Structure, Function, and Expression of Cold-Adapted Tubulins and Microtubule-Dependent Motors from Antarctic Fishes

南极鱼类冷适应微管蛋白和微管依赖性运动的结构、功能和表达

• 批准号: 9420712
• 负责人: Harry Detrich
• 金额: $ 58.33万
• 依托单位: Northeastern University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-06-01 至 1999-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9420712&HistoricalAwards=false
• 关键词: Structure; Function; Expression; Cold; Adapted

Abstract Microtubules, which are subcellular "pipe-like" filaments composed of the protein tubulin, and their associated motors, dynein and kinesin, participate in many fundamental cellular processes, including cell division, nerve growth and regeneration, cell motility, and the organization and transport of organelles within cells. In these processes, microtubules serve as rigid "railroad tracks" for the movement of motors and their cargoes ("trains"), and the motors themselves are efficient, high-velocity, regulative "locomotives." The microtubles and microtubule motors of cold-adapted Antarctic fishes are unique in their capacity to assemble and to function at body temperatures (-2 to +2 oC) well below those of warm- blooded and temperate organisms. The long-range goals of the proposed research are to determine, at the molecular level, the adaptations that enhance the assembly of microtubules, the expression of tubulin genes, and the activity of microtubule motors from Antarctic fishes in this extreme thermal regime. The objectives of this project are: 1) to determine the structural features (e.g., changes in their amino acid sequences) that enable the tubulins of Antarctic fishes to polymerize efficiently at low temperatures; 2) to characterize the structure, organization, and expression of an alpha-tubulin gene cluster from an Antarctic rockcod, Notothenia coriiceps; and 3) to examine the biochemical adaptations required for efficient function of Antarctic fish flagellar dynein motors at low temperatures. Molecular adaptations of the Antarctic fish tubulins will be determined by comparing the amino acid sequences of alpha- and beta-tubulins from Notothenia coriiceps, to those from its temperate relative, the New Zealand black cod, Notothenia angustata. Gene expression in N. coriiceps will be analyzed by defining the organization and transcription- regulating elements of an alpha-tubulin gene cluster. The structure of the cluster will be characterized b y sequence analysis using standard DNA sequencing methods. Features of N. coriicepsalpha-tubulin transcription-regulating elements (promoters) that support efficient gene expression at cold temperatures will be assessed by use of reporter gene constructs in primary cell cultures derived from N. coriiceps tissues. Comparison to results obtained with N. angustata promoters should help to delineate elements of alpha-tubulin promoter regions that are important for high-level expression at low temperatures. Finally, the biochemical adaptations required for efficient function of N. coriiceps flagellar dyneins at low temperatures will be assayed by measurement of the temperature dependence of the motor activities of purified inner- and outer-arm dyneins. For comparison, flagellar dyneins from the temperate trout Salmo gairdneri (and possibly from N. angustata) will also be analyzed. Taken together, these studies should reveal the molecular adaptations of Antarctic fishes that maintain efficient microtubule assembly, mechano-chemical motor function, and gene expression at low temperatures. In the broadest sense, this research program should advance the molecular understanding of the cold-adapted mode of life,

微管是由微管蛋白及其相关马达、动力蛋白和动力蛋白组成的亚细胞“管状”细丝，参与许多基本的细胞过程，包括细胞分裂、神经生长和再生、细胞运动以及细胞内细胞器的组织和运输。在这些过程中，微管充当刚性的“铁轨”，用于马达及其货物（“火车”）的运动，而马达本身则是高效、高速、调节的“机车”。适应寒冷的南极鱼类的微管和微管马达是独一无二的，它们在体温（-2到+2摄氏度）远低于温血和温带生物的情况下组装和工作的能力。本研究的长期目标是在分子水平上确定南极鱼类在这种极端热环境中增强微管组装、微管蛋白基因表达和微管马达活性的适应性。该项目的目标是：1)确定使南极鱼类微管在低温下有效聚合的结构特征（例如，氨基酸序列的变化）；2)研究南极岩鳕鱼(Notothenia coriiceps) α -微管蛋白基因簇的结构、组织和表达特征；3)研究南极鱼类鞭毛动力蛋白马达在低温下高效工作所需的生化适应。南极鱼类微管蛋白的分子适应性将通过比较Notothenia coriiceps和其温带亲戚新西兰黑鳕鱼Notothenia angustata的α -和β -微管蛋白的氨基酸序列来确定。通过定义α -微管蛋白基因簇的组织和转录调控元件，将分析山蕨的基因表达。该簇的结构将通过使用标准DNA测序方法进行序列分析来表征。在原代细胞培养物中，利用报告基因构建物来评估在低温下支持高效基因表达的海苔小管蛋白转录调节元件（启动子）的特性。将其与N. angustata启动子的结果进行比较，应该有助于描述α -微管蛋白启动子区域的元件，这些元件对低温下的高水平表达很重要。最后，通过测量纯化的内臂和外臂动力蛋白的运动活性对温度的依赖性，分析了在低温下毛羽鞭毛动力蛋白有效功能所需的生化适应。为了比较，我们还将分析温带鳟鱼Salmo gairdneri（也可能来自N. angustata）的鞭毛动力蛋白。综上所述，这些研究将揭示南极鱼类在低温下维持高效微管组装、机械化学运动功能和基因表达的分子适应性。从最广泛的意义上说，这个研究项目应该促进对冷适应生命模式的分子理解，