利用原子力显微镜研究细胞膜结构
文献类型：学位论文
| 作者 | 田咏梅 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-09 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 中国科学院长春应用化学研究所 |
| 导师 | 王宏达 |
| 关键词 | 细胞膜 原子力显微镜 膜结构 不对称性 |
| 中文摘要 | 细胞膜对于保持细胞结构的完整性以及维持细胞正常的生命活动具有至关重要的作用。对于细胞膜在生理条件下的超微结构形态和细胞膜各组份的整体结构组装还缺乏一致的结论。原子力显微镜（atomic force microscopy, AFM）兼具高分辨、液相成像以及实时动态的优点，在液相条件下研究细胞膜具有突出的优势。本论文主要利用AFM高分辨成像和单分子力谱（single-molecule force spectroscopy, SMFS）研究接近生理条件下细胞膜的微观结构，从生物进化角度，按照从低等到高等、从简单到复杂的顺序研究了鲫鱼、乌龟、鸡血红细胞，哺乳动物体细胞及细胞器的膜结构，揭示了细胞膜结构的相似性和膜蛋白分布的不对称性规律。主要内容如下： 1. 以鲫鱼为代表，研究了鱼类的血红细胞膜结构。观察到了鲫鱼椭圆形双面凸的盘状红细胞，中间有椭圆形的细胞核，不同于人类双面凹的盘状无核红细胞。鲫鱼红细胞的尺寸比人类红细胞大，细胞膜厚度接近人类红细胞的两倍。揭示了鲫鱼红细胞膜上蛋白的不对称性分布：细胞膜外侧比较光滑没有蛋白颗粒分布；而细胞膜内侧比较粗糙，分布有致密的蛋白颗粒。这种不对称性与人类红细胞膜结构的半镶嵌模型是一致的，说明半镶嵌模型也适用于阐述鱼类红细胞的膜结构。 2. 以乌龟为代表，研究了爬行类的血红细胞膜结构。乌龟红细胞形状与鲫鱼红细胞相似，尺寸更大一些。细胞膜厚度与鲫鱼无明显差别，都比人类红细胞膜厚。高分辨AFM成像揭示了乌龟红细胞膜光滑的外膜和蛋白覆盖的内膜。SMFS检测到内膜上大量膜蛋白暴露的氨基而外膜几乎没有暴露的氨基，证实了蛋白分布的不对称性。乌龟红细胞膜不对称的膜结构与人类、鱼类红细胞膜结构的半镶嵌模型相符，使半镶嵌模型从哺乳动物、鱼类拓展到爬行类。 3. 以鸡血红细胞为代表，研究了鸟类的血红细胞膜结构。鸡血红细胞与鱼类、爬行类红细胞形状相似，都是有核红细胞。细胞尺寸上，从乌龟、鲫鱼到鸡，逐渐减小。鸡血红细胞膜的厚度比鲫鱼、乌龟小一些，但比人类红细胞膜更厚。它们在磷脂双分子层的厚度上无明显差异。高分辨AFM成像表明，鸡血红细胞膜外膜比较光滑，膜蛋白并没有突出于细胞表面；而内膜比较粗糙，大量蛋白紧密排列于磷脂双分子层之上。SMFS进一步证实了蛋白的不对称性分布。原位实时AFM观察到了环糊精对细胞内膜中脂筏区域的溶解作用，第一次为鸡血红细胞膜中脂筏的存在提供了直接的证据。鸡血红细胞膜不对称的膜结构完全符合人类、鱼类、爬行类红细胞膜结构的半镶嵌模型。红细胞膜结构的半镶嵌模型从哺乳类、鱼类、爬行类进一步拓展到鸟类。这个结果对于从生物进化的观点揭示半镶嵌模型的广泛适用性具有重要的意义。 4. 结合三种单分子技术包括AFM、SMFS、随机光学重建显微镜(stochastic optical reconstruction microscopy, STORM)研究了哺乳动物有核体细胞的膜结构。我们发现：（1）在细胞膜胞外侧（外表面），蛋白位于磷脂双分子层之上，形成一层致密的4 nm的蛋白层；（2）在胞质侧，蛋白形成均匀分散的岛状的聚集体，厚度10-12 nm；（3）细胞膜中存在胆固醇富含的区域；（4）在细胞膜外表面糖类形成微区；（5）细胞膜内外两侧暴露的氨基是不对称分布的。据此，我们提出了一个新的细胞膜结构模型，PLLPI模型，可以用来解释有核哺乳动物细胞基本的膜结构。这个模型有助于更好地理解细胞膜信号转导、膜运输、病毒融合机理等一系列细胞膜相关的功能。 5. 研究了从大鼠肝脏分离提取的线粒体膜的超微结构。线粒体外膜表面没有明显的凹凸不平和蛋白颗粒分布，膜内包含膜结合蛋白，平均粗糙度为1.0±0.3 nm。我们第一次用高分辨AFM成像了完整的脱去外膜的线粒体及其形成的多层膜。单层线粒体内膜厚度为7.1±0.9 nm，与人类红细胞膜的厚度差别不大。线粒体内膜膜间隙侧比较光滑，没有蛋白颗粒，而基质侧是包含蛋白颗粒的，平均粗糙度分别为0.6±0.2 nm和1.5±0.4 nm，这与红细胞膜的结构模型相似，证明了细胞生物膜结构的一致性。 |
| 英文摘要 | Cell membranes are essential in keeping the integrity of the whole cell and maintaining normal cellular activities. Research on the ultrastructure of cell membranes and the elaborate organization of membrane components under near native conditions is still a great challenge. With the advantages of high resolution, real-time, dynamic imaging in physiological buffer, AFM has been a powerful tool in cell membrane biology. Here we combined AFM high resolution image and SMFS to study the structure of cell membrane with molecular resolution in a quasi-native state. Following the evolutionary law, we chose crucian carp, turtle and chicken as the representatives of fish, reptiles and birds, and systematically investigated erythrocyte membrane structure of these species, revealing the similar asymmetry of membrane structure with human erythrocyte. The nucleated mammalian cell and organelle membrane were further explored. The main results are summarized as follows: 1. As a representive of fish, crucian carp erythrocyte and erythrocyte membrane were investigated. It was observed that crucian carp erythrocytes are oval, biconvex discs with elliptical nucleus protruding in the central region, very different from biconcave disc-like, non-nucleated human erythrocytes. The size of crucian carp erythrocyte is much larger than that of human erythrocyte, with the membrane thickness nearly twice. The result revealed the asymmetric distribution of proteins in crucian carp erythrocyte membrane: the outer leaflet of membrane is rather smooth without any proteins, whereas the inner leaflet is very rough with dense proteins. This asymmetry fits well with the semi-mosaic model of human erythrocyte membrane structure, indicating that the semi-mosaic model can also be applied to illustrate fish erythrocyte membrane structure. 