Combining coherent Raman scattering with fluorescence microscopy to investigate sugar uptake in living cells

相干拉曼散射与荧光显微镜相结合研究活细胞中的糖摄取

基本信息

  • 批准号:
    426814631
  • 负责人:
  • 金额:
    --
  • 依托单位:
  • 依托单位国家:
    德国
  • 项目类别:
    Research Grants
  • 财政年份:
    2019
  • 资助国家:
    德国
  • 起止时间:
    2018-12-31 至 2023-12-31
  • 项目状态:
    已结题

项目摘要

Sugars are essential molecules in life. Being actively transported over the cell membrane by specialized membrane transporters, they represent a major source of energy and carbon in glycan biosynthesis. Glycosylation, as ubiquitous post-translational modification has been known for a long time. All cells are covered with a dense and complex layer of glycans. This so-called glycocalyx is involved in various processes including signalling, cell-adhesion and immunity based on glycan-driven pattern recognition. Despite its importance, only little is known about the composition, structure and precise function of the glycocalyx due to the extraordinary variability of glycan structures as well as dynamic nature of glycosylation. It is unanswered, how sugar uptake influences the chemical composition of the cellular glycocalyx. Combined structural approaches have identified a growing catalogue of monosaccharides and glycan structures however they are restricted to ex vivo analysis. Metabolic oligosaccharide engineering allowed for fluorescence imaging of sugar moieties in vivo. Nevertheless, labelling of sugars remains challenging. Fluorescent labels can interfere with the sugar’s biological function. Sugars species are present at intracellular concentrations ranging from nM to mM range under physiological conditions. Single-molecule imaging is not suited to simultaneously visualize low and high concentrations of different sugar species, or the involved membrane transporter and substrate at the same time. Despite its immense biomedical importance, we lack a high-speed, multiplexed imaging technology that allows for studying natural sugars during uptake, metabolism and storage as glycans in situ. We propose to establish stimulated Raman microscopy (SRS), which I pioneered during my PhD, as tool for chemical mapping of sugars. I will design a cutting-edge imaging platform, that combines single-molecule live cell imaging with broadband stimulated Raman microscopy. It is dedicated for multiplexed in vivo imaging with chemical specificity at high concentrations and simultaneous single-molecule sensitivity. I will establish the developed methodology to investigate uptake and storage of sugar within the cell. The combined approach serves for the quantification of sugar uptake mediated by GLUT transporters with cellular resolution. Moreover, we will study whether inserted Raman-active moieties in MOE affect the affinity and uptake and if unnatural sugars are taken up actively or via passive diffusion. By investigating the cellular storage of sugars, we ultimately aim to establish this approach as a chemical characterization method for the cellular glycocalyx.
糖是生命中必不可少的分子。它们通过专门的膜转运体在细胞膜上主动运输,是糖聚糖生物合成中能量和碳的主要来源。糖基化作为一种普遍存在的翻译后修饰,早已为人所知。所有的细胞都覆盖着一层密集而复杂的聚糖。这种所谓的糖萼参与了多种过程,包括信号传导、细胞粘附和基于糖驱动模式识别的免疫。尽管它很重要,但由于多糖结构的异常可变性以及糖基化的动态性质,人们对糖萼的组成、结构和精确功能知之甚少。糖摄取如何影响细胞糖萼的化学成分尚无答案。结合结构方法已经确定了单糖和聚糖结构的不断增长的目录,但它们仅限于离体分析。代谢寡糖工程允许荧光成像的糖部分在体内。然而,糖的标签仍然具有挑战性。荧光标记会干扰糖的生物学功能。生理条件下,细胞内的糖浓度从nM到mM不等。单分子成像不适合同时显示低浓度和高浓度的不同糖种,或同时显示相关的膜转运蛋白和底物。尽管天然糖具有巨大的生物医学意义,但我们缺乏一种高速、多路成像技术来研究天然糖在摄取、代谢和储存过程中的原位聚糖。我们建议建立受激拉曼显微镜(SRS),这是我在博士期间首创的,作为糖化学制图的工具。我将设计一个尖端的成像平台,将单分子活细胞成像与宽带刺激拉曼显微镜相结合。它专门用于多重体内成像,具有高浓度的化学特异性和同时的单分子敏感性。我将建立已开发的方法来研究细胞内糖的摄取和储存。该联合方法用于定量由GLUT转运体介导的糖摄取,具有细胞分辨率。此外,我们将研究MOE中插入的拉曼活性基团是否会影响亲和力和摄取,以及非天然糖是主动吸收还是通过被动扩散吸收。通过研究糖的细胞储存,我们最终的目标是建立这种方法作为细胞糖萼的化学表征方法。

项目成果

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Dr. Evelyn Plötz其他文献

Dr. Evelyn Plötz的其他文献

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{{ truncateString('Dr. Evelyn Plötz', 18)}}的其他基金

Linking structure and function of individual virus particles
单个病毒颗粒的连接结构和功能
  • 批准号:
    238802862
  • 财政年份:
    2013
  • 资助金额:
    --
  • 项目类别:
    Research Fellowships

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    数学天元基金项目
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    40574067
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    2005
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    36.0 万元
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