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 Inoue . 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 (cu), respectively . These values for active mitotic metaphase are quite comparable to those obtained by Inoue 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 Inoue, a treatment which theoretically involves first-order polymerization and dissociation kinetics . Treatment of metaphase cells with D20 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 D20 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 Inoue's earlier contention that D 20 treatment can modify the available spindle pool .

Microtubule assembly: Some possible regulatory mechanisms

“…The fibers of the mitotic apparatus have been described as existing in a dynamic equilibrium with monomers (1 5), and by assuming that the birefringence of the spindle was a measure of the extent of polymerization, the thermodynamic parameters for this assembly reaction have been determined (28,29). Since the majority of the birefringence in the mitotic spindle is thought to be due to the ordered arrangement of microtubules Purified protein (C, S, 3.2 mg/ml) was prepared in P solution containing 1 .O mM GTP.…”

Flow fields around ciliated larvae: effects of natural artificial tethers

“…During filming, the whole preparation was kept a t 12 to 13OC by a continuous flow of water through a hollow cooling slide beneath the specimen. The cooling slide was modified from a design by Stevens (1973). The mean swimming speeds of larvae in 2 sequences were 406 pm S-' (106 Lu-n S-' SD, n = 19 frame intervals) and 304 pm S-' (83 bun S-' SD, n = 18 frame intervals).…”

Molecular basis of physical and chemical probes for spindle assembly.

“…Various conditions and agents have been tested that systematically and reversibly alter spindle morphology and birefringence. From a series of observations and experiments (1,13,14,48,57), we postulated that spindle fibers are composed mainly of parallel arrays of microtubules formed by a reversible association of tubulin molecules, and there exists a dynamic equilibrium between the labile spindle fibers and a pool of unassociated tubulin (13). The assembly of microtubules is more than likely controlled by kinetic centers such as centrosomes, MTOCs and kinetochores, and the available concentration of polymerizable tubulin dimers.…”