A THERMODYNAMIC ANALYSIS OF MITOTIC SPINDLE EQUILIBRIUM AT ACTIVE METAPHASE

Abstract

The mitotic apparatus of first-division metaphase eggs of the sea urchin Strongylocentrotus drobachiensis was observed by means of polarization microscopy under controlled temperature conditions. Eggs were fertilized and grown at two temperature extremes in order to produce two different sizes of available spindle pool. Slow division time allowed successive samples of such cells to be observed at the same point in metaphase but at different equilibrium temperatures, yielding curves of metaphase equilibrium birefringence vs. observational temperature. Using the plateau value of birefringence at higher temperatures as a measure of total available spindle pool and the observed birefringence at lower temperatures as a measure of polymerized material at equilibrium, the spindle protein association was evaluated according to the method of Inoué. Both pool conditions produced linear van't Hoff functions. Analysis of these functions yielded enthalpy and entropy changes of +55-65 kcal/mol and +197-233 entropy units (eu), respectively. These values for active mitotic metaphase are quite comparable to those obtained by Inoué and co-workers for arrested meiotic metaphase cells. When other equilibrium treatments were considered, the best fit to the experimental data was still that of Inoué, a treatment which theoretically involves first-order polymerization and dissociation kinetics. Treatment of metaphase cells with D(2)O by direct immersion drove the equilibrium to completion regardless of temperature, attaining or exceeding a birefringence value equal to the cell's characteristic pool size; perfusion with D(2)O appeared to erase the original temperature-determined pool size differences for the two growth conditions, attaining a maximum value characteristic of the larger pool condition. These data confirm Inoué's earlier contention that D(2)O treatment can modify the available spindle pool.