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Vol. 14, Issue 5, 2192-2200, May 2003

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Spindle Formation in Aspergillus Is Coupled to Tubulin Movement into the Nucleus

Yulia Ovechkina ^* , Paul Maddox , C. Elizabeth Oakley ^*, Xin Xiang , Stephen A. Osmani ^*, Edward D. Salmon , and Berl R. Oakley ^* ||

^* Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210; Department of Biology, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina 27599; and Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814

Submitted October 10, 2002; Accepted January 16, 2003
Monitoring Editor: Richard McIntosh

In many important organisms, including many algae and most fungi, the nuclear envelope does not disassemble during mitosis. This fact raises the possibility that mitotic onset and/or exit might be regulated, in part, by movement of important mitotic proteins into and out of the nucleoplasm. We have used two methods to determine whether tubulin levels in the nucleoplasm are regulated in the fungus Aspergillus nidulans. First, we have used benomyl to disassemble microtubules and create a pool of free tubulin that can be readily observed by immunofluorescence. We find that tubulin is substantially excluded from interphase nuclei, but is present in mitotic nuclei. Second, we have observed a green fluorescent protein/-tubulin fusion in living cells by time-lapse spinning-disk confocal microscopy. We find that tubulin is excluded from interphase nuclei, enters the nucleus seconds before the mitotic spindle begins to form, and is removed from the nucleoplasm during the M-to-G₁ transition. Our data indicate that regulation of intranuclear tubulin levels plays an important, perhaps essential, role in the control of mitotic spindle formation in A. nidulans. They suggest that regulation of protein movement into the nucleoplasm may be important for regulating mitotic onset in organisms with intranuclear mitosis.

Abbreviations used: DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescent protein; SPB, spindle pole body.

Online version of this article contains video material for some figures. Online version available at www.molbiolcell.org.

Present address: Department of Physiology and Biophysics, University of Washington, 1959 NW Pacific St., Seattle, Washington, 98195.

^¶ Corresponding author. E-mail address: oakley.2{at}osu.edu.

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