活细胞中内吞囊泡和自吞噬囊泡的荧光成像研究

发布时间：2018-03-19 12:33

本文选题：绿色荧光蛋白　切入点：生长因子受体结合蛋白2　出处：《华中科技大学》2013年博士论文　论文类型：学位论文

【摘要】：荧光显微成像技术极大地增强了人们了解生物功能分子信息的能力。绿色荧光蛋白(Green fluorescent protein, GFP)以及类GFP荧光蛋白(GFP-like fluorescent proteins)对于推动这种进步的技术层面的创新起到了极大的作用。各类荧光探针的开发和现代显微技术的进步,使我们能够以更高的时空分辨率将生物功能直接可视化,这包括基因表达、蛋白和细胞内结构/细胞器动力学、蛋白间相互作用、蛋白构象改变、蛋白翻译后修饰、胞内离子和小分子的浓度(改变)和酶活性等。细胞的分泌途径、内吞途径和自吞噬途径等过程中的囊泡结构吸引了很多研究者的兴趣。人们虽然已经揭示了这些生物膜结构的分子基础并提出了各种各样的假说,但是,对于与这些结构相联系的蛋白的动力学性质及其机制仍不清楚。本研究采用FRAP (fluorescence recovery after photobleaching)、FRAPa (fluorescence redistribution after photoactivation)和FRET (fluorescence resonance energy transfer)等动态荧光显微成像技术,对选取的内吞囊泡和自吞噬囊泡上的蛋白质分子进行了动力学的研究,为深入理解生命过程中的蛋白功能提供了新的视角。主要结果如下： 1)利用发射光谱扫描、受体光漂白和荧光寿命等三种不同检测方法比较基于荧光蛋白mCerulean/mCitrine的不同FRET对间的FRET效率。亦用受体光漂白法检测到了EGF刺激后内吞囊泡上EGFR-mCerulean与GRB2-mCitrine之间相互作用的FRET信号。 2)以内吞囊泡上募集的GRB2-mCitrine为模型,进行了定量的FRAP分析。结果显示质膜、早期内涵体膜及晚期内涵体／溶酶体膜上GRB2-mCitrine与胞浆存在快速交换过程,这表明GRB2-mCitrine与EGFR-mCerulean间的相互作用是瞬时的。在质膜内吞囊泡处,我们也测得SHC4蛋白与胞浆存在较大程度交换。而据文献报道包被蛋白小泡(coated vesicles)上的包被蛋白(如clathrin轻链及重链蛋白)及其它组分如AP1、AP2和GGA1也存在交换,即便这些囊泡与本体膜的脱离过程被抑制也是如此。所有这些关于蛋白动态交换的现象,提示着内吞囊泡表面分子的解离与亲和是高度动态的。 3)使用FRAP、FRAPa和FRET等动态显微成像技术,发现LC3与蛋白聚集体有快速的亲和与解离,但是与自吞噬囊泡的结合很稳定。利用LC3在这两种不同结构上的动力学差异性,可通过活细胞中定性的FRAP分析来判定一个FP-LC3斑点是在蛋白聚集体上还是自吞噬囊泡结构。 4)发现自吞噬早期结构上,PtdIns3P的效应蛋白ZFYVE1仅展示了极少的恢复,而另一个PtdIns3P效应蛋白WIPI1却表现出快的和很大程度的恢复。这些结果提示虽然很多ATG蛋白募集到自吞噬囊泡形成部位,但是它们的动力学特性可能很不相同。 5)证明了IBs中成分组织涉及到了有序的分子相互作用。FRET和FRAP分析结果表明在活细胞中LC3/LC3G12OA与SQSTM1C端的LIR (LC3-interacting region)有作用并且这种亲和是瞬时的。FRET结果表明蛋白聚集体中SQSTM1的排列是有规则的,即SQSTM1的N端之间很靠近,在有效的FRET半径内,而N端和C端间则距离较远,超出了有效的FRET半径。 6)通过将SQSTM1蛋白突变体导入到内质网,建立了一个内质网自吞噬体系。另外,结果表明内质网自吞噬是通过将ATG蛋白募集到特定的内质网局部来诱导自吞噬囊泡的形成。综上所述,本研究阐明了EGFR内吞囊泡上接头蛋白GRB2的动力学性质,发现了LC3在蛋白聚集体和自吞噬囊泡这两种不同结构上的动力学差异性,进一步阐释了蛋白聚集体成分的动态性和组成的有序性。本文的研究确立了定量自吞噬过程动力学的有趣和有用的光学成像方法,也表明了内吞囊泡的高度动态性,为进一步的研究提供了技术上和研究对象上的基础。
[Abstract]:Fluorescence microscopic imaging techniques have greatly enhanced the ability of people to understand the biological function of molecular information. Green fluorescent protein (Green fluorescent, protein, GFP) and GFP (GFP-like fluorescent proteins) fluorescent protein has great effect to the innovation of technology level to promote the progress of the development. All kinds of fluorescent probes and modern micro technology that allows us to higher temporal and spatial resolution will direct visualization of biological functions, including gene expression, protein and cellular structure / organelle dynamics, protein-protein interactions, protein conformational changes, protein post-translational modifications, the intracellular concentration of ions and small molecules (change) and enzyme activity.
The secretory pathway of the cell, the endocytic pathway and self consuming ways in the vesicle structure has attracted much research interest. Although people have revealed the molecular basis of these biofilm structure and put forward various hypothesis, however, for the dynamic properties associated with the structure of the protein and its mechanism is still not sure. This study uses FRAP (fluorescence recovery after photobleaching), FRAPa (fluorescence redistribution after photoactivation) and FRET (fluorescence resonance energy transfer) dynamic fluorescence microscopic imaging technique, the selection of endocytic vesicles and autophagy vesicles on the protein dynamics research, provides a new perspective for deep understanding of life in the process of protein function. The main results are as follows:
