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Study of cellular mechanotransduction by visualizing actin and nucleoskeleton dynamics in real-time.

机译：通过实时观察肌动蛋白和核骨架动态研究细胞机械转导。

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摘要

Cellular mechanotransduction, the tight coupling between biochemical and mechanical properties of the cytoskeleton and nucleus, drives a large range of cellular processes including cell mobility, cytokinesis, vesicle transportation, or even cell fate determination affected by alterations in gene expression. In this study, we study cellular mechanotransduction by direct imaging of the actin cytoskeleton and component of nucleoskeleton during normal cellular processes and under different stresses.;A novel class of cell permeable actin filament free barbed end dye was developed to achieve high spatial and temporal resolution of actin dynamic imaging. the generation of free barbed ends of actin filaments during lamellipodia protrusion, cytokinesis and endocytosis were monitored in this study, providing new insights on the regulation of free barbed end of actin filaments during these actin-driven processes. By distinguished visualization of the filaments and barbed end pool of actin in cells, the controversial views of whether new actin polymerization is involved during cleavage furrow closure and scission were resolved, we show that there is a surge of new actin polymerization during telophase, beginning with ingression of the contractile ring and ending with the separation of daughter cells, supporting the view that new actin polymerization is involved in cytokinesis.;We further developed a triple labeling system with the genetically encoded florescent protein (FP) fused with nuclear component including emerin, Sun1, Sun2 and lamin A. The FP was carefully choosing so the excitation and emission spectrums of FP and (Si)TMR actin dyes are far apart enough to allow spontaneous imaging of the three components. Several chemicals were applied to disrupt cytoskeleton of the cells, the corresponding responses of the nucleus components were visualized real-time, providing insights on the interaction between nucleus and cytoskeleton.;We also developed a label-free optical biosensor that employs a silicon-based high-contrast grating (HCG) resonator with a spectral linewidth of ~500pm sensitive to ligand-induced changes in surface properties in collaboration with Professor Constance Chang-Hasnain's group. The device is used to generate thermodynamic and kinetic data on surface-attached antibodies with their respective antigens. The device can detect serum cardiac troponin I, a biomarker of cardiac disease to 100pg/ml within 4-minutes, which is much faster than and as sensitive as current enzyme-linked immuno-assays for cTnI.
机译：细胞机械转导是细胞骨架和细胞核的生物化学和机械性质之间的紧密结合，它驱动着广泛的细胞过程，包括细胞迁移，胞质分裂，囊泡运输，甚至受基因表达改变影响的细胞命运测定。在这项研究中，我们通过在正常细胞过程和不同压力下通过对肌动蛋白细胞骨架和核骨架成分的直接成像来研究细胞机械转导。;开发了一种新型的细胞渗透性肌动蛋白丝无刺末端染料，以实现高时空分辨率肌动蛋白的动态成像。在这项研究中监测了片状脂蛋白突出，胞质分裂和内吞作用过程中肌动蛋白丝自由刺端的产生，为这些肌动蛋白驱动过程中肌动蛋白丝自由刺端的调控提供了新见解。通过细胞中肌动蛋白丝和倒刺末端池的显着可视化，解决了在裂沟闭合和分裂过程中是否涉及新肌动蛋白聚合的争议观点，我们表明，在末期有新肌动蛋白聚合激增，始于收缩环的进入并以子代细胞的分离而结束，从而支持了新的肌动蛋白聚合反应参与胞质分裂的观点。我们进一步开发了一种三重标记系统，其遗传编码的荧光蛋白（FP）与核成分包括Emerin， Sun1，Sun2和laminA。FP精心选择，因此FP和（Si）TMR肌动蛋白染料的激发和发射光谱相距足够远，可以对这三个成分进行自发成像。几种化学物质被用于破坏细胞的细胞骨架，实时可视化细胞核成分的相应反应，从而提供有关细胞核与细胞骨架之间相互作用的见解。；我们还开发了一种无标记的光学生物传感器，该传感器采用基于硅的与Constance Chang-Hasnain教授的小组合作，光谱线宽为〜500pm的高对比度光栅（HCG）谐振器，对配体诱导的表面性质变化敏感。该设备用于生成表面附着抗体及其各自抗原的热力学和动力学数据。该设备可以在4分钟内检测出血清心肌肌钙蛋白I（一种心脏病的生物标志物）至100pg / ml，它比cTnI的当前酶联免疫测定更快，更灵敏。

著录项

  • 作者

    Kan, Shu.;

  • 作者单位

    University of California, Berkeley.;

  • 授予单位 University of California, Berkeley.;
  • 学科 Biochemistry.;Biomedical engineering.;Biomechanics.;Cellular biology.
  • 学位 Ph.D.
  • 年度 2015
  • 页码 70 p.
  • 总页数 70
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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