The buffering of calcium with quin2 reversibly forestalls anaphase onset in stamen hair cells of Tradescantia.

Cells from immature stamen hairs of the spiderwort plant Tradescantia virginiana cv. Zwanenburg Blue exhibit remarkable precision in the timing of their mitotic events: anaphase onset occurs 33 +/- 4 min after nuclear envelope breakdown, and cell plate vesicle aggregation occurs approximately 19 +/- 4 min after anaphase onset. To test the hypothesis that altered calcium levels might affect mitotic events, we incubated stamen hairs with the acetoxymethyl ester of the Ca-chelator, quin2 (quin2AM), or the K+-salt of quin2 free acid, and found that mitotic progression was blocked in metaphase and late anaphase. The inhibition of mitotic progression was dependent upon the quin2AM or quin2 concentration and the duration of incubation with the probe. Metaphase arrest could be reversed within several min by the addition of 100 microM CaCl2 to the extracellular medium, while lower concentrations of Ca2+ or the presence of Mg2+ in the buffer were insufficient to reverse the block. A second perfusion of 100 microM CaCl2 was usually necessary to promote cell plate vesicle aggregation. The effective concentration of quin2AM for reversible mitotic arrest was 50 microM (30 min) or 500 microM (12 min); a 30-min incubation in 50 microM quin2 acted similarly. The similarity of dose responses of Ca2+-reversal of quin2AM- and quin2-induced metaphase arrest suggests that the active species is the free acid and that the mode of inhibition is through Ca2+-chelation. Because the free acid is not permeant, and because extracellular esterase activity exists in the cell wall, the site of quin2 activity is probably outside the plasma membrane, in the wall space.(ABSTRACT TRUNCATED AT 250 WORDS)