Oegema K, Marshall W F, Sedat J W, Alberts B M
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448, USA.
J Cell Sci. 1997 Jul;110 ( Pt 14):1573-83. doi: 10.1242/jcs.110.14.1573.
Both the nucleus and the centrosome are complex, dynamic structures whose architectures undergo cell cycle-specific rearrangements. CP190 and CP60 are two Drosophila proteins of unknown function that shuttle between centrosomes and nuclei in a cell cycle-dependent manner. These two proteins are associated in vitro, and localize to centrosomes in a microtubule independent manner. We injected fluorescently labeled, bacterially expressed CP190 and CP60 into living Drosophila embryos and followed their behavior during the rapid syncytial blastoderm divisions (nuclear cycles 10-13). Using quantitative 3-D wide-field fluorescence microscopy, we show that CP190 and CP60 cycle between nuclei and centrosomes asynchronously with the accumulation of CP190 leading that of CP60 both at centrosomes and in nuclei. During interphase, CP190 is found in nuclei. Immediately following nuclear envelope breakdown, CP190 localizes to centrosomes where it remains until telophase, thereafter accumulating in reforming nuclei. Unlike CP190, CP60 accumulates at centrosomes primarily during anaphase, where it remains into early interphase. During nuclear cycles 10 and 11, CP60 accumulates in nuclei simultaneous with nuclear envelope breakdown, suggesting that CP60 binds to an unknown nuclear structure that persists into mitosis. During nuclear cycles 12 and 13, CP60 accumulates gradually in nuclei during interphase, reaching peak levels just before nuclear envelope breakdown. Once in the nucleus, both CP190 and CP60 appear to form fibrous intranuclear networks that remain coherent even after nuclear envelope breakdown. The CP190 and CP60 networks do not co-localize extensively with each other or with DNA. This work provides direct evidence, in living cells, of a coherent protein network that may represent a nuclear skeleton.
细胞核和中心体都是复杂的动态结构，其结构会经历细胞周期特异性的重排。CP190和CP60是两种功能未知的果蝇蛋白，它们以细胞周期依赖的方式在中心体和细胞核之间穿梭。这两种蛋白在体外相互关联，并以微管独立的方式定位于中心体。我们将荧光标记的、细菌表达的CP190和CP60注射到活的果蝇胚胎中，并追踪它们在快速合胞体胚盘分裂（核周期10 - 13）过程中的行为。通过定量三维宽场荧光显微镜，我们发现CP190和CP60在细胞核和中心体之间循环，CP190的积累在中心体和细胞核中均先于CP60。在间期，CP190存在于细胞核中。核膜破裂后，CP190立即定位于中心体，并一直保持到末期，之后在重新形成的细胞核中积累。与CP190不同，CP60主要在后期积累于中心体，并一直持续到早间期。在核周期10和11期间，CP60在核膜破裂时同时积累于细胞核中，这表明CP60与一种持续到有丝分裂期的未知核结构结合。在核周期12和13期间，CP60在间期逐渐积累于细胞核中，在核膜破裂前达到峰值水平。一旦进入细胞核，CP190和CP60似乎都形成了纤维状的核内网络，即使在核膜破裂后仍保持连贯。CP190和CP60网络彼此之间以及与DNA都没有广泛共定位。这项工作在活细胞中提供了直接证据，证明了一个可能代表核骨架的连贯蛋白网络。
