TOGp, the human homolog of XMAP215/Dis1, is required for centrosome integrity, spindle pole organization, and bipolar spindle assembly.
The XMAP215/Dis1 MAP family is thought to regulate microtubule plus-end assembly in part by antagonizing the catastrophe-promoting function of kin I kinesins, yet XMAP215/Dis1 proteins localize to centrosomes. We probed the mitotic function of TOGp (human homolog of XMAP215/Dis1) using siRNA. Cells lacking TOGp assembled multipolar spindles, confirming results of Gergely et al. (2003. Genes Dev. 17, 336-341). Eg5 motor activity was necessary to maintain the multipolar morphology. Depletion of TOGp decreased microtubule length and density in the spindle by approximately 20%. Depletion of MCAK, a kin I kinesin, increased MT lengths and density by approximately 20%, but did not disrupt spindle morphology. Mitotic cells lacking both TOGp and MCAK formed bipolar and monopolar spindles, indicating that TOGp and MCAK contribute to spindle bipolarity, without major effects on MT stability. TOGp localized to centrosomes in the absence of MTs and depletion of TOGp resulted in centrosome fragmentation. TOGp depletion also disrupted MT minus-end focus at the spindle poles, detected by localizations of NuMA and the p150 component of dynactin. The major functions of TOGp during mitosis are to focus MT minus ends at spindle poles, maintain centrosome integrity, and contribute to spindle bipolarity.
Cassimeris
Lynne
L
Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania 18015, USA. lc07@lehigh.edu
Morabito
Justin
J
D002469:Cell Separation; D018385:Centrosome / Q000648:ultrastructure*; D000072159:Dynactin Complex; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D015151:Immunoblotting; D008853:Microscopy; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000378:metabolism / Q000502:physiology*; D008938:Mitosis; D010641:Phenotype; D011108:Polymers / Q000737:chemistry; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D008941:Spindle Apparatus; D013997:Time Factors; D014162:Transfection
C454180:CKAP5 protein, human; D000072159:Dynactin Complex; D008869:Microtubule-Associated Proteins; D011108:Polymers; D034741:RNA, Small Interfering