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Use of GFP Fusions to a Microtubule Motor Protein to Analyze Spindle Dynamics in Live Oocytes and Embryos of Drosophila

Published online by Cambridge University Press:  02 July 2020

S. A. Endow  and
D. J. Komma
S. A. Endow
Affiliation:
Department of Microbiology, Duke University Medical Center, Durham, NC27710
D. J. Komma
Affiliation:
Department of Microbiology, Duke University Medical Center, Durham, NC27710
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Extract

Ncd is a kinesin-related microtubule motor protein of Drosophila that plays essential roles in spindle assembly and function during meiosis in oocytes and mitosis in early embryos. Antibody staining experiments have localized the Ned motor protein to spindle fibers and spindle poles throughout the meiotic and early mitotic divisions, demonstrating that Ncd is a spindle motor.

We have made ncd-gfp gene fusions with wild-type and S65T gfp and expressed the chimaeric genes in Drosophila to target GFP to the spindle. Transgenic Drosophila carrying the ncd-gfp gene fusions in an ncd null mutant background are wild type with respect to chromosome segregation, indicating that the Ncd-GFP fusion proteins can replace the function of wild-type Ncd. The Ncd-GFP fusion proteins in transgenic Drosophila are expressed under the regulation of the native ncd promoter.

Analysis of live Drosophila oocytes and early embryos shows green fluorescent spindles, demonstrating association of Ncd-GFP with meiotic and mitotic spindles. In mitotic spindles, Ncd-GFP localizes to centrosomes (Fig. 1a) and spindle fibers (Fig. 1b).

Type
Cell Biology Applications of Green Fluorescent Protein and Other Vital Labeling Probes
Information
Microscopy and Microanalysis , Volume 3 , Issue S2: Proceedings: Microscopy & Microanalysis '97, Microscopy Society of America 55th Annual Meeting, Microbeam Analysis Society 31st Annual Meeting, Histochemical Society 48th Annual Meeting, Cleveland, Ohio, August 10-14, 1997 , August 1997 , pp. 127 - 128
Copyright
Copyright © Microscopy Society of America 1997

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References

1.Hatsumi, M. and Endow, S.A., J. Cell Sci., 101(1992)547.Google Scholar
2.Endow, S.A. and Komma, D.J., J. Cell Sci., 109(1996)2429.Google Scholar
3. This work was supported by grants from the National Institutes of Health and American Cancer Society. We thank Dr. T. Salmon for help with initial imaging, and the Duke University Cancer Center for use of confocal microscope and computer facilities.Google Scholar