Intracellular free calcium and mitosis in mammalian cells: anaphase onset is calcium modulated, but is not triggered by a brief transient.

Abstract

Abstract. Swiss 3T3 fibroblasts and LLC-PK epithelial cells in prometaphase or metaphase were either injected with fura-2 or loaded with the acetoxymethyl ester derivative of fura-2 (fura-2 AM) and monitored by microspectrofluorimetry. With both methods of loading, we observed two aspects of intracellular free calcium (Ca~) metabolism. in calcium-free medium, but not in calcium containing medium, blocked both anaphase and the sustained Ca~ elevation in almost all cases. Blocked cells were rescued by returning calcium to the medium, whereupon Ca~ slowly but steadily rose as the cell entered anaphase. Spindle microtubules persisted through the EGTA block. Depolymerization of spindle microtubules by nocodazole also reversibly blocked anaphase onset and the sustained Ca~ elevation, but did not block transients. This study has revealed the following: (a) anaphase in mammalian fibroblasts and epithelial cells is not triggered by brief calcium transients; (b) anaphase is a calcium-modulated event, usually accompanied by a sustained elevation of Cai above 50 nM; (c) the elevation of Ca~ is dependent upon an intact spindle; and (d) fibroblasts progress through mitosis by drawing upon either intracellular or extracellular sources of calcium.

Calcium ions function as a booster of chromosome condensation

“…Intracellular Ca 2+ concentrations increase from mitotic entry in response to ionositol-1,4,5-triphosphate (InsP 3 ), which releases Ca 2+ from intracellular storage organelles38. Increasing Ca 2+ concentrations during mitosis guide the cellular progression through mitosis, by mechanisms including NEB, chromosome condensation, spindle fibre formation and sister chromatid separation212639. Ca 2+ is a universal intracellular secondary messenger; therefore, the prolonged prometaphase stage caused by Ca 2+ depletion might be explained by effects other than the compaction of chromosomes.…”

“…Divalent cations, particularly Mg 2+ and Ca 2+ , are known to increase and shift from their storage organelles to chromatin during the mitotic phase of the cell cycle2122, implicating them in mitosis. Unlike Mg 2+ , free intracellular Ca 2+ is present in low concentrations (nanomolar level) and is tightly regulated.…”

Ca2+ triggers premature inactivation of the cdc2 protein kinase in permeabilized sea urchin embryos.

“…In vertebrate oocytes arrested at the second meiotic metaphase, a transient increase in the concentration of cytosolic Ca2+ ([Ca2W]I) at fertilization induces cdc2 kinase inactivation and progression into anaphase (10)(11)(12). Elevated [Ca2li also has been implicated as an inducer of anaphase onset in mitotic cells, since [Ca2+Ji increases transiently at the metaphase/anaphase transition (13,14) and manipulations that raise or lower [Ca2W]i accelerate or delay anaphase onset in some experimental systems (15)(16)(17)(18)(19). [Ca2+]i is controlled by intracellular membrane compartments concentrated in the mitotic spindle (20)(21)(22)(23).…”

Calcium sensitivity of α-actinin is required for equatorial actin assembly during cytokinesis

“…Our observation suggests that calcium sensitivity of α-actinin is required for its proper accumulation at the equator during cytokinesis. Therefore, calcium transients associated with anaphase onset 16 most likely trigger the dissociation of α-actinin from nonequatorial F-actin, facilitating its movement to and accumulation at the equator, where it would subsequently cross-link equatorial F-actin.…”

“…In mammalian cells, calcium transients, which may be global rather than localized, have been observed at anaphase onset, 16 although the functional role of the calcium signals have not been determined. Our findings suggest that calcium transients most likely regulate α-actinin dynamics outside the equator, facilitating the delivery of both α-actinin and F-actin into the equator.…”