Using Microdensitometry to Correlate Cell Morphology with the Nuclear Cycle in Ustilago maydis

Abstract

Nuclear cycle events are usually studied using 3H-thymidine, which cells take up from culture medium and incorporate into DNA. Because fungi do not incorporate exogenously supplied thymidine, other methods must be used for these organisms. The experiments described here utilized nuclear staining and computer-assisted microdensitometry. Vegetative Ustilago maydis cells were taken from liquid cultures at various points along the growth curve, attached to microscope slides, stained with 4′,6-diamidino-2-phenylindole, and viewed using Differential Interference Contrast and epifluorescence microscopy. Digital images of fluorescing nuclei were captured and inverted (negated) so that densitometry software could be used to determine the relative DNA content of the nuclei. When nuclei were grouped by relative density, two peaks presumably corresponding to groups of nuclei before (1C) and after (2C) DNA synthesis were observed. Correlating nuclear density with cell morphology showed that cells complete DNA synthesis before beginning to form buds. The presence of a number of unbudded cells with the 1C amount of DNA indicated that there was a clearly defined G1 period. During early log phase, approximately half of the cells in a culture were in G1. As the growth rate slowed, the percentage of cells in G1 increased dramatically. This indicates that the length of time spent in G1 varies with growth conditions in U. maydis. When cells from late log phase populations were subcultured into fresh medium, a lag period preceded resumption of cell growth. In addition, a significant percentage of these cells underwent a round of nuclear division and formed a central septum prior to bud formation. This resulted in a population of binucleate cells that budded asynchronously at both poles.