Detection of the membrane-calcium distribution during mitosis in Haemanthus endosperm with chlorotetracycline.

Abstract

The distribution of membrane-associated Ca was determined at various stages of mitosis in Haemanthus endosperm cells with the fluorescent chelate probe chlortetracycline (CTC). CTC fluorescence in Haemanthus has 2 components: punctate, because of mitochondrial and plastid membrane-Ca2+; and diffuse, primarily because of Ca2+ associated with endoplasmic reticulum membranes. Punctate fluorescence assumes a polar distribution throughout mitosis. Cones of diffuse fluorescence in the chromosome-to-pole regions of the metaphase spindle appear to coincide with the kinetochore fibers; during anaphase, the cones of fluorescence coalesce and this region of the spindle exhibits uniform diffuse fluorescence. Perturbation of the cellular Ca2+ distribution by treatment with lanthanum, procaine, or EDTA [ethylene glycol bis(2-aminoethyl ether) tetraacetic acid] results in a loss of diffuse fluorescence with no accompanying change in the intensity of punctate fluorescence. Detergent extraction of cellular membranes causes a total elimination of CTC fluorescence. CTC fluorescence of freshly teased crayfish claw muscle sarcoplasmic reticulum coincides with the A bands and is reduced by perfusion with lanthanum, procaine, and EDTA in a manner similar to that for diffuse fluorescence in the endosperm cells. These results are consistent with the hypothesis that a membrane system in the chromosome-to-pole region of the mitotic apparatus functions in the localized release of sequestered Ca2+, thereby regulating the mechanochemical events of mitosis.