Morcillo G, Gorab E, Tanguay R M, Díez J L
Department of Organic Chemistry and Biology, Universidad Nacional de Educación a Distancia, Madrid, Spain.
Exp Cell Res. 1997 Nov 1;236(2):361-70. doi: 10.1006/excr.1997.3726.
Hsp70, the most abundant and conserved heat shock protein, has been described as strongly concentrating in the nucleolus during heat shock. The important metabolic processes that take place in the nucleolus, rDNA transcription, processing, and assembling with ribosomal proteins, and the nucleolar architecture itself are very sensitive to temperature changes. In this work, we have analyzed in detail the nucleolar changes, in structure and activity, induced by temperature in Chironomus thummi salivary gland cells and the fine subnucleolar localization of Hsp70 during heat shock. The optimum temperature chosen to induce the heat shock response was 35 degrees C. Under these conditions transcription of heat shock genes, inactivation of previously active genes and maximum synthesis of Hsps take place, while survival of larvae and recovery were ensured. After 1 h at 35 degrees C, nucleoli change from a uniform control pattern to a segregated pattern of nucleolar components that can be observed even at the light microscopic level. The dense fibrillar component (DFC) and the granular component appeared perfectly differentiated and spatially separated, the former occupying mainly the central inner region surrounded by a rim of granular component. Hsp70 was specifically localized within the DFC upon heat shock as shown by immunolocalization by both light and electron microscopy. Pulse labeling with [3H]uridine proves that rRNA transcription continues during heat shock. The pattern of Hsp70 distribution within the nucleolus correlates with that of newly produced rRNA transcripts. Hsp70 also colocalizes with RNA polymerase I, both being restricted to the DFC. These data show that the DFC seems to be the intranucleolar target for Hsp70 in heat-shocked cells. We discuss these results in relation to the possible function of Hsp70 in the first steps of preribosome synthesis.
热休克蛋白70（Hsp70）是最丰富且保守的热休克蛋白，据描述在热休克期间会强烈聚集于核仁。核仁中发生的重要代谢过程，即核糖体DNA（rDNA）转录、加工以及与核糖体蛋白的组装，以及核仁结构本身对温度变化非常敏感。在这项研究中，我们详细分析了温度诱导的嗜热四斑摇蚊唾液腺细胞的核仁结构和活性变化，以及热休克期间Hsp70在核仁内的精细亚定位。选择35摄氏度作为诱导热休克反应的最佳温度。在这些条件下，热休克基因转录、先前活跃基因失活以及热休克蛋白（Hsps）的最大合成会发生，同时确保幼虫存活和恢复。在35摄氏度处理1小时后，核仁从均匀的对照模式转变为核仁成分的分离模式，甚至在光学显微镜水平就能观察到。致密纤维成分（DFC）和颗粒成分呈现出完美的分化且在空间上分离，前者主要占据由颗粒成分边缘包围的中央内部区域。通过光学和电子显微镜免疫定位显示，热休克时Hsp70特异性定位于DFC内。用[3H]尿苷脉冲标记证明热休克期间rRNA转录持续进行。Hsp70在核仁内的分布模式与新产生的rRNA转录本的模式相关。Hsp70也与RNA聚合酶I共定位，两者都局限于DFC。这些数据表明，DFC似乎是热休克细胞中Hsp70在核仁内的靶点。我们结合Hsp70在核糖体前体合成第一步中可能的功能来讨论这些结果。
