REGULATION OF SUBCELLULAR ORGANIZATION OF EXCITABLE CELLS

兴奋细胞亚细胞组织的调节

• 批准号: 6111884
• 负责人: Evelyn Ralston
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6111884
• 关键词: Golgi apparatus; axon; cell line; cellular polarity; dendrites; developmental neurobiology; genetic translation; glucose transporter; immunofluorescence technique; intracellular transport; laboratory rat; messenger RNA; muscle cells; nerve /myelin protein; neuromuscular junction; neurons; organelles; protein transport; tissue /cell culture; transfection; transferrin receptor; vesicle /vacuole

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.

特异性的靶向差异定位 mRNA和蛋白质的不同结构域是重要的， 神经系统中细胞的功能特化。的 建立这种差异定位是一个关键部分， 神经系统细胞的发育。这个目标 项目是了解如何域组织在肌肉和 神经元在过去的几年里，我们进行了一些实验， 旨在了解mRNA是如何分布的虽然 一些mRNA在神经元树突中发现，大多数mRNA似乎 被限制在细胞体中，通过一个似乎 需要持续的蛋白质合成。我们研究的 培养的大鼠海马神经元铁蛋白mRNA的定位。 在对照培养物中，通过原位杂交观察到铁蛋白mRNA 仅限于神经元细胞体，但在培养物中， 蛋白质合成抑制剂，它延伸到神经元树突。 用两种物质中的任何一种处理培养物进行的实验 蛋白质合成抑制剂（放线菌酮和嘌呤霉素）对它们的 拥有或与RNA转录抑制剂（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 铁蛋白mRNA翻译的特异性敏感性的优点， 介质中的铁浓度，指出存在一个 额外的、可饱和的mRNA汇。这些结果提供了一个工作 用于解释mRNA定位数据的模型。我们现在 集中力量调查 高尔基复合体的分布，发生在肌肉 分化编码高尔基体定位的 用荧光蛋白GFP标记的酶已经被 导入肌细胞系C2的成肌细胞中。永久 转染的细胞系已被克隆和表征， 用来追踪活细胞中的高尔基复合体因为高尔基体 在大多数细胞中，复合体通过微管会聚定位 我们还计划跟踪中心体的变化， 肌肉分化过程中中心体的分布。到 因此，已经克隆了C2细胞系， GFP标记的中心体蛋白，pericentrin，在一个 诱导型启动子我们目前正在鉴定这些细胞系。 初步结果表明，高尔基复合体的变化， 在分化过程中发生的变化类似于 在有丝分裂过程中，一个非常有趣的平行现象。