Development and disintegration of phragmoplasts in living cultured cells of a GFP?TUA6 transgenic Arabidopsis thaliana plant

Abstract

Cultured suspension cells of Arabidopsis thaliana that stably express a green-fluorescent protein-alpha-tubulin 6 fusion protein were used to follow the development and disintegration of phragmoplasts. The development and disintegration of phragmoplasts in the living cultured cells could be successively observed by detecting the green-fluorescent protein fluorescence of the microtubules. In the early telophase spindle, where two kinetochore groups and two daughter chromosome groups had completely separated from one another, fluorescence appeared in the interzone between the two chromosome groups. The fluorescent region was gradually condensed at the previous equator and increased in fluorescence intensity, and finally it formed the initial phragmoplast. The initial phragmoplast moved from the cell center towards the cell periphery, and it lost fluorescence at its center and became double rings in shape. The expansion orientation of the phragmoplast was not always the same as that of the future new cell wall before it came in contact with the cell wall. The phragmoplast did not usually come in contact with the cell wall simultaneously with its entire length. A portion of the phragmoplast which was earlier in contact with the cell wall disappeared earlier than other portions of the phragmoplast. The duration of contact between any portions of the phragmoplast and the plasma membrane of the cell wall was 15-30 min. The fluorescence intensity of the cytoplasm did not seem to be elevated by the disintegration of the strongly fluorescent phragmoplast.

Quantitative analysis of changes in spatial distribution and plus-end geometry of microtubules involved in plant-cell cytokinesis

“…The similarity of this modeled image to published micrographs of fluorescently labeled somatic-type phragmoplasts is striking [compare Fig. 6B with published images (Gunning, 1982;Zang and Hepler, 1990;Asada et al, 1991;Hush et al, 1994;Gu and Verma, 1997;Ueda et al, 2003;Muller et al, 2004)]. …”

“…Many excellent light-microscope analyses of somatic-type cell-plate formation using fluorescent tubulin antibodies (Gunning, 1982;Asada et al, 1991;Gu and Verma, 1997;Muller et al, 2004), injected fluorescent tubulin (Zang and Hepler, 1990;Hush et al, 1994) or green-fluorescent-proteintagged tubulin in living cells (Ueda et al, 2003) have also failed to provide evidence for interdigitating phragmoplast microtubules. However, because most of the authors have Journal of Cell Science 118 (17) 5.…”

“…We postulate that the syncytial-type phragmoplasts have interdigitating microtubules to help to stabilize their (mini)phragmoplasts mechanically, which consist of only about ten microtubules, as opposed to >1000 in somatic-type phragmoplasts (Hepler and Newcomb, 1967;Gunning, 1982;Otegui et al, 2001). Furthermore, the problem of phragmoplast stabilization is even more important during syncytial-type cytokinesis, because the simultaneous production of about six syncytial walls requires much more time (>1 hour) (Otegui and Staehelin, 2001) than the formation of one somatic-type cross-wall (10-20 min) (Zhang et al, 1990;Ueda et al, 2003).…”

Hypocotyl Directional Growth in Arabidopsis: A Complex Trait

“…We show here that this BR-induced change in microtubule organization was attenuated by Glc. The microtubule organization of seedlings exhibiting BR reset was determined using a GFP-TUA6 transgenic line (Ueda et al, 2003). Epidermal cells of hypocotyls grown without Glc in the dark displayed a network of microtubules across the hypocotyl while horizontal organization of microtubules was observed in BR-treated hypocotyls.…”

Synthetic Lipid (DOPG) Vesicles Accumulate in the Cell Plate Region But Do Not Fuse

“…The phragmoplast is initiated at late anaphase from the antiparallel overlapping microtubule and actin filament arrays at the spindle midzone, the plus-ends of cytoskeleton polymers facing the accumulating vesicles that form the cell plate (Kakimoto and Shibaoka, 1988;Baskin and Cande, 1990;Zhang et al, 1990Zhang et al, , 1993Wick, 1991;Cleary et al, 1992;Hepler et al, 1993;Sano et al, 2005). During expansion of the cell plate, microtubules disappear from the center of the phragmoplast as soon as a cell plate has formed (Zhang et al, 1990Cleary et al, 1992;Hepler et al, 1993;Granger and Cyr, 2000;Ueda et al, 2003), whereas actin filaments become shorter but remain present in the whole area throughout cell plate formation Zhang et al, 1993).…”