Reticulon Function in ER Morphology

描述（由申请人提供）：网状蛋白是普遍存在的高度保守的整体膜蛋白，存在于所有真核生物中。在真核细胞中，网状蛋白通过将外周内质网（ER）的膜双分子层塑造成一个小管网络，从而产生ER的结构。尽管由核膜、外周内质网片和小管组成的内质网膜是连续的，但这些蛋白质只局限于内质网小管。该计划的总体目标是发展我们对网状蛋白如何分裂并将内质网膜双层塑造成小管的理解。该项目的具体目标包括脊椎动物和酵母网状蛋白的结构/功能研究，以确定它们如何定位于管状内质网，以及它们的哪些结构特征是形成膜活动所必需的。我们将突变网状蛋白的各个区域，以确定对蛋白质定位，寡聚，扩散迁移率和膜曲率的影响。在这些研究中，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技术分析爪蟾卵提取物体外形成的ER小管的三维结构和表面。这项工作的动力是寻找内质网小管周围的网状支架，这可以解释这些蛋白质如何直接塑造确定尺寸的内质网小管。然而，即使没有在内质网小管上发现网状支架，这项工作也将是内质网小管的第一个高分辨率结构分析，并可能揭示该细胞器的大量形态学特性。最后，我们将讨论网状细胞产生的管状内质网形态对细胞分化是否重要。我们正在研究神经元的分化，这使得神经元细胞的形态发生了巨大的变化，并变得高度极化。神经元分化导致轴突和树突的延伸，如图所示的长突起充满了网状蛋白染色的管状内质网。我们将讨论网状蛋白水平的增加和减少是否会影响神经元分化，以及管状内质网的哪些功能对轴突/神经突生长是必要的。我们将寻找网状相互作用因素，这些因素有助于管状内质网在轴突/神经突中的生长、包装和组织，并通过电子显微镜分析神经突/轴突内内质网的结构。这项工作将解决细胞器形态与细胞分化的功能相关性。

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.