Zatsepina O V, Todorov I T, Philipova R N, Krachmarov C P, Trendelenburg M F, Jordan E G
A. N. Belozersky Institute of Physical and Chemical Biology, Moscow State University, Russia.
Eur J Cell Biol. 1997 May;73(1):58-70.
A major nucleolar phosphoprotein, B23, is thought to play several apparently unrelated roles and appears to be associated with other cell compartments besides the nucleolus. However, characteristic properties of B23 variants still remain to be established. Here, we raised a new monoclonal antibody against B23 (20B2) and used it to address the issue particularly focusing on the events during mitosis. Also, we made an attempt to generalize the data on the cell cycle-dependent translocations of B23 by the use of three mammalian cell lines (HeLa, PK, RAMT) which were found to be immunoreactive for 20B2. In all the cell strains studied, B23 was chiefly located within the nucleolus at interphase, and was associated with a few cellular domains during mitosis. They were: the nucleoplasm (at prophase before the nuclear envelope breakdown), the cytoplasm (from prometaphase until mid telophase), the perichromosomal layer (from prometaphase till early telophase), cytoplasmic B23-containing bodies (at anaphase and telophase) and prenucleolar bodies, PNBs (at telophase). On Western blots, electrophoretic mobility of B23 was found to be the same at G1, S and G2 periods of interphase, but became slower at mitosis. In situ and cell extraction experiments showed that like the nucleolar B23, B23 of the perichromosomal layer and that of PNBs was highly resistant to extraction with Triton X-100, but could be released with Triton X-100/RNase A. These findings indicated that these portions of B23 were most likely to be associated with RNA. The cytoplasmic B23 was the major intracellular variant of B23 during mitosis. It had a slightly lower electrophoretic mobility than the perichromosomal B23 and could readily be extracted with Triton X-100 without addition of RNase A, a fact indicating that the cytoplasmic B23 was mainly in an RNA-free state. Mitosis-like translocations of B23 from the nucleolus to the nucleoplasm induced by actinomycin D increased its extractability but did not affect the electrophoretic mobility. The phosphorylation status of different B23 variants at interphase and mitosis both in controls and following the drug, is discussed.
一种主要的核仁磷蛋白B23，被认为发挥着几种明显不相关的作用，并且似乎除了核仁之外还与其他细胞区室相关联。然而，B23变体的特征特性仍有待确定。在此，我们制备了一种针对B23的新单克隆抗体（20B2），并使用它来解决该问题，特别关注有丝分裂期间的事件。此外，我们尝试通过使用三种对20B2具有免疫反应性的哺乳动物细胞系（HeLa、PK、RAMT）来概括关于B23细胞周期依赖性易位的数据。在所研究的所有细胞系中，B23在间期主要位于核仁内，并且在有丝分裂期间与一些细胞结构域相关联。它们是：核质（在核膜破裂前的前期）、细胞质（从前中期直到末期中期）、染色体周围层（从前中期直到末期早期）、含B23的细胞质体（在后期和末期）以及前核仁体，PNBs（在末期）。在蛋白质免疫印迹法中，发现B23在间期的G1、S和G2期的电泳迁移率相同，但在有丝分裂时变慢。原位和细胞抽提实验表明，与核仁B23一样，染色体周围层的B23和PNBs的B23对Triton X - 100抽提具有高度抗性，但可以用Triton X - 100/核糖核酸酶A释放。这些发现表明，B23的这些部分最有可能与RNA相关联。细胞质B23是有丝分裂期间B23的主要细胞内变体。它的电泳迁移率比染色体周围的B23略低，并且在不添加核糖核酸酶A的情况下很容易被Triton X - 100抽提，这一事实表明细胞质B23主要处于无RNA状态。放线菌素D诱导的B23从核仁到核质的类有丝分裂易位增加了其可抽提性，但不影响电泳迁移率。讨论了在对照以及用药后不同B23变体在间期和有丝分裂时的磷酸化状态。
