Effects of cycloheximide on preprophase bands and prophase spindles in onion (Allium cepa L.) root tip cells

Abstract

Effects of cycloheximide (CHM) on preprophase bands (PPBs) of microtubules (MTs) and on prophase spindle MTs in root tip cells of onion (Allium cepa L.) were examined. When root tip cells were treated with 36 μM CHM for 0.5–4 h, the population of cells with a PPB did not decrease markedly although the population of mitotic cells and that of prophase cells with a PPB gradually decreased to half of the control root tips. In prophase cells treated with 11 and 36 μM CHM for 2 h, the width of the PPB was 1.4 times broader than that in the prophase PPB without CHM. Electron microscopic observation on the cross section of the PPB showed that the number of MTs and the distance between adjacent MTs in prophase PPBs treated with CHM were similar to those in the early developmental stage of PPBs without CHM. The bipolar spindle, that appeared in late prophase was not seen in prophase cells treated with 11 μM or higher concentrations of CHM for 2 h. In order to examine differences of perinuclear MT arrangement between CHM treated and non-treated prophase cells, arrangement of perinuclear MTs was examined by confocal laser scanning microscopy. In control cells without CHM, MTs appeared on the nuclear surface with several “branched” or “cross over” type MT foci in the cytoplasm when broad PPB formation started. These MT foci were replaced by the “aster” type MT foci, from which several MTs radiated along the nuclear surface. The “aster” type MT foci gradually gathered to form a bipolar spindle. MTs connecting the spindle pole region and the PPB were seen in late prophase. In CHM-treated cells (11-360 μM for 2 h), “branched” and “cross over” type MT foci were prominent, even in prophase cells with well condensed chromosomes. Neither linkages of MTs between the spindle pole region and the PPB nor “aster” type MT foci were seen. These observations showed that CHM prevents the bundling of MTs in the PPB and also inhibits the formation of “aster” type MT foci that is essential for bipolar spindle development.