Changes in the centrin and microtubule cytoskeletons after metaphase arrest of the Chlamydomonas reinhardtii met1 mutant

The temperature-sensitive conditional met1 Chlamydomonas reinhardtii mutant arrests in metaphase at the restrictive temperature (33°C) with an intact spindle and high cell division kinase levels. In this study, met1 was investigated with respect to changes in the microtubule and centrin-based cytoskeletons after arrest at 33°C. Immunofluorescence microscopy revealed that, initially on arrest, the microtubule spindle and centrin-based cytoskeleton appeared as previously reported for wild-type metaphase cells; crescent-shaped spindles were seen with two brightly labelled centrin foci at each spindle pole in the basal body region at the cell surface. Observation of met1 held at the restrictive temperature reveals spindles can detach from one spindle pole and chromosomes eventually detach from the spindles. Moreover, a pseudo-anaphase event of spindle and nucleus elongation occurs in the absence of chromosome separation. Electron microscopy confirms that cytokinesis is initiated, the nuclei maintain a crescent shape but are distended and multiple pyrenoids are detected, suggesting chloroplast division also continues. Interestingly, prolamellar-like bodies usually present in etioplasts of dark-grown plants appear at the nuclear envelope. These results are discussed in relation to the coordination of division events in Chlamydomonas reinhardtii and the loss of viability in arrested cells of this mutant.