Live imaging of Drosophila brain neuroblasts reveals a role for Lis1/dynactin in spindle assembly and mitotic checkpoint control.
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.
Siller
Karsten H
KH
Howard Hughes Medical Institute, Eugene, OR 97403, USA.
Serr
Madeline
M
Steward
Ruth
R
Hays
Tom S
TS
Doe
Chris Q
CQ
D000818:Animals; D030801:Animals, Genetically Modified; D001921:Brain / Q000166:cytology / Q000196:embryology; D029742:Caenorhabditis elegans Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D018385:Centrosome / Q000378:metabolism; D004330:Drosophila / Q000235:genetics*; D029721:Drosophila Proteins / Q000378:metabolism*; D000072159:Dynactin Complex; D004398:Dyneins / Q000502:physiology; D004843:Epistasis, Genetic; D066298:In Vitro Techniques; D018386:Kinetochores / Q000378:metabolism; D007814:Larva; D018715:Microscopy, Video; D008869:Microtubule-Associated Proteins / Q000378:metabolism* / Q000502:physiology*; D008938:Mitosis; D008954:Models, Biological; D008941:Spindle Apparatus / Q000235:genetics / Q000502:physiology*
D029742:Caenorhabditis elegans Proteins; D018797:Cell Cycle Proteins; C105393:DCTN1-p150 protein, Drosophila; D029721:Drosophila Proteins; D000072159:Dynactin Complex; C513192:Lis-1 protein, Drosophila; D008869:Microtubule-Associated Proteins; C064993:ROD protein, Drosophila; C513193:lis-1 protein, C elegans; D004398:Dyneins