Synchronous nuclear-envelope breakdown and anaphase onset in plant multinucleate cells

Abstract

Multinucleate plant cells with genetically balanced nuclei can be generated by inhibiting cytokinesis in sequential telophases. These cells can be used to relate the effect of changes in the distribution of nuclei in the cytoplasm to the control of the timing of cell cycle transitions. Which mitotic cell cycle events are sensitive to differences in the amount of cytoplasm surrounding each chromosomal complement has not been determined. To address this, we maximized the cell size by transiently inhibiting replication, while cell growth was not affected. The nuclei of 93% of the elongated cells reached prophase asynchronously compared to 46% of normal-sized multinucleate cells. The asynchronous prophases of normal-sized cells became synchronous at the time of nuclear-envelope breakdown, and the ensuing metaphase plate formation and anaphase onset and progression occurred synchronously. The elongated multinucleate cells were also very efficient in synchronizing the prophases at nuclear-envelope breakdown, in the prophase-to-prometaphase transition. However, 2.4% of these cells broke down the nuclear envelope asynchronously, though they became synchronous at the metaphase-to-anaphase transition. The kinetochore-microtubular cycle, responsible for coordinating the metaphase-to-anaphase transition and for the rate of sister segregation to opposite spindle poles during anaphase, remained strictly controlled and synchronous in the different mitoses of a single cell, independently of differences in the amount of cytoplasm surrounding each mitosis or its ploidy. Moreover, the degree of chromosome condensation varied considerably within the different mitotic spindles, being higher in the mitoses with the largest surrounding cytoplasm.

Inter- and intrachromosomal asynchrony of cell division cycle events in root meristem cells of Allium cepa: possible connection with gradient of cyclin B-like proteins

“…The higher the number of nuclei (and the longer the cell), the greater the frequency of evident differences between the various states of chromosome condensation or between the discrete stages at which individual nuclei can be observed passing through mitosis. According to the data presented by Giménez-Abián et al (2001), the advancement of chromosome condensation has been often noticed within the outlying parts of giant multinucleate cells (in more than 90% of cases; Fig. 1a), indicating that acceleration of mitosis is due mainly to relatively high amount of surrounding cytoplasm, compared with the perinuclear cytoplasm located in middle part of the cell.…”

“…Repeated incubations of primary root meristems of Allium cepa with caffeine (to inhibit cell plate formation) and then with hydroxyurea (to stop DNA replication and to increase the length of the cell) have been shown effective in producing polykaryotic cells that advance the metaphase–anaphase breakpoint synchronously, despite previous differences in chromosome condensation (Giménez-Abián et al 2001). Our results indicate that using a modified version of this method (with extended periods of HU and CF treatment) two dominant types of cells can be formed: one, comprising various numbers of nuclei (1, 2, 4, 6, and sporadically 8; each nucleus containing up to 4C DNA content; Fig.…”

“…To obtain multinucleate cells, seedlings were treated according to the protocol of Giménez-Abián et al (2001) using alternate incubations with 0.75 mM HU and 5 mM CF, except that additional third treatment with CF (3 h) and post-incubation with water (12 h) were introduced before fixation. Induction of biphasic cells showing gradual changes of chromatin condensation (intrachromosomal asynchrony) were obtained by incubation of seedlings with 0.75 mM HU (12 h), followed by continuous treatment with 0.5 mM HU (total time ranging from 24 up to 120 h).…”

“…In plants, axially elongated cells may result from administration of chemical agents that, respectively, either strongly delay progression through DNA replication [but allow cell growth to continue (Navarrete et al 1983; Giménez-Abián et al 2001)] or affect formation of the phragmoplast, a highly specific cytokinetic apparatus made up of microtubules and microfilaments (Samuels and Staehelin 1996; Nishihama and Machida 2001). In order to assess the extent to which particular cell nuclei located in the same cytoplasm may display mitotic autonomy, two experimental systems effective in producing extremely large cells have been used.…”

“…In order to assess the extent to which particular cell nuclei located in the same cytoplasm may display mitotic autonomy, two experimental systems effective in producing extremely large cells have been used. The first method, based on the modified procedure advanced by Giménez-Abián et al (2001), relied on alternate incubations of primary root meristems of Allium cepa with hydroxyurea (HU, an inhibitor of ribonucleotide reductase) and caffeine (CF, a well-known inhibitor of cytokinesis in plants). Some of abnormally long polykaryotic cells, with two or more nuclei fused together, gave rise to atypical images of mitotic divisions, including asynchronous metaphase-to-anaphase transitions.…”