Role of the Kinesin-like Protein KipB in Aspergillus nidulans

Molecular motors are protein machines, which power almost all forms of movement in the living world. Among the best known are the motors that hydrolyze ATP and use the derived energy to generate force. They are involved in a variety of diverse cellular functions as vesicle and organelle transport, cytoskeleton dynamics, morphogenesis, polarized growth, cell movements, spindle formation, chromosome movement, nuclear fusion, and signal transduction. Three superfamilies of molecular motors, kinesins, dyneins, and myosins, have so far been well characterized. These motors use microtubules (in the case of kinesines and dyneins) or actin filaments (in the case of myosins) as tracks to transport cargo materials within a cell. Analysis of fungal genomes revealed at least 10 distinct kinesins in filamentous fungi, some of which are not found in yeasts. We used the motor domain of conventional kinesin (KinA) from Aspergillus nidulans to perfom BLAST searches at the public A. nidulans genome database, at the Whitehead Center for Genome Research (Cambridge USA), and identified eleven putative kinesin motors. They grouped into nine of the eleven families, two kinesins being found in the Unc104 familiy and interestingly, one did not fall into any of the known families. The present work analyses the function of a kinesin-like protein in A. nidulans, KipB, which is a member of the Kip3 kinesin family. This family includes one representative in Saccharomyces cerevisiae (Kip3, the family founding member), two in Schizosaccharomyces pombe, Klp5 and Klp6 and one in Drosophila, Klp67A, the single one reported so far for higher eukaryotes in this family. Kip3 kinesins are implicated in microtubule disassembly and are required for chromosome segregation in mitosis and meiosis. To assess the function of KipB kinesin in A. nidulans, a kipB disruption strain was constructed. Analysis of the DkipB mutant revealed new features concerning the cellular functions of Kip3 proteins, but also some conserved ones. kipB is not essential for vegetative growth, and meiosis and ascospore formation were not affected in the DkipB mutant. The KipB protein was shown to be involved in the turnover of interphase cytoplasmic, mitotic and astral microtubules. DkipB mutants are less sensitive to the microtubule-destabilizing drug benomyl, and the microtubule cytoskeleton of interphase cells in DkipB mutants appears altered. Interestingly, spindle morphology and positioning were severely affected. Spindles were highly mobile, could overpass each other, moved over long distances through the cytoplasm, and displayed in 64% of the cases an extremely bent shape, latter feature being the first time reported for Kip3 kinesins. Mitotic progression was delayed in the DkipB mutant and a higher number of cytoplasmic microtubules remained intact during mitosis. DkipB heterozygous strains showed an increased instability of diploid nuclei, which proved once more KipB involvement in mitosis, along with DkipB clear genetic interaction with a mutation in another mitotic kinesin in A. nidulans, bimC4. An N-terminal GFP-KipB construct localized to cytoplasmic microtubules in interphase cells and to spindle and astral microtubules during mitosis, in a discontinuous pattern. Speckles of GFP-KipB appeared to be aligned in the cell. Time-lapse video microscopy indicated that the spots were moving independently towards the microtubule plus ends. This advanced the hypothesis that KipB could display processivity and intrinsic motility along microtubules, or that other kinesins involved in organelle motility are able to target the KipB protein to the microtubule plus ends. In the case of C-terminally truncated GFP-KipB protein versions, a stronger GFP signal was obtained and colocalization with a-tubulin-GFP revealed that they uniformly stain cytoplasmic, mitotic and astral microtubules. This suggests that the C-terminus is important for the correct localization and the movement of KipB protein along microtubules