Eg5 is static in bipolar spindles relative to tubulin: evidence for a static spindle matrix.
We used fluorescent speckle microscopy to probe the dynamics of the mitotic kinesin Eg5 in Xenopus extract spindles, and compared them to microtubule dynamics. We found significant populations of Eg5 that were static over several seconds while microtubules flux towards spindle poles. Eg5 dynamics are frozen by adenylimidodiphosphate. Bulk turnover experiments showed that Eg5 can exchange between the spindle and the extract with a half life of <55 s. Eg5 distribution in spindles was not perturbed by inhibition of its motor activity with monastrol, but was perturbed by inhibition of dynactin with p50 dynamitin. We interpret these data as revealing the existence of a static spindle matrix that promotes Eg5 targeting to spindles, and transient immobilization of Eg5 within spindles. We discuss alternative interpretations of the Eg5 dynamics we observe, ideas for the biochemical nature of a spindle matrix, and implications for Eg5 function.
Kapoor
T M
TM
Laboratory of Chemistry and Cell Biology, Rockefeller University, New York, NY 10021, USA. kapoor@mail.rockefeller.edu
Mitchison
T J
TJ
D000251:Adenosine Triphosphatases / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D003593:Cytoplasm / Q000737:chemistry; D000072159:Dynactin Complex; D006801:Humans; D016547:Kinesins / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000494:pharmacology; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D020409:Molecular Motor Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology; D009068:Movement; D011743:Pyrimidines / Q000494:pharmacology; D008941:Spindle Apparatus / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D013871:Thiones / Q000494:pharmacology; D014981:Xenopus; D029867:Xenopus Proteins
C000606361:DCTN2 protein, human; D000072159:Dynactin Complex; C088335:KIF11 protein, Xenopus; D008869:Microtubule-Associated Proteins; D020409:Molecular Motor Proteins; D011743:Pyrimidines; D013871:Thiones; D029867:Xenopus Proteins; D000266:Adenylyl Imidodiphosphate; C400223:monastrol; D000251:Adenosine Triphosphatases; D016547:Kinesins