Experimental manipulation of the amount of tubulin available for assembly into the spindle of dividing sea urchin eggs
Experimental manipulation of the amount of tubulin available for assembly into the spindle of dividing sea urchin eggs
复制标题
可组装成分裂海胆卵纺锤体的微管蛋白量的实验操作
作者：
G. Sluder
通过实验改变卵内可聚合微管蛋白的量，研究了海胆 Lytechinus variegatus 卵中纺锤体的组装。在第一次核膜破裂前至少 20 分钟，将来自同一雌性的受精卵的等分试样分别用 1 X 10(-6) M Colcemid 脉冲 1-6 分钟。这种处理在纺锤体形成之前使部分细胞微管蛋白失活。进入有丝分裂后，经过处理的卵形成功能性纺锤体，其长度和双折射延迟减少，但宽度不变。随着秋水仙胺暴露量的增加，中期纺锤体的长度和延迟逐渐减少。在受精前用秋水酰胺脉冲卵子也得到了类似的结果，这表明未受精海胆卵中发现的微管蛋白后来用于纺锤体的形成。纺锤体一旦组装好，就会对细胞内可聚合微管蛋白数量的增加做出反应。通过用 366 nm 光照射细胞，可以通过实验实现经秋水仙胺处理的细胞内可聚合微管蛋白数量的快速增加。这种处理以光化学方式使秋水仙胺失活，从而释放微管蛋白进行聚合。照射后，经秋水仙胺处理的细胞的中期小纺锤体的长度和延迟立即增加。在这些受辐射的细胞中，纺锤体长度和延迟的增加速度比正常纺锤体在中期的速度快四倍。这表明正常前期延迟和纺锤体大小增加的速率可能是由细胞中微管蛋白聚合的最大速率以外的因素决定的。
Spindle assembly is studied in the eggs of the sea urchin Lytechinus variegatus by experimentally varying the amount of polymerizable tubulin within the egg. Aliquots of fertilized eggs from the same female are individually pulsed for 1-6 min with 1 X 10(-6) M Colcemid at least 20 min before first nuclear envelope breakdown. This treatment inactivates a portion of the cellular tubulin before the spindle is formed. Upon entering mitosis, treated eggs form functional spindles that are reduced in length and birefringent retardation but not width. With increased exposure to Colcemid, the length and retardation of the metaphase spindles are progressively reduced. Similar results are obtained by pulsing the eggs with Colcemid before fertilization, which demonstrates that the tubulin found in unfertilized sea urchin eggs is later used in spindle formation. Spindles, once assembled, are responsive to increases in the amount of polymerizable tubulin within the cell. Rapid increases in the amount of polymerizable tubulin within a Colcemid-treated cell can be experimentally effected by irradiating the cells with 366-nm light. This treatment photochemically inactivates the Colcemid, thereby freeing the tubulin to polymerize. Upon irradiation, the small prometaphase spindles of Colcemid-treated cells immediately increase in length and retardation. In these irradiated cells, spindle length and retardation increase as much as four times faster than they do during prometaphase for normal spindles. This suggests that the rate of the normal prometaphase increase in retardation and spindle size may be determined by factors other than the maximum rate of tubulin polymerization in the cell.