Cellular mechanics of particle binding and phagocytosis investigated by photonic force microscopy and high-speed imaging

通过光子力显微镜和高速成像研究颗粒结合和吞噬作用的细胞力学

• 批准号: 189771364
• 负责人: Professor Dr. Alexander Rohrbach
• 金额: --
• 依托单位: Institut für Mikrosystemtechnik (IMTEK)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/189771364?language=en
• 关键词: Cellular; mechanics; particle; binding; phagocytosis

The binding of particles such as bacteria, viruses or debris to living cells and the possible uptake into the cell interior – phagocytosis – represent central processes in cell biology and immunology. The observation of such binding and uptake processes, which often occur stepwise, is limited by the spatial and temporal resolution of light microscopes. Despite modern fluorescence techniques, fluctuation based processes, such as the (re-)organization of molecular bonds can hardly be observed or analyzed. Therefore, many partial processes like binding, particle transport or engulfment into the cell body are not sufficiently understood, especially not regarding the cell mechanics, since they occur on scales of nanometers and milliseconds, which are difficult to address by most optical methods.This proposal bases on the results of the first proposal and aims to extend the measurement and analysis methods developed therein. Using the established technique of Photonic Force Microscopy, interaction processes between particle and living cells shall be induced and then controlled variably in space and time. Binding, particle transport at the surface and the actual uptake consist of many multi-scale processes (10^-9 to 10^-5 meters and 10^-5 to 10^2 seconds) and can be better understood, if these processes can be measured on a large temporal and spatial bandwidth. This shall be accomplished by using fast, coherent, i.e. label-free methods such as interferometric 3D particle tracking (at 1 MHz sampling rate) or a novel live-cell super-resolution microscopy method, based on rotating coherent scattered light (ROCS, 100Hz sampling rate). On the other side, fluorescence-based confocal spinning disc scanning shall be extended by an electro-optical tunable lens to enable fast acquisition of 3D image stacks.In this proposal, two PhD students, PhD1 and PhD2, shall work on the following projects: using the above mentioned optical methods, PhD1 shall investigate the direct binding and uptake of particles at flat cell membranes of mouse macrophages. PhD1 shall research, when and how a cell can sense and respond to an approaching particle (e.g. by actin reorganization). Furthermore, PhD1 shall measure the stepwise engulfment by the cell membrane, and analyze the local change of the 3D binding stiffness and friction extracted from the particle’s fluctuations. PhD2, on the other side, shall investigate binding and uptake of the particle mediated by filopodia and lamellipodia. PhD2 shall analyze to what extent the retraction of filopodia can be self-optimized. In addition, the frequently observed, discontinuous transport of particles along filopodia and lamellipodia and the underlying mechanical coupling processes to the dynamic cytoskeleton shall be researched and analyzed.

诸如细菌、病毒或碎片的颗粒与活细胞的结合以及可能被摄取到细胞内部-吞噬作用-代表细胞生物学和免疫学中的中心过程。这种结合和吸收过程的观察，这往往是逐步发生的，是有限的光学显微镜的空间和时间分辨率。尽管有现代荧光技术，基于波动的过程，如分子键的（重新）组织，几乎无法观察或分析。因此，许多局部过程，如结合，颗粒运输或吞噬到细胞体内，没有得到充分的理解，特别是没有关于细胞力学，因为它们发生在纳米和毫秒的尺度上，这是很难解决的大多数光学方法。这个建议的基础上的结果的第一个提案，旨在扩展其中开发的测量和分析方法。利用已建立的光子力显微镜技术，诱导粒子与活细胞之间的相互作用过程，然后在空间和时间上进行控制。结合、表面的粒子输运和实际的吸收由许多多尺度过程（10^-9到10^-5米和10^-5到10 ^2秒）组成，如果这些过程能够在大的时间和空间带宽上测量，就可以更好地理解。这应通过使用快速、相干（即无标记）方法来实现，例如干涉测量3D颗粒跟踪（1 MHz采样率）或基于旋转相干散射光（ROCS，100 Hz采样率）的新型活细胞超分辨率显微镜方法。另一方面，基于荧光的共聚焦旋转盘扫描将通过电光可调透镜进行扩展，以实现3D图像堆栈的快速获取。在此提案中，两位博士生PhD 1和PhD 2将进行以下项目：使用上述光学方法，PhD 1将研究颗粒在小鼠巨噬细胞平坦细胞膜上的直接结合和摄取。PhD 1将研究细胞何时以及如何感知和响应接近的粒子（例如通过肌动蛋白重组）。此外，PhD 1将测量细胞膜的逐步吞噬，并分析从颗粒波动中提取的3D结合刚度和摩擦力的局部变化。另一方面，PhD 2将研究由丝状伪足和板状伪足介导的颗粒结合和摄取。PhD 2应分析丝状伪足的收缩可以自我优化到何种程度。此外，应研究和分析经常观察到的颗粒沿着丝状伪足和片状伪足的不连续运输以及与动态细胞骨架的潜在机械耦合过程。