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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 organe...
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| Format: | Daten Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2004
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| Zusammenfassung: | 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. |
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| Umfang: | 125 Seiten |
| DOI: | 10.17192/z2004.0104 |