MEMBRANE FUSION IN RETINAL ROD OUTER SEGMENTS

描述（改编自申请人的摘要）：维持一个 恒定的长度是视杆细胞对光反射能力的本质。 这些有丝分裂后的细胞包含一堆封闭的扁平膜盘 在杆外段（ROS）内以不连续的横向阵列组织。 而外部分部更新和处置计划被定义为 花盘形态发生（在基部）和花盘脱落（在顶端）的协调过程， 细胞如何组装和更新其建筑特征仍然是一个谜， 分子水平。研究人员建议，在这两个步骤中， 过程，新的圆盘封闭和圆盘膜包形成之前， 脱落，需要相对膜的融合。生化成分 ROS中介导的融合过程在很大程度上是未知的， 是这项研究的长期重点。迄今为止，最具特色的 ROS内融合的蛋白质介体是外周蛋白/rds。探讨 这些融合过程的分子机制，特别强调 外周蛋白/RDS的作用，研究者已经开发了一系列无细胞的 融合分析系统，旨在增加目标的复杂性 膜在融合过程中利用生物物理学、分子生物学和细胞 生物技术，研究人员有一个补充的技术，允许 她来描述整个融合过程，并描绘个人 过程中的步骤，并识别外围设备/RDS的功能区域， 该蛋白质的融合能力所必需的结构要求。它 目的：（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）确定椎间盘边缘的蛋白质成分是否有助于 （4）阐明脂质和蛋白质如何介导融合 途径会聚以调节融合过程;和（5）确定什么是融合过程。 融合机制可能参与椎间盘形态发生。使用无细胞融合 试验，研究者将研究涉及的总体融合反应 在盘包形成和盘形态发生中起重要作用。使用COS细胞表达 系统，研究人员将研究各种突变的影响， peripherin/RDS和ROM-1对融合过程的影响。识别功能 参与该过程的蛋白质区域，研究人员将 研究各种肽对细胞中各个步骤的影响， 聚变反应使用FTIR和NMR，研究者将确定 外周蛋白/RDS和膜双层的结构要求 融合发生。对视杆细胞的分子机制的理解 保持恒定的长度对于解开细胞的机制是重要的。 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.