Reticulon Function in ER Morphology

内质网形态中的网状功能

• 批准号: 8204707
• 负责人: Gia Voeltz
• 金额: $ 29.07万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8204707
• 关键词: 3-Dimensional; Address; Affect; Architecture; Axon; Binding; Caliber; Cell Line; Cells; Cellular Morphology; Collaborations; Colorado; Complex; Data; Dendrites; Dimensions; Electron Microscopy; Endoplasmic Reticulum; Eukaryota; Eukaryotic Cell; Fluorescence Microscopy; Fluorescence Recovery After Photobleaching; Goals; Growth; Growth Cones; Health; Higher Order Chromatin Structure; Housing; Immunoprecipitation; In Vitro; Integral Membrane Protein; Label; Laboratories; Length; Measures; Membrane; Metals; Morphology; Mutate; Neurites; Neuronal Differentiation; Neurons; Nuclear Envelope; Organelles; Pattern; Peripheral; Play; Process; Property; Protein Family; Proteins; Relative (related person); Resolution; Role; Shadowing (Histology); Shapes; Site; Site-Directed Mutagenesis; Staining method; Stains; Structure; Surface; Techniques; Testing; Time; Transmembrane Domain; Trees; Tube; Tubular formation; Universities; Work; Xenopus; Yeasts; egg; experience; interest; neurite growth; neuronal cell body; programs; protein complex; protein function; research study; scaffold; tomography

DESCRIPTION (provided by applicant): Reticulons are ubiquitous, highly conserved integral membrane proteins present in all eukaryotes. Reticulon proteins generate the structure of the peripheral endoplasmic reticulum (ER) by shaping the membrane bilayer into a network of tubules in eukaryotic cells. Despite the continuity of the ER membrane, which is composed of the nuclear envelope as well as peripheral ER sheets and tubules, these proteins localize exclusively to ER tubules. The overall goal of the proposed program is to develop our understanding of how the reticulon proteins partition into and shape the ER membrane bilayer into tubules. The specific aims of the program include structure/function studies of vertebrate and yeast reticulon proteins to determine how they localize to tubular ER and which of their structural features are required for their membrane shaping activities. We will mutate various regions of the reticulon protein to determine the effect on protein localization, oligomerization, diffusional mobility, and membrane curvature. For these studies, we will be using fluorescence microscopy, immunoprecipitations, fluorescence recovery after photobleaching, and electron microscopy with immunogold labeling. These experiments will reveal how these integral membrane proteins localize to and shape the membrane bilayer of an organelle, the ER. We propose to use high-resolution EM techniques to analyze the 3-D architecture and surface of ER tubules formed in vitro from Xenopus egg extract. The impetus for this work is to look for a reticulon scaffold around ER tubules that could explain how these proteins directly shape ER tubules of defined dimensions. However, even in the absence of discovering a reticulon scaffold on ER tubules, this work would be the first high-resolution structural analysis of ER tubules and could reveal a great deal about the morphological properties of this organelle. Finally, we will be addressing whether reticulon-generated tubular ER morphology is important for cellular differentiation. We are studying the differentiation of neurons, which causes the morphology of neuronal cells to change quite dramatically and become highly polarized. Neuronal differentiation results in the extension of axons and dendrites, long processes that we show here are filled with tubular ER stained with the reticulon protein. We will address whether increasing and decreasing reticulon protein levels affects neuronal differentiation and what functions of the tubular ER are necessary for axon/neurite growth. We will look for reticulon interacting factors that contribute to the growth, packaging, and organization of the tubular ER into the axon/neurite and analyze the structure of the ER within neurite/axon by electron microscopy. This work will address the functional relevance of organelle morphology to cellular differentiation. PUBLIC HEALTH RELEVANCE: The proposed program to study reticulon protein function will further our understanding of how integral membrane proteins shape the membrane bilayer of an organelle to create functional subdomains. This work will also address the importance of reticulon-generated ER shape to the differentiation and the uniquely polarized cellular morphology of neurons.

描述（由申请人提供）：网状蛋白是所有真核生物中普遍存在的、高度保守的整合膜蛋白。网状蛋白通过将膜双层塑造成真核细胞中的小管网络来产生外周内质网 (ER) 的结构。尽管由核膜以及外周 ER 片和小管组成的 ER 膜具有连续性，但这些蛋白质仅定位于 ER 小管。该计划的总体目标是加深我们对网织蛋白如何分割成内质网双层膜并将其塑造成小管的理解。该计划的具体目标包括脊椎动物和酵母网织蛋白的结构/功能研究，以确定它们如何定位于管状内质网，以及它们的膜成形活动需要哪些结构特征。我们将对网织蛋白的各个区域进行突变，以确定对蛋白质定位、寡聚化、扩散迁移率和膜曲率的影响。对于这些研究，我们将使用荧光显微镜、免疫沉淀、光漂白后的荧光恢复以及带有免疫金标记的电子显微镜。这些实验将揭示这些整合膜蛋白如何定位并塑造细胞器内质网的膜双层。我们建议使用高分辨率 EM 技术来分析非洲爪蟾卵提取物体外形成的 ER 小管的 3-D 结构和表面。这项工作的动力是寻找内质网小管周围的网状支架，它可以解释这些蛋白质如何直接塑造特定尺寸的内质网小管。然而，即使没有发现内质网小管上的网状支架，这项工作也将是首次对内质网小管进行高分辨率结构分析，并且可以揭示有关该细胞器形态特性的大量信息。最后，我们将讨论网织蛋白产生的管状内质网形态对于细胞分化是否重要。我们正在研究神经元的分化，这会导致神经元细胞的形态发生相当大的变化并变得高度极化。神经元分化导致轴突和树突的延伸，我们在这里展示的长过程充满了用网织蛋白染色的管状 ER。我们将讨论增加和减少网织蛋白水平是否会影响神经元分化，以及管状 ER 的哪些功能对于轴突/神经突生长是必需的。我们将寻找有助于管状 ER 生长、包装和组织成轴突/神经突的网织相互作用因子，并通过电子显微镜分析神经突/轴突内 ER 的结构。这项工作将解决细胞器形态与细胞分化的功能相关性。 公共健康相关性：拟议的研究网织蛋白功能的计划将进一步了解整合膜蛋白如何塑造细胞器的膜双层以创建功能子域。这项工作还将解决网状蛋白生成的内质网形状对神经元分化和独特极化细胞形态的重要性。