MEMBRANE FUSION IN RETINAL ROD OUTER SEGMENTS

描述(改编自申请人的摘要)：保持 恒定的长度是杆状细胞传光能力的本质。 这些有丝分裂后细胞含有一堆闭合的扁平的膜盘。 在杆外段(ROS)内以不连续的横向阵列组织。 鉴于外段更新和处置计划被定义为 视盘形态发生(在底部)和视盘脱落(在顶端)的协调过程， 关于细胞如何组装和更新其架构特征，目前仍不清楚 一个分子水平。调查人员提出，在这两个过程中， 工艺、新的盘片闭合和盘膜包形成 脱落，需要相互对立的膜融合。生化成分 调节ROS中的融合过程在很大程度上是未知的，它们的阐明 是本研究长期关注的焦点。到目前为止，最具特色的 ROS内融合的蛋白中介是外周蛋白/RDS。为了调查 这些融合过程的分子机制，特别强调 由于外周蛋白/RDS的作用，研究人员开发了一系列无细胞 融合分析系统，旨在增加目标的复杂性 融合过程中的细胞膜。利用生物物理学、分子生物学和细胞 生物学技术，调查者有一套补充的技术，可以 她来描述整个融合过程并描绘出个体 过程中的步骤并确定外围设备/RDS的功能区和 这种蛋白质的融合能力所必需的结构要求。它 提出：(1)确定外周蛋白/RDS在ROS膜中的作用 融合：(2)确定磷酸化外周蛋白/RDS在ROS膜中的作用 融合；(3)确定盘沿的蛋白质成分是否有贡献 对融合过程的影响；(4)阐述了脂类和蛋白质如何介导融合 通路汇聚以调节融合过程；以及(5)决定什么 融合机制可能参与了椎间盘的形态发生。使用无细胞融合 化验，调查员将调查所涉及的整体聚变反应 在盘包形成和盘形态发生中。使用COS单元格表达式 系统中，研究人员将观察体内各种突变的影响 外设/rds和rom-1的融合进程。要确定功能 参与这一过程的蛋白质区域，研究人员将 研究不同多肽对小鼠不同步幅的影响。 聚变反应。使用FTIR和核磁共振，调查者将确定 所需的外围/RDS和膜双层的结构要求 核聚变即将发生。对杆状细胞的分子机制的认识 保持恒定的长度对解开细胞的机制很重要 生存能力、退行性疾病过程和适应。

DESCRIPTION (Adapted from applicant's abstract): The ability to maintain a constant length is the essence of the rod cell's ability to transduce light. These post-mitotic cells contain a stack of closed flattened membranous discs organized in a discontinuous lateral array within the rod outer segment (ROS). Whereas the program of outer segment renewal and disposal is defined as the coordinated process of disc morphogenesis (at base) and disc shedding (at tip), how the cell assembles and renews its architectural features remains obscure on a molecular level. The investigator proposes that a step during both of these processes, new disc closure and disc membrane packet formation prior to shedding, require the fusion of opposing membranes. The biochemical components mediating fusion processes in the ROS are largely unknown and their elucidation is the long-term focus of this research. To date, the best-characterized protein mediator of fusion within the ROS is peripherin/rds. To investigate the molecular mechanisms of these fusion processes with specific emphasis on the role of peripherin/rds, the investigator has developed a series of cell-free fusion assay systems, designed to increase the complexity of the target membrane during fusion. With the use of biophysical, molecular biology and cell biology techniques, the investigator has a complement of techniques that allows her to characterize the overall fusion process and to delineate the individual steps in the process and identify functional regions of the peripherin/rds and the structural requirements necessary for fusion competency of this protein. It is proposed to: (1) determine the role of peripherin/rds in ROS membrane fusion: (2) determine the role of phospho-peripherin/rds in ROS membrane fusion; (3) ascertain whether the protein components of the disc rim contribute to the fusion process; (4) address how the lipid and protein mediated fusion pathways converge to regulate the fusion proc3ess; and (5) determine what fusion machinery may be involved in disc morphogenesis. Using cell-free fusion assays, the investigator will investigate the overall fusion reaction involved in disc packet formation and disc morphogenesis. Using a COS cell expression system, the investigator will look at the effect of various mutations within peripherin/rds and rom-1 on the fusion process. To identify the functional regions of the proteins involved in the process, the investigator will investigate the effects of various peptides on the individual steps in the fusion reaction. Using FTIR and NMR, the investigator will determine the structural requirement of the peripherin/rds and membrane bilayer required for fusion to occur. An understanding of the molecular mechanism by which rod cells maintain a constant length is important to unraveling the mechanism of cell viability, degenerative disease processes and adaptation.