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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Probing myosin structural conformation in vivo by second-harmonic generation microscopy
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Probing myosin structural conformation in vivo by second-harmonic generation microscopy

机译:通过二次谐波显微镜在体内探测肌球蛋白的结构构象

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摘要

Understanding of complex biological processes requires knowledge of molecular structures and measurement of their dynamics in vivo. The collective chemomechanical action of myosin molecules (the molecular motors) in the muscle sarcomere represents a paradigmatic example in this respect. Here, we describe a label-free imaging method sensitive to protein conformation in vivo. We employed the order-based contrast enhancement by second-harmonic generation (SHG) for the functional imaging of muscle cells. We found that SHG polarization anisotropy (SPA) measurements report on the structural state of the actomyosin motors, with significant sensitivity to the conformation of myosin. In fact, each physiological/biochemical state we probed (relaxed, rigor, isometric contraction) produced a distinct value of polarization anisotropy. Employing a full reconstruction of the contributing elementary SHG emitters in the actomyosin motor array at atomic scale, we provide a molecular interpretation of the SPA measurements in terms of myosin conformations. We applied this method to the discrimination between attached and detached myosin heads in an isometrically contracting intact fiber. Our observations indicate that isometrically contracting muscle sustains its tetanic force by steady-state commitment of 30% of myosin heads. Applying SPA and molecular structure modeling to the imaging of unstained living tissues provides the basis for a generation of imaging and diagnostic tools capable of probing molecular structures and dynamics in vivo.
机译:了解复杂的生物过程需要了解分子结构并测量其在体内的动力学。在这方面,肌球蛋白中肌球蛋白分子(分子马达)的集体化学机械作用代表了一个典型的例子。在这里,我们描述了对体内蛋白质构象敏感的无标记成像方法。我们通过二次谐波生成(SHG)为肌肉细胞的功能成像采用基于顺序的对比度增强。我们发现SHG极化各向异性(SPA)测量报告了肌动球蛋白马达的结构状态,对肌球蛋白的构型具有显着的敏感性。实际上,我们探测到的每个生理/生化状态(松弛,严谨,等轴测收缩)都会产生不同的极化各向异性值。通过以原子尺度完全重建放线菌素马达阵列中贡献的基本SHG发射体,我们以肌球蛋白构象提供了SPA测量的分子解释。我们将这种方法应用于等轴测收缩完整纤维中附着和分离的肌球蛋白头之间的区分。我们的观察结果表明,等轴测收缩的肌肉通过30%的肌球蛋白头的稳态摄取来维持其强直作用力。将SPA和分子结构建模应用于未染色的活组织的成像,为生成能够探测体内分子结构和动力学的成像和诊断工具提供了基础。

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  • 作者单位

    Laboratory of Physiology, Department of Evolutionary Biology, University of Florence, 50125 Florence, Italy;

    rnEuropean Laboratory for Non-Linear Spectroscopy, University of Florence, 50019 Sesto Fiorentino, Italy Department of Physics, University of Trento, 38123 Povo, Italy;

    rnEuropean Laboratory for Non-Linear Spectroscopy, University of Florence, 50019 Sesto Fiorentino, Italy;

    rnEuropean Laboratory for Non-Linear Spectroscopy, University of Florence, 50019 Sesto Fiorentino, Italy;

    rnLaboratory of Physiology, Department of Evolutionary Biology, University of Florence, 50125 Florence, Italy;

    rnLaboratory of Physiology, Department of Evolutionary Biology, University of Florence, 50125 Florence, Italy;

    rnLaboratory of Physiology, Department of Evolutionary Biology, University of Florence, 50125 Florence, Italy;

    rnLaboratory of Physiology, Department of Evolutionary Biology, University of Florence, 50125 Florence, Italy European Laboratory for Non-Linear Spectroscopy, University of Florence, 50019 Sesto Fiorentino, Italy;

    rnEuropean Laboratory for Non-Linear Spectroscopy, University of Florence, 50019 Sesto Fiorentino, Italy Department of Physics, University of Florence, 50019 Sesto Fiorentino, Italy;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    hyper-rayleigh scattering; nonlinear microscopy; protein organization;

    机译:超瑞利散射非线性显微镜蛋白质组织;

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