NEBULIN AS A MOLECULAR RULER OF THIN FILAMENTS

描述 拟议研究的长期目标是了解 星云蛋白异构体在脊椎动物中的生理和发育作用 骨骼肌，这已经确定了分子的变异性， 质量（600-900 kD）。 这种巨大的模块化蛋白质被认为是 蛋白质的尺子，以调节细丝的长度，作为一个 钙-钙调蛋白介导的肌动球蛋白相互作用的调节蛋白， 并作为在制造过程中组装细丝网格的模板 发展 具体目标是：（1）确定星云蛋白的异构体 在胎儿、新生儿和成年兔的广泛骨骼肌中， 大鼠 星云蛋白的表达将与 其它肌丝蛋白如肌联蛋白，肌球蛋白，C-蛋白， 肌动蛋白、原肌球蛋白、肌钙蛋白、α-肌动蛋白、Cap Z和原调节蛋白;（2）至 鉴定和制备同种型特异性和泛抗星云蛋白抗体。 单克隆抗星云蛋白的表位谱将针对每个 同种型 抗肽抗体将产生共同的和可变的 星云蛋白序列的区域;（3）关联星云蛋白异构体的大小 和肌动蛋白丝的长度在选定的胎儿，新生儿和成人 家兔和大鼠骨骼肌。 扫描透射电子 显微镜和质谱将用于确定大小， 星云蛋白异构体的轮廓长度。 星云蛋白亚型的免疫定位， 肌动蛋白、原肌球蛋白、原调节蛋白和cap Z将用于确定 细胞分布和细丝的长度和跨度;和（4）至 描述星云蛋白异构体多样性的分子基础。 星云蛋白外显子的发育调节表达将由以下决定： 聚合酶链反应和核苷酸测序。 这项拟议中的研究将定义星云蛋白的生理功能， 它对人类的发育和生理基础 骨骼肌中细丝的结构、调节和组装 健康和疾病状态。

DESCRIPTION The long-term goal of the proposed research is to understand the physiological and developmental roles of nebulin isoforms in vertebrate skeletal muscles, which have been identified by variability in the molecular mass (600-900 kD). This giant modular protein has been proposed to act as a protein ruler to regulate the length of thin filaments, as a calcium-calmodulin mediated regulatory protein of actomyosin interaction, and as a template for the assembly of thin filament lattice during development. The specific aims are: (1) to characterize nebulin isoforms in a wide range of skeletal muscles of fetal, neonatal and adult rabbits and rats. Nebulin expression will be compared and contrasted with the transition of other myofilament proteins such as titin, myosin, C-protein, actin, tropomyosin, troponin, alpha-actin, Cap Z and tropomodulin; (2) to identify and prepare isoform-specific and pan anti-nebulin antibodies. Epitope profiles of monoclonal anti-nebulin will be characterized for each isoform. Anti-peptide antibodies will be produced to common and variable regions of nebulin sequences; (3) to correlate the size of nebulin isoforms and the length of actin filaments in selected fetal, neonatal and adult skeletal muscles of rabbit and rats. Scanning transmission electron microscopy and mass spectroscopy will be used to determine the size and contour length of nebulin isoforms. Immunolocalization of nebulin isoforms, actin, tropomyosin, tropomodulin and cap Z will be used to determine the cellular distribution and length and span of thin filaments; and (4) to characterize the molecular basis of nebulin isoform diversity. Developmentally regulated expression of nebulin exons will be determined by polymerase chain reaction and nucleotide sequencing. The proposed research would define the physiological function of nebulin and the developmental and physiological basis for its contribution to the structure, regulation, and assembly of thin filaments in skeletal muscles in healthy and diseased states.