Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes.
Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes.
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DOI：
10.1083/jcb.135.6.1701
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发表时间：
1996-12
影响因子：
7.8
通讯作者：
Nigg, EA
中科院分区：
文献类型：
--
作者：
Lane, HA;Nigg, EA
哺乳动物polo-like激酶1（Plk 1）与果蝇波罗基因产物、酿酒酵母Cdc 5 p和粟酒裂殖酵母plo 1+结构相关，后者是一个新出现的丝氨酸-苏氨酸激酶家族，参与细胞周期调控。基于在无脊椎动物和酵母中获得的假定同源物的数据，人类Plk 1被怀疑调节有丝分裂的一些基本方面，但以前没有直接的实验证据支持这一假设。在这项研究中，我们已经使用了细胞复制，显微注射试验，以探讨在体内功能的Plk 1在永生化（HeLa）和nonimmortalized（Hs 68）人类细胞。在细胞周期的各个阶段注射抗Plk 1抗体（Plk 1+）对DNA复制的动力学没有影响，但严重损害了细胞分裂的能力。通过荧光显微镜分析Plk 1（+）注射的有丝分裂停滞的HeLa细胞，发现浓缩的染色质和单星微管阵列的异常分布，这些微管阵列是从重复但未分离的中心体成核的。最引人注目的是，Plk 1（+）注射细胞的中心体的大小急剧减少，γ-微管蛋白和MPM-2免疫反应性的积累受到损害。这些数据表明，Plk 1活性是必要的中心体的功能成熟在G2晚期/早期前期，因此，建立一个双极纺锤体。不排除Plk 1在有丝分裂后期的其他作用，尽管在纺锤体形成完成后注射Plk 1+不会干扰胞质分裂。将Plk 1+注射到非永生化Hs 68细胞中产生了定性相似的表型，但绝大多数注射的Hs 68细胞在G2期停滞为单个单核细胞。后一种观察结果提示，在非永生化细胞中，存在一个对Plk 1功能受损敏感的中心体成熟检查点。
Mammalian polo-like kinase 1 (Plk1) is structurally related to the polo gene product of Drosophila melanogaster, Cdc5p of Saccharomyces cerevisiae, and plo1+ of Schizosaccharomyces pombe, a newly emerging family of serine-threonine kinases implicated in cell cycle regulation. Based on data obtained for its putative homologues in invertebrates and yeasts, human Plk1 is suspected to regulate some fundamental aspect(s) of mitosis, but no direct experimental evidence in support of this hypothesis has previously been reported. In this study, we have used a cell duplication, microinjection assay to investigate the in vivo function of Plk1 in both immortalized (HeLa) and nonimmortalized (Hs68) human cells. Injection of anti-Plk1 antibodies (Plk1+) at various stages of the cell cycle had no effect on the kinetics of DNA replication but severely impaired the ability of cells to divide. Analysis of Plk1(+)-injected, mitotically arrested HeLa cells by fluorescence microscopy revealed abnormal distributions of condensed chromatin and monoastral microtubule arrays that were nucleated from duplicated but unseparated centrosomes. Most strikingly, centrosomes in Plk1(+)-injected cells were drastically reduced in size, and the accumulation of both gamma-tubulin and MPM-2 immunoreactivity was impaired. These data indicate that Plk1 activity is necessary for the functional maturation of centrosomes in late G2/early prophase and, consequently, for the establishment of a bipolar spindle. Additional roles for Plk1 at later stages of mitosis are not excluded, although injection of Plk1+ after the completion of spindle formation did not interfere with cytokinesis. Injection of Plk1+ into nonimmortalized Hs68 cells produced qualitatively similar phenotypes, but the vast majority of the injected Hs68 cells arrested as single, mononucleated cells in G2. This latter observation hints at the existence, in nonimmortalized cells, of a centrosome-maturation checkpoint sensitive to the impairment of Plk1 function.
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DOI：
10.1083/jcb.129.6.1617
发表时间：
1995-06
期刊：
The Journal of cell biology
影响因子：
--
作者：
Golsteyn RM;Mundt KE;Fry AM;Nigg EA
通讯作者：
Nigg EA
影响因子：
11.4
作者：
BAILLY, E;DOREE, M;BORNENS, M
通讯作者：
BORNENS, M
DOI：
10.1073/pnas.91.14.6388
发表时间：
1994-07-05
影响因子：
11.1
作者：
FODE, C;MOTRO, B;DENNIS, JW
通讯作者：
DENNIS, JW
影响因子：
4.8
作者：
HAMANAKA, R;SMITH, MR;FERRIS, DK
通讯作者：
FERRIS, DK
DOI：
10.1073/pnas.80.10.2926
发表时间：
1983-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
DAVIS, FM;TSAO, TY;RAO, PN
通讯作者：
RAO, PN