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MBC in Press, published online ahead of print August 17, 2005
Mol. Biol. Cell 10.1091/mbc.E05-04-0338

A more recent version of this article appeared on November 1, 2005
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Submitted on April 22, 2005
Revised on July 8, 2005
Accepted on August 10, 2005

Live Imaging of Drosophila Brain Neuroblasts Reveals a Role for Lis1/Dynactin in Spindle Assembly and Mitotic Checkpoint Control

Karsten H. Siller,* Madeline Serr,{dagger} Ruth Steward,{ddagger} Tom S. Hays,{dagger} and Chris Q. Doe*

*Howard Hughes Medical Institute, Institute of Molecular Biology, University of Oregon, Eugene, OR 97403; {dagger}Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455; {ddagger}Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854

Monitoring Editor: Erika Holzbaur

Lis1 is required for nuclear migration in fungi, cell cycle progression in mammals, and the formation of a folded cerebral cortex in humans. Lis1 binds dynactin and the dynein motor complex, but the role of Lis1 in many dynein/dynactin-dependent processes is not clearly understood. Here we generate and/or characterize mutants for Drosophila Lis1 and a dynactin subunit, Glued, to investigate the role of Lis1/dynactin in mitotic checkpoint function. In addition, we develop an improved time-lapse video microscopy technique that allows live imaging of GFP-Lis1, GFP-Rod checkpoint protein, GFP-labeled chromosomes, or GFP-labeled mitotic spindle dynamics in neuroblasts within whole larval brain explants. Our mutant analyses show that Lis1/dynactin have at least two independent functions during mitosis: first promoting centrosome separation and bipolar spindle assembly during prophase/prometaphase, and subsequently generating interkinetochore tension and transporting checkpoint proteins off kinetochores during metaphase, thus promoting timely anaphase onset. Furthermore, we show that Lis1/dynactin/dynein physically associate and colocalize on centrosomes, spindle MTs, and kinetochores, and that regulation of Lis1/dynactin kinetochore localization in Drosophila differs from both C. elegans and mammals. We conclude that Lis1/dynactin act together to regulate multiple, independent functions in mitotic cells, including spindle formation and cell cycle checkpoint release.

Address correspondence to: Chris Q. Doe (cdoe{at}uoneuro.uoregon.edu)




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