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

摘要微管是由微管蛋白及其相关的马达、动力蛋白和驱动蛋白组成的亚细胞“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，微管作为刚性的“铁轨”，用于发动机及其货物（“火车”）的运动，而发动机本身是高效、高速、可调节的“火车头”。“适应寒冷的南极鱼类的微管和微管马达在其组装和功能方面具有独特的能力，其体温（-2至+2 ℃）远低于温血和温带生物。拟议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状态下增强微管组装、微管蛋白基因表达和微管马达活动的适应性。 该项目的目标是：1）确定结构特征（例如，其氨基酸序列的变化），使南极鱼类的微管蛋白在低温下有效地消化; 2）表征的结构，组织和表达的α-微管蛋白基因簇从南极rockcod，Notothenia coriiceps;和3）检查所需的生物化学适应南极鱼类鞭毛动力蛋白马达在低温下的有效功能。 南极鱼微管蛋白的分子适应性将通过比较南极鱼的α-和β-微管蛋白的氨基酸序列与其温带亲戚新西兰黑鳕鱼的氨基酸序列来确定。 基因在N.将通过定义α-微管蛋白基因簇的组织和转录调节元件来分析coriiceps。 将使用标准DNA测序方法通过B y序列分析表征簇的结构。 N的特点。 通过在来源于N.皮层组织 与N. angustata启动子应该有助于描绘α-微管蛋白启动子区域的元件，这些元件对于低温下的高水平表达是重要的。 最后，探讨了氮素高效发挥作用所需的生化适应性。通过测量纯化的内臂和外臂动力蛋白的运动活性的温度依赖性来分析低温下的革蜱鞭毛动力蛋白。 作为比较，来自温带鳟鱼萨尔莫gairdneri（和可能来自N. angustata）也将被分析。 总之，这些研究应该揭示南极鱼类在低温下维持有效微管组装、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，这项研究计划应该促进对寒冷适应生命模式的分子理解，

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,