REGULATION OF SUBCELLULAR ORGANIZATION OF EXCITABLE CELLS

特定的、有针对性的差异化本地化 不同结构域的mRNAs和蛋白质对于 神经系统中细胞的功能特化。这个 建立这种差异化的本地化是 神经系统细胞的发育。这样做的目的是 项目是了解领域是如何在肌肉中组织的， 神经元。在过去的几年里，我们进行了一些实验 旨在了解mRNAs是如何分布的。虽然 在神经元树突中发现了一些mRNAs，似乎大多数mrna 被限制在细胞体内，通过一个似乎是 需要持续的蛋白质合成。我们已经研究了 培养的大鼠海马神经元中铁蛋白mRNA的定位。 在对照培养中，用原位杂交观察铁蛋白mRNA 仅限于神经元细胞体，但在经过 蛋白质合成抑制剂，它延伸到神经元树突。 在用两种方法中的任何一种处理的培养上进行的实验 蛋白质合成抑制剂(放线菌酮和嘌呤霉素)对它们的影响 拥有或与RNA转录抑制物(DRB或 放线菌素D)指出核糖体结合是 将mRNA保留在细胞体中。然而，实验采取了 铁蛋白信使核糖核酸翻译的特异性敏感性优势 介质中的铁浓度，表明存在一种 额外的、可饱和的信使核糖核酸池。这些结果提供了一个有效的 解释信使核糖核酸本地化数据的模型。我们现在是 把重点放在调查新时期的变化上 肌肉过程中高尔基复合体的分布 差异化。高尔基体定位编码基因的构建 用荧光蛋白GFP标记的酶已经被 将其导入肌肉细胞系C2的成肌细胞。永久 已经克隆和鉴定了转基因细胞系，并将 用于在活细胞中跟随高尔基复合体。因为高尔基人 在大多数细胞中，复合体由聚集的微管定位。 在中心体上，我们还计划跟踪 肌肉分化过程中中心体的分布。至 因此，C2细胞株已经被克隆出来，表达一种 GFP标记的中心体蛋白，周中心素，在一种 诱导型启动子。我们目前正在对这些细胞系进行鉴定。 初步结果表明，高尔基复合体改变了 在分化过程中发生的变化类似于发生的变化 在有丝分裂期间，有一个非常有趣的相似之处。

The targeted, differential localization of specific mRNAs and proteins into distinct domains is important for the functional specialization of cells in the nervous system. The establishment of this differential localization is a crucial part of the development of the cells of the nervous system. The goal of this project is to understand how domains are organized in muscle and neurons. Over the past years we have carried out experiments aimed at understanding how mRNAs are distributed. Although some mRNAs are found in neuronal dendrites, most mRNAs seem to be confined to the cell bodies, by a process which seems to require ongoing protein synthesis. We have examined the localization of ferritin mRNA in cultured rat hippocampal neurons. In control cultures, ferritin mRNA observed by in situ hybridization is confined to the neuronal cell bodies, but in cultures treated by protein synthesis inhibitors, it extends into neuronal dendrites. Experiments carried out on cultures treated with either of two protein synthesis inhibitors (cycloheximide and puromycin) on their own or together with an inhibitor of RNA transcription (DRB or actinomycin D) pointed to ribosome binding as the mechanism for retaining mRNAs in the cell bodies. However, experiments taking advantage of the specific sensitivity of ferritin mRNA translation to the iron concentration in the medium, pointed to the existence of an additional, saturable mRNA sink. These results provide a working model with which to interpret mRNA localization data. We are now focusing our efforts on the investigation of the changes in the distribution of the Golgi complex that take place during muscle differentiation. cDNA constructs encoding Golgi- localized enzymes tagged with the fluorescent protein GFP have been introduced into myoblasts of the muscle cell line C2. Permanently transfected cell lines have been cloned and characterized and will be used to follow the Golgi complex in live cells. Because the Golgi complex, in most cells, is positioned by microtubules converging onto the centrosome, we also plan to follow changes in the distribution of the centrosome during muscle differentiation. To that effect, C2 cell lines have been cloned that express a GFP-tagged centrosomal protein, pericentrin, under control of an inducible promoter. We are presently characterizing these cell lines. Preliminary results suggest that the Golgi complex changes that occur during differentiation resemble the changes that take place during mitosis, a very interesting parallel.