Incorporation and turnover of labeled exogenous tubulin in the mitotic spindles of Chaetopterus oocytes and HeLa cells.
Incorporation and turnover of labeled exogenous tubulin in the mitotic spindles of Chaetopterus oocytes and HeLa cells.
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标记的外源微管蛋白在毛毛虫卵母细胞和 HeLa 细胞有丝分裂纺锤体中的掺入和周转。
DOI：
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发表时间：
1986
影响因子：
--
通讯作者：
V. Sarma
中科院分区：
文献类型：
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作者：
D. Goode;V. Sarma
微管蛋白掺入有丝分裂纺锤体原位研究孵育透化的有丝分裂细胞的解决方案含有[3 H] GTP标记或二氯三嗪氨基荧光素（DTAF）标记的微管蛋白。将来自毛翅目卵母细胞的中期HeLa细胞或含纺锤体的“小细胞”在微管组装缓冲液加0.5%Nonidet P-40、lmg/ml 120，000 g哺乳动物脑微管蛋白上清液和[3 H]GTP中裂解。在不同的孵育时间后，在含有2 M-甘油的组装缓冲液中分离有丝分裂纺锤体，并通过4 M-甘油垫离心将其与未结合的计数分离;每mg蛋白质的3 H计数线性增加8-12分钟，然后在毛翅目卵母细胞和HeLa细胞中达到平台或稳定状态。加入4 mM CaCl 2可阻断或逆转掺入。如果从组装混合物中省略外源性微管蛋白或裂解细胞，则很少或没有[3 H]GTP掺入。为了测量[3 H] GTP-微管蛋白从有丝分裂纺锤体的损失率，将细胞在微管蛋白加[3 H]GTP中孵育30分钟，并加入20倍过量的冷GTP（2 mM）。孵育不同时间后取出样品，如上所述分离纺锤体并计数3 H含量。[3 H]GTP以约16%/min的速率从纺锤体中丢失，直到约8分钟内达到新的稳态。这些结果与这些溶解细胞模型的纺锤体微管中[3 H] GTP-微管蛋白的掺入和周转一致。通过将荧光DTAF-微管蛋白掺入这些裂解细胞类型的有丝分裂纺锤体中，研究了这种新掺入的微管蛋白在纺锤体中的位置。（250字处删节）
The incorporation of tubulin into mitotic spindles in situ was studied by incubating permeabilized mitotic cells in solutions containing [3H]GTP-labeled or dichlorotriazinylamino fluorescein (DTAF)-labeled tubulin. Metaphase HeLa cells or spindle-containing "minicells" from Chaetopterus oocytes were lysed in a microtubule-assembly buffer plus 0.5% Nonidet P-40, 1 mg/ml 120,000g supernatant mammalian brain tubulin, and [3H]GTP. After different periods of incubation, mitotic spindles were isolated in 2 M-glycerol-containing assembly buffer and separated from unbound counts by centrifugation through a 4 M-glycerol cushion; 3H counts per mg protein increase linearly for 8-12 min and then reach a plateau or steady state in both Chaetopterus oocytes and HeLa cells. Addition of 4 mM CaCl2 blocks or reverses incorporation. Little or no [3H]GTP is incorporated if exogenous tubulin or lysed cells are omitted from the assembly mixture. To measure the loss rate of [3H]GTP-tubulin from mitotic spindles, cells were incubated in tubulin plus [3H]GTP for 30 min, and a 20-fold excess of cold GTP (2 mM) was added. Samples were removed after incubation for different periods, and spindles were isolated as described above and counted for 3H content. [3H]GTP is lost from spindles at a rate of about 16%/min until a new steady state is reached in about 8 min. These results are consistent with an incorporation and turnover of [3H]GTP-tubulin in spindle microtubules of these lysed-cell models. The location of this newly incorporated tubulin in the spindle was investigated by incorporating fluorescent DTAF-tubulin into mitotic spindles of these lysed cell types.(ABSTRACT TRUNCATED AT 250 WORDS)
在稳态下将放射性微管蛋白掺入微管中。
DOI：
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发表时间：
1980
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Zeeberg,B;Reid,R;Caplow,M
通讯作者：
Caplow,M