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首页> 外文学位 >Characterization of membrane organization and dynamics in giant unilamellar vesicles assembled from rat kidney membrane extracts and fragments.
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Characterization of membrane organization and dynamics in giant unilamellar vesicles assembled from rat kidney membrane extracts and fragments.

机译：从大鼠肾脏膜提取物和片段组装而成的巨大单层囊泡中膜组织和动力学的表征。

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摘要

In renal proximal tubular cells, the plasma membrane is polarized in terms of composition, structure and function into distinct regions: the brushborder (BBM) and the basolateral (BLM) membrane. The purpose of our work is to gain insight into the complex structure of biological membranes by applying the techniques that have been used in characterizing artificial membrane systems to natural membranes reconstituted into giant unilamellar vesicles (GUVs).; In GUVs made from natural lipid extracts from BBM and BLM, we observed phase separation through the formation of non-fluorescent domains at physiological temperatures, implying that domain formation may be driven by lipid-lipid interactions. In intact membrane GUVs, the morphology and membrane fluidity of the vesicles demonstrate that membrane proteins also play an important role in the organization of natural membranes. The presence of domains in these membrane preparations gives support to the presence of "rafts" or raft-like membrane domains in natural membranes.; GUV studies of natural membranes would be incomplete without the determination that the membrane in the vesicles closely approximates the structure and composition of the plasma membrane. Thus the incorporation of proteins in the GUVs assembled from intact membrane fragments was confirmed through primary and secondary antibody detection of various membrane proteins: type IIa Na/Pi cotransporter (NaPi-IIa), beta-actin and Na+/K+ ATPase.; Fluctuation correlation spectroscopy (FCS) measurements performed on the intact BBM reveal that the protein-lipid dynamics in renal BBM is complex with a large range of diffusing species. In particular, the dynamics of NaPi-IIa was related to the perturbations in the BBM as a result of dietary potassium deficiency. By calculating the diffusion coefficient of NaPi-IIa and using photon counting histogram (PCH) analysis to measure protein aggregation, we determined that the lateral diffusion of NaPi-IIa is slowed down and its aggregation is increased in potassium deficiency, both of which are associated with the decreased Na/Pi cotransport activity. This model was proposed as a means for posttranslational regulation of Na/Pi cotransport activity that is mediated by the increased sphingomyelin and glycosphingolipid content in potassium-deficient BBM, although the mechanisms by which these lipids may modulate NaPi-IIa activity have yet to be determined.
机译：在肾近端肾小管细胞中，质膜在组成，结构和功能方面极化为不同的区域：刷状缘膜（BBM）和基底外侧（BLM）膜。我们的工作目的是通过将用于表征人造膜系统的技术应用于重构为单层大囊泡（GUV）的天然膜，从而深入了解生物膜的复杂结构。在由BBM和BLM的天然脂质提取物制成的GUV中，我们观察到在生理温度下通过形成非荧光结构域而发生相分离，这表明结构域的形成可能是由脂质-脂质相互作用驱动的。在完整的膜GUV中，囊泡的形态和膜流动性表明，膜蛋白在天然膜的组织中也起着重要作用。这些膜制品中结构域的存在为天然膜中“筏”或筏状膜结构域的存在提供了支持。如果没有确定囊泡中的膜与质膜的结构和组成非常接近，那么天然膜的GUV研究将是不完整的。因此，通过各种膜蛋白的一级和二级抗体检测，证实了蛋白质从完整的膜片段组装而成的GUV中的结合：IIa型Na / Pi共转运蛋白（NaPi-IIa），β-肌动蛋白和Na + / K + ATPase。对完整BBM进行的波动相关光谱（FCS）测量表明，肾BBM中的蛋白质-脂质动力学非常复杂，具有多种扩散物质。特别是，NaPi-IIa的动力学与饮食中钾缺乏引起的BBM摄动有关。通过计算NaPi-IIa的扩散系数并使用光子计数直方图（PCH）分析来测量蛋白质聚集，我们确定NaPi-IIa的侧向扩散减慢并且其聚集在钾缺乏症中增加，这两者均与Na / Pi共转运活性降低。该模型被提议作为Na / Pi共转运活性的翻译后调控手段，其由缺钾的BBM中鞘磷脂和糖鞘脂含量的增加介导，尽管这些脂质调节NaPi-IIa活性的机制尚待确定。 。

著录项

  • 作者

    Cheng, Melanie Anne B.;

  • 作者单位

    University of Illinois at Urbana-Champaign.;

  • 授予单位 University of Illinois at Urbana-Champaign.;
  • 学科 Chemistry Biochemistry.; Biology Cell.
  • 学位 Ph.D.
  • 年度 2005
  • 页码 126 p.
  • 总页数 126
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;细胞生物学;
  • 关键词

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