Nanotechnology In Muscle And Motility Systems

在过去的十年里，在纳米尺度上操纵、制造和改变微小物体的能力(纳米技术)取得了惊人的进步，使材料科学发生了革命性的变化。最近纳米技术在生命科学中的应用在广泛的学科领域显示出令人振奋的前景。激光技术的发展(共聚焦显微镜、激光镊子、激光剪刀、多光子激发共聚焦显微镜、近场显微镜等)现在可以对厚组织中的活细胞进行功能成像；操纵单分子、单细胞器和单细胞；测定DNA与其他单分子的结合力和相互作用速度；对活细胞中的染色体和细胞器进行手术，以及制造微型医疗设备。扫描探针显微镜(原子力、扫描隧道和电化学探针、近场显微镜)的发展使单分子的操纵、新型生物芯片和生物传感器的制备以及活细胞中单分子的物理和光谱性质的测量成为可能。我们的工作组成功地建立了最先进的纳米技术设施(原子力显微镜、细胞力显微镜、单纤维力站、实时共焦显微镜、单分子荧光显微镜和数字图像分析)，以应用、适应和发展生物纳米技术。这些技术正被应用于研究单个马达，如肌球蛋白和动蛋白，以及弹性蛋白，如肌蛋白和星云蛋白，肌肉细丝，细胞骨架细丝，细胞膜和细胞器中的受体，如肌原纤维，核糖体和染色质。更具体地说，我们的目标是：1.测量单分子、细胞器和单细胞的机械性能和光谱。2.测量相互作用的单分子/细胞器/细胞的强度、速度和运动。3.操纵和改变活细胞内的细胞器。4.制造和设计纳米技术中的新型仪器。这些对单个分子、细丝和细胞器的直接测量和操作将为肌肉和非肌肉细胞收缩机械的组装和功能中的事件提供独特和重要的见解。这些研究还将揭示设计具有规定机械性能的组织的重要工程原理。在过去的一年里，我们成功地将原子力显微镜应用于成像和测量单个单体蛋白质、寡聚蛋白质和基因工程Titin和星云蛋白分子的弹性性质。我们还应用单分子荧光显微镜在单分子水平上研究了肌动蛋白、肌球蛋白、肌动蛋白和肌球蛋白马达的形态和动态相互作用。 在这一年的报告中，我们在1.通过将单分子力学测量与构象状态相关联来了解titin PEVK、星云蛋白和肌球蛋白如何发展和调节弹性方面取得了重大进展。2.研制了一种能同时测量水溶液中的动力学、刚度和力的原子力显微镜。3.以选定的PEVK模块为基础的蛋白质聚合物工程，以获得定义的弹性性能。这些聚合物也构成了药物输送水凝胶的基础。4.利用自组装单聚体和纳米刻蚀技术开发蛋白质纳米粒子技术。这些技术极大地加快了对单个蛋白质的准确和可重复性的纳米机械测量。

Spectacular advances in the ability to manipulate, fabricate and alter tiny subjects at the nanometer scale (Nanotechnology) have revolutionized material science in the past decade. The recent application of nanotechnology to life sciences has shown exciting promises in a wide range of disciplines. The development of laser-based technology (confocal microscope, laser tweezers, laser scissors, mutilphoton excitation confocal microscope, near field microscope, etc.) now allows functional imaging of living cells in thick tissues; the manipulation of single molecules, single organelles and single cells; the determination of the binding force and rate of interaction of DNA and other single molecules; the surgery of chromosomes and organelles in a living cell and the fabrication of miniature medical devices. The development of scanning probe microscopy (atomic force, scanning tunneling and electrochemical probe, near field microscopy) has enabled the manipulation of single molecules, the preparation of novel biochips and biosensors, and the measurement of physical and spectroscopic properties of single molecules in living cells. Our workgroup have succeeded in establishing state-of-the-art nanotechnology facilities (atomic force microscope, cellular force microscope, single fiber force station, real time confocal microscope, single molecule fluorescence microscope, and digital image analysis) to apply, adapt and develop bio-nanotechnology. These techniques are being applied to study single motors such as myosin and kinesin as well elastic proteins such a titin and nebulin, muscle filaments, cytoskeletal filaments, receptors in cellular membranes and cellular organelles such as myofibril, ribosome and chromatin. More specifically, our goals are: 1. Measuring the mechanical property and spectroscopy of single molecules, organelles and single cells. 2. Measuring the strength, speed and movement of interacting single molecules/organelles/cells. 3. Manipulating and altering intracellular organelles in living cells. 4. Fabricating and designing new instrumentation in nanotechnology. These direct measurement and manipulation of single molecule and filaments and organelles will provide unique and important insights of the events in the assembly and function of contractile machinery in muscle and nonmuscle cells. These studies will also reveal important engineering principles for designing tissues with prescribed mechanical properties. In the past year, we have successfully applied atomic force microscopy to image and measure the elastic properties of single monomeric protein, oligomeric protein and genetically engineered titin and nebulin molecules. We are also applying the single molecule fluorescence microscope to study the morphology and dynamic interactions of titin, nebulin, actin and myosin motors at the single molecule level. In this report year, we have made major progress in 1. Understanding how titin PEVK , nebulin and myosin develops and modulates elasticity by correlating single molecule mechanical measurements with conformational states. 2. The development of an AFM that measure dynamics stiffness and force simultaneously in aqueous solutions. 3. The engineering of protein polymers based on selected PEVK modules to obtain defined elastic properties. These polymers also formed the basis for drug delivery hydrogels. 4. The development of protein nanopatterning techniques using self assembled monolyaers and nanolithograpgy. These techniques have greatly expedited the accurate and reproducible nanomechanical measurement of single proteins.