The present experiments were designed to test whether the simple equilibrium assembly model proposed by Inoue could predict variations in spindle microtubule assembly in response to changes in hydrostatic pressure as it does for changes in temperature. The results were also analyzed according to a model based on nucleated condensation polymerization since this recently appears to be the mechanism by which purified brain microtubules are assembled in vitro. Equilibrium birefringence (BR) of the meiotic metaphase-arrested spindle was measured in vivo as a function of hydrostatic pressure and temperature in Chaetopterus oocytes using a miniature microscope pressure chamber. Increasing pressure in steps to 3,000 psi at temperatures below 22 degrees C did produce decreases in spindle equilibrium BR predictable directly from the simple equilibrium model of spindle assembly. Thermodynamic analysis of the pressure data yielded a value of delta V congruent to 400 ml/mol of polymerizing unit. Theoretical curves based on the nucleated condensation model can also be made to fit the data, but semilog plots of the dependence of the equilibrium constant versus pressure and versus reciprocal temperature are biphasic, suggesting that either the size of the polymerizing unit changes or more than one equilibrium constant governs the assembly reaction. That the same value of delta V, 90 ml/mol, was estimated from both the majority of the spindle BR data and data for the assembly of neural microtubules in vitro supports the possibility that spindle microtubules are assembled by a nucleated condensation mechanism.
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机译:本实验旨在测试Inoue提出的简单平衡装配模型是否可以预测纺锤体微管装配中的变化,以响应静水压力的变化(如温度变化)。还根据基于核缩合聚合反应的模型对结果进行了分析,因为最近这似乎是在体外组装纯化的脑微管的机制。使用微型显微镜压力室,在体内测量了减数分裂中期停滞的纺锤体的平衡双折射(BR)与Chaetopterus卵母细胞中静水压力和温度的关系。在低于22摄氏度的温度下将压力逐步升高至3,000 psi,确实会导致主轴平衡BR的降低,这可以直接从主轴组件的简单平衡模型直接预测到。压力数据的热力学分析得出与400ml / mol聚合单元一致的ΔV值。也可以制作基于成核冷凝模型的理论曲线以拟合数据,但是平衡常数与压力和相对温度的依存关系的半对数图是两相的,这表明聚合单元的大小发生了变化或大于一个平衡常数控制组装反应。从大多数纺锤体BR数据和体外神经微管的组装数据中估计出相同的delta V值90 ml / mol,支持了通过有核冷凝机制组装纺锤体微管的可能性。
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