The effect of monastrol on the processive motility of a dimeric kinesin-5 head/kinesin-1 stalk chimera.
Controlled activity of several kinesin motors is required for the proper assembly of the mitotic spindle. Eg5, a homotetrameric bipolar kinesin-5 from Xenopus laevis, can cross-link and slide anti-parallel microtubules apart by a motility mechanism comprising diffusional and directional modes. How this mechanism is regulated, possibly by the tail domains of the opposing motors, is poorly understood. In order to explore the basic unregulated kinesin-5 motor activity, we generated a stably dimeric kinesin-5 construct, Eg5Kin, consisting of the motor domain and neck linker of Eg5 and the neck coiled coil of Drosophila melanogaster kinesin-1 (DmKHC). In single-molecule motility assays, we found this chimera to be highly processive. In addition, we studied the effect of the kinesin-5-specific inhibitor monastrol using single-molecule fluorescence assays. We found that monastrol reduced the length of processive runs, but strikingly did not affect velocity. Quantitative analysis of monastrol dose dependence suggests that two bound monastrol molecules are required to be bound to an Eg5Kin dimer to terminate a run.
Lakämper
Stefan
S
Drittes Physikalisches Institut, Georg-August-Universität Göttingen, Göttingen, Germany. lakaemper@physik3.gwdg.de
Thiede
Christina
C
Düselder
André
A
Reiter
Stefanie
S
Korneev
Mikhail J
MJ
Kapitein
Lukas C
LC
Peterman
Erwin J G
EJ
Schmidt
Christoph F
CF
D000595:Amino Acid Sequence; D000818:Animals; D004330:Drosophila / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D008969:Molecular Sequence Data; D011743:Pyrimidines / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013871:Thiones / Q000494:pharmacology*; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D029721:Drosophila Proteins; C088335:KIF11 protein, Xenopus; D011743:Pyrimidines; D011993:Recombinant Fusion Proteins; D013871:Thiones; D029867:Xenopus Proteins; C400223:monastrol; C515209:Khc protein, Drosophila; D016547:Kinesins