STRUCTURE/FUNCTION OF MYOSIN IN SKELETAL MUSCLE

说明(申请人摘要)：肌球蛋白是骨骼中的分子马达 肌肉粗丝与含有肌动蛋白的细丝结合以产生 机械力和位移。所有脊椎动物都表达一个多基因家族 肌球蛋白的两个亚基：肌球蛋白重链和肌球蛋白轻链 (MLC)。已知MHC和MLC亚型的表达变异是 肌肉力学性能的主要决定因素。然而，其影响 MHC和MLC亚型对力-速度关系各部分的影响 仍然存在争议，而且从未在完整的肌肉细胞中被确定。在……里面 此外，经常发现单独的纤维表达一种以上的异构体 MHC。机械后果，因此它的功能目的是 共表达，实际上是未知的。青蛙肌肉提供了一个很好的模型 肌球蛋白结构/功能的研究，因为它是唯一一种 可以分离出保持完整机械结构的单一完整的“活”纤维 稳定性。这种机械稳定性允许高分辨率测量 机械功能、肌节长度瞬变与肌球蛋白交叉桥 运动学。对蛙单纤维中肌球蛋白结构/功能的研究 由于对MHC和MLC异构体的定义不充分，迄今仅限于此。 最近，我们克隆了四个新的成蛙MHC，鉴定了四个 并建立了相应的蛋白质异构体及其表达模式 单个肌肉细胞。在这个提案中，我们将使用类似的方法 (包括分子克隆)以鉴定和确定表达模式 各种纤维类型中的MLC的数量。这种对MHC和MHC的准确识别 所有纤维类型的MLC亚型将使我们能够完成 该提案的以下两个主要目标：(1)准确的相关性将是 MHC和MLC异构体含量与机械性能之间的关系 单一完整的青蛙肌肉纤维。单个肌肉纤维将从 成蛙和力-速度特性的测量将在1 Mm沿纤维的全长分段。遵循机械原理 实验中，MHC和MLC的异构体组成将在每个 用十二烷基硫酸钠-PAGE进行分段。这一分析将包括确定如何 MHC和MLC多种异构体在单个纤维中的共表达影响 机械性能。(2)我们将确定MHC和MLC的异构体 在正常情况下对机械功能的影响 移动。单个青蛙纤维将在体内穿过纤维长度 测量时跳跃过程中出现的漂移和刺激条件 力量产生和肌节长度瞬变。这一范例将得到改进 我们对慢速和快速纤维类型在世代中如何发挥作用的理解 刻板的弹道动作。肌球蛋白亚型表达的改变 模式发生在肌肉停用/过度使用、直接创伤、失神经 和衰老。对MHC和MLC异构体影响的基本认识 在完整的蜂窝环境中的机械功能将有助于 了解这些表型改变的潜在后果。此外，一个 对收缩功能是如何受 肌球蛋白亚型沿长度方向的共表达和非均一表达 纤维很重要，因为给定肌肉中的大多数纤维可能 共表达多种肌球蛋白亚型。

DESCRIPTION (Applicant's abstract): Myosin is the molecular motor in skeletal muscle thick filaments that binds to actin-containing thin filaments to produce mechanical force and displacement. All vertebrates express a multigene family of the two myosin subunits; myosin heavy chain (MHC) and myosin light chain (MLC). Variations in expression of both MHC and MLC isoforms is known to be a primary determinant of a muscle's mechanical properties. However, the influence of MHC and MLC isoforms on the various parts of the force-velocity relationship remains controversial and has never been determined in intact muscle cells. In addition, individual fibers are often found to express more than one isoform of MHC. The mechanical consequences, and hence the functional purpose of this coexpression, is virtually unknown. Frog muscle provides an excellent model for studies of myosin structure/function because it is the only organism from which single intact "living" fibers can be isolated that retain complete mechanical stability. This mechanical stability permits high resolution measurements of mechanical function, sarcomere length transients and myosin cross-bridge kinetics. Studies of myosin structure/function in frog single fibers have been limited to date because of inadequate definition of MHC and MLC isoforms. Recently, we have cloned four novel adult frog MHCs, identified the four corresponding protein isoforms and established their expression pattern in individual muscle cells. In this proposal we will use a similar approach (including molecular cloning) to identify and determine the expression patter of MLCs in the various fiber types. This precise identification of both MHC and MLC isoforms across the full range of fiber types will allow us to complete the following two major aims of the proposal: (1) A precise correlation will be established between MHC and MLC isoform content and mechanical function of single intact frog muscle fibers. Single muscle fibers will be isolated from adult Rana pipiens and the force-velocity properties will be measured within 1 mm segments along the full length of the fiber. Following the mechanics experiments, the MHC and MLC isoform composition will be quantified in each segment using SDS-PAGE. This analysis will include a determination of how coexpression of multiple isoforms of MHC and MLC in individual fibers affects mechanical performance. (2) We will determine how MHC and MLC isoforms influence mechanical function under conditions experienced during normal locomotion. Single frog fibers will driven through in vivo fiber length excursions and stimulus conditions that occur during jumping while measuring force production and sarcomere length transients. This paradigm will improve our understanding of how slow and fast fiber types function in the generation of stereotypical ballistic movements. Altered myosin isoform expression patterns occur in response to muscle disuse/overuse, direct trauma, denervation and aging. A fundamental understanding of how MHC and MLC isoforms influence mechanical function in an intact cellular environment will be useful for understanding potential consequences of these altered phenotypes. Further, a fundamental understanding of how contractile function is influenced by coexpression and non-uniform expression of myosin isoforms along the length of fibers is important, because a majority of fibers in a given muscle may coexpress multiple myosin isoforms.