Reticulon Function in ER Morphology

内质网形态中的网状功能

• 批准号: 7741713
• 负责人: Gia Voeltz
• 金额: $ 29.77万
• 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/7741713
• 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; 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; public health relevance; 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）：网织红细胞是存在于所有真核生物中的普遍存在的高度保守的整合膜蛋白。网织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。尽管内质网膜的连续性，这是由核膜以及周边ER片和小管，这些蛋白质定位专门的ER小管。该计划的总体目标是发展我们对reticulon蛋白如何分配并将ER膜双层塑造成小管的理解。该计划的具体目标包括脊椎动物和酵母reticulon蛋白的结构/功能研究，以确定它们如何定位于管状ER以及它们的膜成形活动所需的结构特征。我们将对reticulon蛋白的各个区域进行突变，以确定对蛋白定位、寡聚化、扩散流动性和膜曲率的影响。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，和免疫金标记的电子显微镜。这些实验将揭示这些完整的膜蛋白如何定位和形成细胞器的膜双层，ER。我们建议使用高分辨率的EM技术来分析的3-D架构和表面的内质网小管在体外形成的爪蟾卵提取物。这项工作的动力是寻找内质网小管周围的网状支架，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，即使在没有发现ER小管上的reticulon支架的情况下，这项工作也将是ER小管的第一个高分辨率结构分析，并且可以揭示大量关于这种细胞器的形态学特性。最后，我们将讨论是否网织产生的管状ER形态是重要的细胞分化。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显著的变化，并变得高度极化。神经元分化导致轴突和树突的延伸，我们在这里显示的长过程充满了被reticulon蛋白染色的管状ER。我们将讨论是否增加和减少reticulon蛋白水平影响神经元的分化和什么样的功能的管状ER是必要的轴突/神经突的生长。我们将寻找有助于管状ER进入轴突/轴突的生长、包装和组织的reticulon相互作用因子，并通过电子显微镜分析轴突/轴突内ER的结构。这项工作将解决功能相关的细胞器形态细胞分化。 公共卫生相关性：建议的计划，研究reticulon蛋白质的功能将进一步我们的理解，如何完整的膜蛋白的形状膜双层的细胞器，以创建功能子域。这项工作也将解决的重要性，网状产生的ER形状的分化和独特的极化细胞形态的神经元。