1) by emission spectroscopy scanning, acceptor photobleaching and fluorescence lifetime of three different detection methods based on FRET efficiency of different FRET fluorescent protein mCerulean/mCitrine on the acceptor photobleaching method. Also used to detect EGF stimulates FRET signaling interaction between EGFR-mCerulean and GRB2-mCitrine after endocytic vesicles.
2) within the endocytic vesicles by the GRB2-mCitrine model, the quantitative analysis of FRAP. The results showed that the plasma membrane, the early endosome membrane and late endosomes / lysosomes membrane and intracellular GRB2-mCitrine rapid exchange process, which indicates that the interaction between EGFR-mCerulean and GRB2-mCitrine is instantaneous. The endocytic vesicles in the plasma membrane. We also measured SHC4 protein and cytoplasmic large degree of exchange. According to Literature coated vesicles (coated vesicles) on the envelope protein (such as the clathrin light chain and heavy chain protein) and other components such as AP1, AP2 and GGA1 are exchanged, even from these vesicles and the body membrane process the suppressed as well. All of these proteins on dynamic exchange phenomenon, suggests that the dissociation of endocytic vesicle surface molecules with affinity is highly dynamic.
3) the use of FRAP, FRAPa and FRET dynamic imaging technology, found that LC3 and protein aggregates have affinity and dissociation of fast, but the combination of autophagy and vesicles are stable. The use of LC3 in these two kinds of different structure on the dynamics of FRAP through the qualitative analysis of living cells to determine a FP-LC3 dot is in protein aggregates or autophagic vesicles.
4) found that autophagy early on the structure, the effect of the PtdIns3P protein ZFYVE1 demonstrated only minimal recovery, while another PtdIns3P effect protein WIPI1 has shown rapid and largely restored. These results suggest that although many ATG protein raised autophagic vesicle formation sites, but their dynamics are not the same.
5) proved that the components of tissue IBs involved in ordered molecular interaction between.FRET and FRAP analysis showed that LC3/LC3G12OA and SQSTM1C LIR in living cells (LC3-interacting region) and the role of affinity is instantaneous.FRET results showed that SQSTM1 protein aggregates are arranged in a regular, i.e. between SQSTM1 N end very close to the FRET, in the effective radius of the inner side of the C and N, and between the distance, beyond the radius of FRET effectively.
6) by introducing the SQSTM1 protein mutant into the endoplasmic reticulum, an endoplasmic reticulum autophagy system was established. Moreover, the results showed that the endoplasmic reticulum autophagy was induced by the recruitment of ATG protein into the specific endoplasmic reticulum part to induce the formation of autophagic vesicles.
In summary, this study illustrates the dynamic properties of EGFR endocytic vesicles on the adaptor protein GRB2, LC3 was found in protein aggregates and autophagy vesicles of these two kinds of different structure on the dynamics, further explained the order of the dynamic characteristics and composition of protein aggregates composition. This paper established a quantitative self interesting and useful imaging method of phagocytic process dynamics, also shows that the highly dynamic endocytic vesicles, provides technical basis and research object for further research.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R310

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