Gribbon Chris, Dahm Ralf, Prescott Alan R, Quinlan Roy A
School of Life Sciences, MSIWTB, University of Dundee, UK.
Eur J Cell Biol. 2002 Oct;81(10):557-66. doi: 10.1078/0171-9335-00275.
The transcriptional status of cells can be deduced from the staining pattern of various nuclear markers such as the Cajal body, nucleolus and nuclear speckles. In this study we have used these markers to correlate transcriptional status with cell differentiation in the lens. As a closed system with no cell loss and with each stage being spatially preserved, it is particularly well suited to such studies. To confirm that the nuclear markers in lens cells follow the same trends as in other cells, primary bovine lens epithelial cells were cultured and then treated with actinomycin D to inhibit transcription. This reduced the Cajal body markers to one or two foci per nucleus and the nucleoli became compacted as revealed by fibrillarin staining. The nuclear speckles, containing snRNPs (e.g. Sm) and the splicing factor, SC35, also became larger and more numerous while the signal for trimethylguanine (TMG) decreased suggesting a role hierarchy for the various speckle factors during transcriptional shutdown. The signal for survival of motor neurones gene product (SMN) also decreased at this point. In the lens epithelium, postmitotic cells near the equatorial region had one or two Cajal bodies per nucleus, indicating these cells had only basal levels of transcription. Sm was also present as large foci in these cells. Interestingly, both the speckles and Cajal bodies were NuMA-positive in these post-mitotic cells. At the epithelial-fibre cell transition, Cajal body number increased, while their size decreased indicative of increased transcriptional activity. Fibrillarin adopted the open floret pattern indicating increased transcriptional activity. The nuclear speckles adopted a more diffuse nucleoplasmic pattern, although some spots were still observed. All NuMA colocalisation with the Cajal bodies and nuclear speckles was lost at this stage of lens cell differentiation. Transcriptional shutdown occurs at a later stage in fibre cell differentiation, prior to programmed nuclear destruction. In the lens, both the Cajal bodies and nuclear speckles again became NuMA-positive, although separate NuMA spots were also formed during transcriptional shutdown. These data suggest the nuclear matrix is important in the concentration of Cajal body and speckle components into large, distinct spots in transcriptionally inactive nuclei and also suggest a new role for NuMA in post-mitotic cells to assist in these sub-nuclear reorganisations.
细胞的转录状态可通过多种核标记物的染色模式推断出来，如卡哈尔体、核仁及核斑。在本研究中，我们利用这些标记物来关联晶状体中的转录状态与细胞分化。作为一个无细胞丢失且各阶段在空间上得以保留的封闭系统，它特别适合此类研究。为确认晶状体细胞中的核标记物与其他细胞遵循相同趋势，我们培养了原代牛晶状体上皮细胞，然后用放线菌素D处理以抑制转录。这使得每个细胞核中的卡哈尔体标记物减少至一两个斑点，并且通过纤维蛋白原染色显示核仁变得致密。包含小核核糖核蛋白（如Sm）和剪接因子SC35的核斑也变得更大且数量更多，而三甲基鸟嘌呤（TMG）信号降低，这表明在转录关闭期间各种斑点因子存在作用层级关系。此时运动神经元存活基因产物（SMN）的信号也降低。在晶状体上皮中，赤道区域附近的有丝分裂后细胞每个细胞核有一两个卡哈尔体，表明这些细胞仅具有基础水平的转录。Sm在这些细胞中也以大斑点形式存在。有趣的是，在这些有丝分裂后细胞中，核斑和卡哈尔体均为NuMA阳性。在上皮 - 纤维细胞转变处，卡哈尔体数量增加，而其大小减小，这表明转录活性增加。纤维蛋白原呈现开放的小花图案，表明转录活性增加。核斑呈现出更弥散的核质模式，尽管仍观察到一些斑点。在晶状体细胞分化的这个阶段，所有NuMA与卡哈尔体和核斑的共定位均消失。转录关闭发生在纤维细胞分化的后期，即在程序性核破坏之前。在晶状体中，卡哈尔体和核斑再次变为NuMA阳性，尽管在转录关闭期间也形成了单独的NuMA斑点。这些数据表明核基质在将卡哈尔体和斑点成分浓缩到转录不活跃细胞核中的大的、明显的斑点中很重要，并且还表明NuMA在有丝分裂后细胞中具有协助这些核内亚结构重组的新作用。
