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Vol. 16, Issue 11, 5127-5140, November 2005

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

Karsten H. Siller ^*, Madeline Serr , Ruth Steward , Tom S. Hays , and Chris Q. Doe ^*

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

Submitted April 22, 2005; Revised July 8, 2005; Accepted August 10, 2005
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, green fluorescent 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 Caenorhabditis 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.

Abbreviations used: MT, microtubule; kMT, kinetochore microtubule; NEB, nuclear envelope breakdown; ALH, after larval hatching; G1, Glued; MTOC, microtubule organizing center.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

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

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