Dictyostelium centrin B localization during cell cycle progression

Abstract

Recently, we have reported the initial characterization of a novel centrin from Dictyostelium discoideum (DdCenB).1 Sequence and phylogenetic analyses clearly establish DdCenB as a centrin, yet further characterization revealed some interesting peculiarities about this novel centrin. Figure 1 depicts the localization of DdCenB at three points in the cell cycle: interphase, mitosis and cytokinesis. In interphase DdCenB primarily localizes to the nuclear envelope (NE). Although the NE remains intact during mitosis and cytokinesis in Dictyostelium, DdCenB disappears from the NE at these two stages of the cell cycle. In addition to localization at the NE, we also see weak localization in the nucleoplasm and cytoplasm (weakest). Although the nucleoplasmic concentration appears constant throughout the cell cycle, the very faint localization in the cytoplasm does appear to increase to the level of the nucleoplasm during mitosis and cytokinesis. Unlike most centrins characterized to date, we found no evidence of DdCenB at the centrosome at any point in the cell cycle. Here we examine the importance of DdCenB localization in cell cycle progression, as well as several other roles.

Keywords: centrin; centrosome; cytokinesis; dictyostelium; mitosis; nuclear envelope.

Figure 1

Localization of DdCenB at three stages of the cell cycle. DdCenB is shown as gray shading, the darkness of which corresponds to the amount of DdCenB. N, nucleus; NE, nuclear envelope; C, centrosome; PM, plasma membrane.

Figure 2

Cytokinesis defects as a result of DdCenB knock-out. (A) consists of images captured from time-lapse microscopy. The time of capture (minutes) for each frame is indicated in the lower right corners. The arrowheads point to the persistent cytoplasmic bridges observed in dividing cells. In (B) we see a wild-type cell dividing. Here the arrowhead points to a typical cleavage furrow. (C) is a dividing mutant cell stained with DA PI (DNA) and visualized by fluorescence/DIC microscopy. Arrows point to distorted nuclei in the furrow region. (D) shows a mutant cell, with a long cytoplasmic bridge, stained with DAPI. The arrows point to higher magnifications of the respective areas, and highlight the presence of DNA in the cytoplasmic bridge. D, fluorescence; D’, phase contrast. Bars in all cases correspond to 5 µm.