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© The Rockefeller University Press, 0021-9525/1999//125 $5.00
The Journal of Cell Biology, Volume 144, Number 1, , 1999 125-138

Articles

The Bipolar Kinesin, KLP61F, Cross-links Microtubules within Interpolar Microtubule Bundles of Drosophila Embryonic Mitotic Spindles



David J. Sharp*, Kent L. McDonald{ddagger}, Heather M. Brown*, Heinrich J. Matthies*, Claire Walczak§, Ron D. Vale||, Timothy J. Mitchison, and Jonathan M. Scholey*

* Section of Molecular and Cellular Biology, University of California Davis, Davis, California 95616; {ddagger} Electron Microscope Lab, University of California, Berkeley, California 94720-3330; § Medical Sciences Program, Indiana University, Bloomington, Indiana 47405; || Howard Hughes Medical Institute, University of California, San Francisco, California 94143; and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115

Previous genetic and biochemical studies have led to the hypothesis that the essential mitotic bipolar kinesin, KLP61F, cross-links and slides microtubules (MTs) during spindle assembly and function. Here, we have tested this hypothesis by immunofluorescence and immunoelectron microscopy (immunoEM). We show that Drosophila embryonic spindles at metaphase and anaphase contain abundant bundles of MTs running between the spindle poles. These interpolar MT bundles are parallel near the poles and antiparallel in the midzone. We have observed that KLP61F motors, phosphorylated at a cdk1/cyclin B consensus domain within the BimC box (BCB), localize along the length of these interpolar MT bundles, being concentrated in the midzone region. Nonphosphorylated KLP61F motors, in contrast, are excluded from the spindle and display a cytoplasmic localization. Immunoelectron microscopy further suggested that phospho-KLP61F motors form cross-links between MTs within interpolar MT bundles. These bipolar KLP61F MT-MT cross-links should be capable of organizing parallel MTs into bundles within half spindles and sliding antiparallel MTs apart in the spindle midzone. Thus we propose that bipolar kinesin motors and MTs interact by a "sliding filament mechanism" during the formation and function of the mitotic spindle.

Key Words: mitosis • bipolar kinesin • Bim C • microtubule • Drosophila

Abbreviations used in this paper: BCB, unphosphorylated BimC box; cdk1, cyclin-dependent kinase 1; CSF, cytostatic factor; HPF/FS, high-pressure freezing/freeze substitution; HSS, high speed supernatant; immunoEM, immunoelectron microscopy; LB, Luria broth; MAP, microtubule-associated protein; MT, microtubule; p-BCB, phosphorylated BCB; PI, protease inhibitor; Rs, Stokes radius; TEM, transmission EM.

Address correspondence to Dr. Jonathan M. Scholey, Section of Molecular and Cellular Biology, 1 Shields Ave., The University of California, Davis, CA 95616. Tel.: (530) 752-2271. Fax: (530) 752-7522. E-mail: jmscholey @ucdavis.edu

The p-BCB antibody project was initiated while J.M. Scholey was on sabbatical in the Department of Biochemistry and Biophysics at UCSF and he thanks the members of the Alberts, Mitchison, Vale, and Walter labs for their hospitality. The authors gratefully acknowledge Dr. Larry Goldstein for discussions and for supplying anti-recombinant KLP61F antibody. We would also like to thank members of the Scholey lab and particularly Greg Rogers for Fig. 10 and Dana Rashid for help with photographic development. Finally, we would like to thank Rick Harris, director of the UCD Section of Molecular and Cellular Biology Microscopy Center for help in numerous areas of the project.


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