A unique tubulin antiserum attenuates the rate of poleward chromosome movement in anaphase.
An antiserum against tubulin, NS20, was previously shown to specifically attenuate both fast axonal transport in vivo (Johnston, K. M. et al., Brain Res. 385, 38-45 (1986)) and in vitro (Johnston, K. M. et al., Cell Motil. Cytoskel. 7, 110-115 (1987)) and flagellar motility (Goldsmith, M. et al., Cell Motil. Cytoskel. 20, 249-262 (1991)). We hypothesized that NS20 blocked motility by binding to a multifunctional motor binding domain on the microtubules (MTs), or axonemes. Here we have examined the effect of microinjecting NS20, at metaphase, into dividing PtK2 cells. Plotting chromosome separation (CS) as a function of time, we report here that CS rates for anaphase A (chromosome-to-pole movement) were reduced by approximately 50% relative to uninjected controls. CS rates for anaphase B (spindle pole elongation) were unaffected by the NS20 antiserum. The inhibition of CS rate during anaphase A by NS20 was significantly greater than the inhibition caused by a control antitubulin serum (PC5). Two possible mechanisms underlying NS20's inhibition of CS during anaphase A were considered. NS20 could block the binding of a kinetochore-associated motor to kinetochore MTs (kMTs) or, alternatively, NS20 could stabilize kMTs against depolymerization. Our results favor the first alternative. In a cold-induced depolymerization assay, NS20 had no selective stabilizing effect on MTs. Moreover, we show that NS20 can selectively block the binding of a well characterized MT-associated motor (kinesin) to MTs, in vitro. These results suggest that NS20 may be defining a unique tubulin binding domain common to the motors underlying vesicle transport, flagellar motility, and chromosome movements during anaphase A.
Goldsmith
M
M
Department of Anatomy, Faculty of Medicine, University of Toronto, Ontario, Canada.
Leyland
S
S
Connolly
J A
JA
van der Kooy
D
D
D000705:Anaphase / Q000187:drug effects*; D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D001666:Binding Sites, Antibody; D002460:Cell Line; D002875:Chromosomes / Q000187:drug effects; D016547:Kinesins / Q000037:antagonists & inhibitors; D007614:Macropodidae; D008845:Microinjections; D008870:Microtubules / Q000378:metabolism*; D014404:Tubulin / Q000276:immunology* / Q000378:metabolism
D000906:Antibodies; D014404:Tubulin; D016547:Kinesins