STRUCTURE/FUNCTION OF MYOSIN IN SKELETAL MUSCLE

描述（申请人摘要）：肌球蛋白是骨骼肌中的分子马达， 肌肉粗丝，与含肌动蛋白的细丝结合， 机械力和位移。所有脊椎动物都表达一个多基因家族 两种肌球蛋白亚基;肌球蛋白重链（MHC）和肌球蛋白轻链 （MLC）。已知MHC和MLC同种型表达的变化是一个重要因素。 肌肉机械特性的主要决定因素。然而，影响 MHC和MLC亚型对各部位的力-速度关系 仍然存在争议，从未在完整的肌肉细胞中确定过。在 此外，个别纤维往往被发现表达一个以上的同种型 MHC。机械的后果，因此，这一功能的目的， coexpression，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，因为它是唯一的生物体， 可以分离出单个完整的“活”纤维， 稳定这种机械稳定性允许高分辨率测量 肌节长度瞬变和肌球蛋白跨桥 动力学青蛙单纤维肌球蛋白结构/功能的研究已经 由于对MHC和MLC同种型的定义不充分，迄今为止仅限于此。 最近，我们克隆了四种新的成年青蛙MHC， 相应的蛋白质亚型，并建立了他们的表达模式， 单个肌肉细胞。在本提案中，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 各种纤维类型的MLC。这种对MHC和 MLC异构体在整个范围的纤维类型将使我们能够完成 以下两个主要目标的建议：（1）一个精确的相关性将是 建立MHC和MLC亚型含量和机械功能之间的关系， 单个完整的青蛙肌肉纤维将分离单个肌纤维， 成年尖音蛙的力-速度特性将在1 沿着光纤的全长沿着10 mm的段。跟随机械师 在每个实验中，将对MHC和MLC同种型组成进行定量， 使用SDS-PAGE进行片段化。该分析将包括如何确定 单个纤维中MHC和MLC多种亚型的共表达影响 机械性能(2)我们将确定MHC和MLC亚型 在正常情况下影响机械功能 运动将单个青蛙纤维驱动通过体内纤维长度 测量时跳跃过程中发生的偏移和刺激条件 力产生和肌节长度瞬变。这种模式将得到改善 我们对慢纤维和快纤维在生成过程中的作用的理解 典型的弹道运动肌球蛋白亚型表达改变 模式发生在对肌肉废用/过度使用、直接创伤、去神经支配 和衰老。对MHC和MLC亚型如何影响 完整细胞环境中的机械功能将有助于 了解这些改变的表型的潜在后果。此外，A 对收缩功能如何受到 肌球蛋白同工型的共表达和不均匀表达沿长度的肌球蛋白同工型沿着长度的肌球蛋白同工型的共表达和不均匀表达沿长度的肌球蛋白同工型的不均匀表达。 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同种型。

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.