2. As a representive of reptiles, turtle erythrocyte and erythrocyte membrane were studied. In morphology, turtle erythrocytes are similar with fish erythrocyte, except the larger size. The membrane thickness of turtle erythrocyte is not significantly different from that of Crucian carp erythrocyte, both much thicker than human erythrocyte. High resolution images on both leaflets of turtle erythrocyte membranes revealed a smooth outer membrane leaflet and a protein covered inner membrane leaflet. This asymmetry was verified by SMFS, which detects numerous exposed amino groups of membrane proteins in the inner membrane leaflet but much fewer in the outer leaflet. The asymmetric membrane structure of turtle erythrocytes is consistent with the semi-mosaic model of human and fish erythrocyte membrane structure, extending the semi-mosaic model to reptiles. 3. As a representative of birds, chicken erythrocytes and erythrocyte membranes were examined. Chicken erythrocytes are also nucleated, morphologically similar with fish and reptile erythrocyte. The size of erythrocyte decreases from turtle, crucian carp, chicken to human, the membrane thickness decreasing similarly. The thicknesses of the lipid bilayer of these erythrocyte membranes are comparable. The smooth outer membrane leaflet and protein covered inner membrane leaflet were revealed by high resolution image. SMFS confirmed that the amino groups are exposed in the inner leaflet membrane but inaccessible by the AFM tip in the outer leaflet membrane. The erosion of lipid rafts by MβCD was visualized by in situ, real time imaging, providing the first direct evidence of lipid rafts in chicken erythrocyte membranes. The asymmetric membrane structure of chicken erythrocytes corresponds well with the semi-mosaic model of human, turtle and fish erythrocyte membrane structure, which was further extended from mammalian, reptiles, fish to birds. This result may be of significance to verify the universality of the semi-mosaic model from the perspective of the biological evolution. 4. Using a combination of single-molecule techniques, including AFM, SMFS and STORM, to study the structure of nucleated cell membranes, we found that (1) proteins at the ectoplasmic side of the cell membrane form a dense protein layer (4 nm) on top of a lipid bilayer; (2) proteins aggregate to form islands evenly dispersed at the cytoplasmic side of the cell membrane with a height of about 10–12 nm; (3) cholesterol-enriched domains exist within the cell membrane; (4) carbohydrates stay in microdomains at the ectoplasmic side; and (5) exposed amino groups are asymmetrically distributed on both sides. Based on these observations, we proposed a Protein Layer-Lipid-Protein Island (PLLPI) model, to provide a better understanding of cell membrane structure, membrane trafficking and viral fusion mechanisms. 5. Isolated mitochondria and mitochondrial membrane from rat liver were imaged. The outer mitochondrial membrane is slightly rough in the outer surface and protein-embedded inside with an average roughness of 1.0±0.3 nm. For the first time, intact and partly flattened mitoplasts were observed. The thickness of unilamella inner mitochondrial membrane was 7.1±0.9 nm. The native inner mitochondrial membrane is characterized as smooth in the intermembrane space surface and protein-covered in the matrix side with average roughness of 0.6±0.2 nm and 1.5±0.4 nm, respectively. The asymmetry of protein distribution on both sides of the inner mitochondrial membrane is similar with the semi-mosaic model of erythrocyte membrane structure, demonstrating the structural consistency of cellular biomembranes. |
| 语种 | 中文 |
| 公开日期 | 2016-05-03 |
| 源URL | [http://ir.ciac.jl.cn/handle/322003/64469]  |
| 专题 | 长春应用化学研究所_长春应用化学研究所知识产出_学位论文 |
| 推荐引用方式 GB/T 7714 | 田咏梅. 利用原子力显微镜研究细胞膜结构[D]. 中国科学院长春应用化学研究所. 中国科学院研究生院. 2014. |
入库方式： OAI收割
来源：长春应用化学研究所
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