitosis are all highly regulated cell cycle transitions. In all eukaryotes, one of the central triggers for mitotic entry is the activation of the cyclin-dependent kinase CDC2 (12,21,30). In Aspergillus nidulans, the NIMA (never in mitosis A) kinase is also required for mitotic entry, as cells with active CDC2 and inactive NIMA are unable to enter mitosis (32).Two lines of evidence suggest that a NIMA pathway of cell cycle regulation exists in a wide range of organisms. Ectopic expression of NIMA in metazoan cells can induce some of the hallmarks of mitotic onset, including DNA condensation and germinal vesicle breakdown (25). Also, NIMA-related kinases (Nek) have been identified in a range of organisms, including yeast, flies, frogs, mice, and humans (29). Additionally, mitotic regulatory roles have been identified for the Fin1p kinase (15,16,22,23) from Schizosaccharomyces pombe as well as for a number of mammalian Nek kinases, including Nek2...

Caught Nek-ing: cilia and centrioles

“…Taken together with observations of synthetic phenotypes generated by some NimA alleles, these data suggest that NIMA participates in nuclear membrane fission (Osmani et al, 1991;Davies et al, 2004). Do these activities reflect those of Neks in other organisms?…”

“…TINC appears to be a protein specific to filamentous fungi. This mechanism of nuclear membrane fission might therefore be specific to this group (Davies et al, 2004). However, a role for NIMA in nuclear membrane fission might be common to all fungi: the kinase activity of the Schizosaccharomyces pombe NIMA ortholog (Fin1p) peaks at the metaphaseanaphase transition, and fin1-deletion mutants have extensive elaborations of the nuclear envelope (Krien et al, 2002).…”

Systematic Deletion and Mitotic Localization of the Nuclear Pore Complex Proteins ofAspergillus nidulans

“…A link between mitotic regulators and the NPC of A. nidulans came from studies aimed at understanding how the NimA kinase promotes initiation and completion of mitosis. Several approaches have been used in this endeavor including twohybrid Davies et al, 2004) and extragenic suppressor screens (Wu et al, 1998;De Souza et al, 2003). In the extragenic suppressor screen two genes were identified and both encode proteins (SonA Gle2/Rae1 and SonBn Nup98 ).…”

Mycoviruses: future therapeutic agents of invasive fungal infections in humans?

“…As a rule, however, when two fusing hyphae are not compatible, they recognize each other as nonself, which triggers programmed cell death (PCD). In aspergilli, the genes involved in activation of PCD reactions are currently being characterised and regarded as a promising approach to combat invasive aspergillosis [23][24][25][26]. Genetic incompatibility is mostly followed by death of the hyphal fusion cell, the so-called heterokaryon, and often of surrounding cells.…”