SUBCELLULAR MRNA DISTRIBUTION IN MYOCYTES

适应对于超负荷或衰竭的心脏至关重要， 心脏中的总肌原纤维质量决定心脏强度。 我 长期目标是了解横纹肌如何适应其分子 组合物响应其生理需求的变化。 到 解剖刺激和肥大之间的机制，我检查早期 第一个小时发生的变化。 我的假设是， 生长受两个过程控制：一个主要的磷酸化过程， 转录因子和细胞质mRNA的控制。 研究要求 对β-肾上腺素能途径的快速药理学干预。 因此，我使用新生大鼠心脏的原代细胞培养物。 这 多样性，混合细胞群是最好的研究细胞生物学 单细胞水平的技术。 我用免疫化学来监测 通过磷酸化的转录因子，cAMP调节 元件结合蛋白（CREB）。 我用原位杂交技术 亚细胞信息分布的变化。 具体目标1： CREB磷酸化的途径，决定了一个有效的范例， PKA激活细胞肥大，并建立条件， 增加肌原纤维中α-肌球蛋白沉积。 具体目标2 定量mRNA从细胞核的向外迁移， mRNA的分布模式是否是由于扩散、转运或 翻译捕获 具体目标3测试非翻译区， 3 'UTR是微管转运或翻译控制所必需的。 我用β-半乳糖苷酶编码的嵌合构建体转染 区域和α-肌球蛋白3 'UTR。 细胞质mRNA的研究还没有 作为心脏肥大的快速调节事件进行了充分探索。

Adaptation is of utmost importance in overloaded or failing hearts because the total myofibrillar mass in the heart determines cardiac strength. My long-term goal is to understand how striated muscle adapts its molecular composition in response to changes in its physiological demands. To dissect mechanisms between stimulation and hypertrophy, I examine early changes occurring in the first hour. My hypotheses proposes that early growth is governed by two processes; phosphorylation of a major transcription factor and control of cytoplasmic mRNA. The study requires rapid pharmacological interventions to the beta-adrenergic pathway. Therefore, I use a primary cell culture from neonatal rat heart. This diverse, mixed cell population is best studied by cell biological techniques at the single cell level. I use immunochemistry to monitor activation by phosphorylation of the transcription factor, cAMP regulatory element binding protein (CREB). I use in situ hybridization to examine changes in subcellular message distribution. Specific aim 1 characterizes the pathways of CREB phosphorylation, determines an effective paradigm of PKA activation for cell hypertrophy, and establishes conditions that increase alpha-myosin protein deposition in myofibrils. Specific aim 2 quantifies the outward migration of mRNA from the nucleus and distinguishes whether the mRNA distribution pattern is due to diffusion, transport, or translational trapping. Specific aim 3 tests if the untranslated region, 3'UTR, is required for transport by microtubules or control of translation. I use transfection with a chimeric construct of beta-galactosidase coding region and alpha-myosin 3'UTR. Study of cytoplasmic mRNA has not been sufficiently explored as a rapid regulatory event in cardiac hypertrophy.