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

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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 inAspergillus 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. nidulansHETC) are conserved in other filamentous fungi. Neither deletion oftinCnor deletion of bothtinCandA. nidulans hetCis lethal, but deletion oftinCdoes produce cold sensitivity as well as osmotic sensitivity. Expression of an amino-terminal-truncated form of TINC (ΔN-TINC) inhibits colony growth inAspergillusand 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 inA. nidulans. Additional experiments demonstrate that expression of ΔN-TINC results in premature NIMA disappearance in mitotic samples. We propose that TINC's interaction with NIMA and the cell cycle defects produced by ΔN-TINC expression suggest possible roles for TINC and NIMA during nuclear membrane fission.

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