Megator, an essential coiled-coil protein that localizes to the putative spindle matrix during mitosis in Drosophila.
We have used immunocytochemistry and cross-immunoprecipitation analysis to demonstrate that Megator (Bx34 antigen), a Tpr ortholog in Drosophila with an extended coiled-coil domain, colocalizes with the putative spindle matrix proteins Skeletor and Chromator during mitosis. Analysis of P-element mutations in the Megator locus showed that Megator is an essential protein. During interphase Megator is localized to the nuclear rim and occupies the intranuclear space surrounding the chromosomes. However, during mitosis Megator reorganizes and aligns together with Skeletor and Chromator into a fusiform spindle structure. The Megator metaphase spindle persists in the absence of microtubule spindles, strongly implying that the existence of the Megator-defined spindle does not require polymerized microtubules. Deletion construct analysis in S2 cells indicates that the COOH-terminal part of Megator without the coiled-coil region was sufficient for both nuclear as well as spindle localization. In contrast, the NH2-terminal coiled-coil region remains in the cytoplasm; however, we show that it is capable of assembling into spherical structures. On the basis of these findings we propose that the COOH-terminal domain of Megator functions as a targeting and localization domain, whereas the NH2-terminal domain is responsible for forming polymers that may serve as a structural basis for the putative spindle matrix complex.
Qi
Hongying
H
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA.
Rath
Uttama
U
Wang
Dong
D
Xu
Ying-Zhi
YZ
Ding
Yun
Y
Zhang
Weiguo
W
Blacketer
Melissa J
MJ
Paddy
Michael R
MR
Girton
Jack
J
Johansen
Jørgen
J
Johansen
Kristen M
KM
D000818:Animals; D002453:Cell Cycle; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D002868:Chromosomal Proteins, Non-Histone / Q000096:biosynthesis; D003593:Cytoplasm / Q000378:metabolism; D029721:Drosophila Proteins / Q000096:biosynthesis* / Q000235:genetics*; D004331:Drosophila melanogaster / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D015151:Immunoblotting; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D007399:Interphase; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000737:chemistry; D008938:Mitosis; D008957:Models, Genetic; D015739:Nocodazole / Q000494:pharmacology; D034521:Nuclear Matrix-Associated Proteins / Q000096:biosynthesis* / Q000235:genetics*; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D008941:Spindle Apparatus; D013997:Time Factors; D014162:Transfection
C498868:Chro protein, Drosophila; D002868:Chromosomal Proteins, Non-Histone; D029721:Drosophila Proteins; C498867:Mtor protein, Drosophila; D034521:Nuclear Matrix-Associated Proteins; C419556:skeletor protein, Drosophila; D015739:Nocodazole