ULTRASTRUCTURAL-LOCALIZATION OF FILAMENTOUS ACTIN WITHIN NEURONAL INTERPHASE NUCLEI IN-SITU
ULTRASTRUCTURAL-LOCALIZATION OF FILAMENTOUS ACTIN WITHIN NEURONAL INTERPHASE NUCLEI IN-SITU
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DOI：
10.1006/excr.1994.1044
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发表时间：
1994-02-01
影响因子：
3.7
通讯作者：
DEBONI, U
中科院分区：
文献类型：
--
作者：
AMANKWAH, KS;DEBONI, U
先前利用分离的细胞核和核基质的生化研究表明肌动蛋白是间期细胞核的组成部分。此外，最近的超微结构研究表明，间期细胞的细胞核内原位存在肌动蛋白和肌球蛋白。然而，尚不清楚这种核内肌动蛋白是否以未聚合的球状肌动蛋白或丝状（F）-肌动蛋白形式存在。目前的工作使用共聚焦显微镜和超微结构细胞化学技术，在体外和体内证明了完整、未受损的背根神经节神经元的间期核内存在 F-肌动蛋白。FITC-鬼笔环肽标记在体外检测到一小部分细胞中邻近核仁周边的核内 F-肌动蛋白的存在，这一观察结果通过三维重建得到证实。对暴露于重粒肌球蛋白 (HMM) 的细胞进行超微结构分析，结果显示存在典型的“箭头”复合物。这些复合物与核仁相关的观察结果证实了 FITC-鬼笔环肽检测到的核内配体确实代表了 F-肌动蛋白。使用与 20 nm 胶体金 (HMM-Au20) 结合的 HMM 进行嵌入后标记，得到的标记与使用 HMM 获得的标记类似。然而，HMM-Au20 被发现在体外和体内标记的细胞比例比 FITC-鬼笔环肽或 HMM 都要大得多。这一发现表明，HMM-Au20 标记更准确地反映了细胞核中肌动蛋白聚合的程度。 HMM-Au20 和 α-肌节肌动蛋白抗体的双重标记结果证实，只有少量核肌动蛋白呈 F 型。总之，这些结果首次从超微结构上证明了神经元细胞核中存在 F-肌动蛋白。虽然核 F-肌动蛋白的作用尚未确定，但结果表明 F-肌动蛋白可能代表分子马达的一个组成部分，负责将特定染色质结构域动态定位到在许多细胞类型中观察到的组织特异性、非随机模式。
Previous biochemical studies utilizing isolated nuclei and nuclear matrices have shown actin to be a constituent of the interphase nucleus. In addition, recent ultrastructural work has shown the presence of actin and myosin within nuclei of interphase cellsin situ. It was unclear, however, whether this intranuclear actin is present in the unpolymerized globular actin or the filamentous (F)-actin form. The present work, using confocal microscopy and ultrastructural cytochemical techniques, demonstrates the presence of F-actin within interphase nuclei of intact, uncompromised, dorsal root ganglion neuronsin vitroandin vivo.Labeling by FITC-phalloidin detected the presence of intranuclear F-actin adjacent to the nucleolar periphery in a small fraction of cellsin vitro, an observation confirmed by three-dimensional reconstruction. Ultrastructural analyses of cells exposed to heavy meromyosin (HMM), showed the presence of typical "arrowhead" complexes. The observation that these complexes were associated with nucleoli confirms that the intranuclear ligand detected by FITC-phalloidin indeed represents F-actin. Postembedding labeling with HMM conjugated to 20-nm colloidal gold (HMM-Au20) resulted in labeling similar to that obtained with HMM. However, HMM-Au20was found to label a much larger fraction of cells, bothin vitroandin vivo, than did FITC-phalloidin or HMM. This finding indicates that labeling with HMM-Au20more accurately reflects the extent of actin polymerization in nuclei. Results from double labeling with HMM-Au20and an antibody to α-sarcomeric actin confirmed that only a small amount of nuclear actin is in the F-form. Together, these results represent a first ultrastructural demonstration of the presence of F-actin in nuclei of neurons. While the role of nuclear F-actin has yet to be defined, the results suggest that F-actin may represent a component of the molecular motor responsible for the dynamic positioning of specific chromatin domains into the tissue-specific, nonrandom patterns observed in many cell types.