Molecular architecture of lymphocyte cortex

淋巴细胞皮层的分子结构

• 批准号: 7733397
• 负责人: James Shaw
• 金额: $ 38.35万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7733397
• 关键词: Actins; Adhesions; Architecture; Area; Back; Biochemical; Bioinformatics; Biological; Blood; Cell Line; Cell membrane; Cell physiology; Cells; Class; Classification; Collaborations; Cytoskeletal Proteins; Endocytosis; Endothelium; Event; G-substrate; GTP-Binding Proteins; Gelsolin; Genetic; Goals; Hematopoietic; Heterotrimeric GTP-Binding Proteins; Human; Immune system; Immunologic Surveillance; Immunologics; Integral Membrane Protein; Intermediate Filaments; Investigation; Knock-out; Knockout Mice; Label; Lymphocyte; Lymphocyte Function; Lymphoid Cell; Mediating; Membrane; Membrane Proteins; Microfilament Proteins; Microfilaments; Microtubules; Molecular; Monomeric GTP-Binding Proteins; Mouse Strains; Mus; Myosin ATPase; Myosin Type II; Nuclear; Pattern; Play; Pre-B Lymphocyte; Preparation; Procedures; Property; Proteins; Proteomics; Reagent; Regulation; Relative (related person); Rest; Role; Structure-Activity Relationship; T-Lymphocyte; Talin; Testing; Tissues; Western Blotting; base; cell motility; cell type; cellular microvillus; cofilin; ezrin; gastrointestinal microvillus; improved; interest; migration; moesin; peripheral blood; polyclonal antibody; profilin; radixin protein; tool; trafficking

Lymphocyte microvilli mediate initial adhesion to endothelium during lymphocyte transition from blood into tissue but their molecular organization is incompletely understood. We modified a shear-based procedure to prepare biochemical fractions enriched for membrane/microvilli (MMV) from both human peripheral blood T-lymphocytes (PBT) and a mouse pre-B lymphocyte line (300.19). Enrichment of proteins in MMV relative to post nuclear lysate was determined by mass spectrometric (LC/MS/MS) analysis and label-free quantitation. Subsequent analysis emphasized the 291 proteins shared by PBT and 300.19 and estimated by MS peak area to be highest abundance. Validity of the label-free quantitation was confirmed by many internal consistencies and by comparison with Western blot analyses. The MMV fraction was enriched primarily for subsets of cytoskeletal proteins, transmembrane proteins and G-proteins, with similar patterns in both lymphoid cell types. The most enriched cytoskeletal proteins were microfilament-related proteins NHERF1, Ezrin/Radixin/Moesin (ERMs), ADF/cofilin and Myosin1G Myo1G). Other microfilament proteins such as talin, gelsolin, myosin II and profilin were markedly reduced in MMV, as were intermediate filament- and microtubule-related proteins. Heterotrimeric G-proteins and some small G-proteins (especially Ras and Rap1) were enriched in the MMV preparation. Two notable general observations also emerged. There was less overlap between the two cells in their transmembrane proteins than in other classes of proteins, consistent with a special role of plasma membrane proteins in differentiation. Second, unstimulated primary T-lymphocytes have an unusually high concentration of actin and other microfilament related proteins, consistent with the singular role of actin-mediated motility in the immunological surveillance performed by these primary cells. We have chosen for intensive investigation two of more abundant molecules identified as strongly enriched in the membrane/microvillus fraction in the foregoing studies: Myo1G and NHERF1. Myo1G was chosen because class I unconventional myosins in other cells have been shown to play important roles in regulation of microvilli formation, endocytosis, vesicular traffic and other key cellular events but their role in hematopoietic cell function is largely unstudied. Myo1G in particular is completely unstudied, despite its prominent expression in T-cells. We have therefore have initiated systematic investigation of Myo1G. Key reagents have been generated including a specific polyclonal antibody, constructs to study structure-function relationship and a conditional knockout mouse These tools are being used to investigate Myo1G expression, localization, and function in cells and in the mouse. Similarly, NHERF1 is a PDZ-containing adaptor molecule important in regulating molecular localization and function in multiple cells types, but whose function is unstudied in the immune system. We have initiated a collaboration to study knockout mice, and are back-crossing the mouse strain to Bl6 to improve their usefulness for immunologic analysis. Initial results indicate that knockout lymphocytes have alterations in cell spreading and migration. Systematic analysis is underway.

淋巴细胞微绒毛介导了从血液向组织的淋巴细胞过渡期间对内皮的初始粘附，但其分子组织尚不完全了解。 我们修改了一种基于剪切的程序，以制备从人外周血T淋巴细胞（PBT）和小鼠前B淋巴细胞系（300.19）中富含膜/微绒毛（MMV）的生化分数（300.19）。通过质谱（LC/MS/MS）分析和无标记定量确定MMV中蛋白质中蛋白质的富集。 随后的分析强调了PBT共有的291蛋白和300.19，并由MS峰面积估算为最高丰度。许多内部一致性证实了无标签定量的有效性，并与Western印迹分析进行了比较。 MMV馏分主要富集于细胞骨架蛋白，跨膜蛋白和G蛋白的亚群，在两种淋巴样细胞类型中均具有相似的模式。最富集的细胞骨架蛋白是与微丝相关的蛋白NHERF1，Ezrin/radixin/Moesin（ERMS），ADF/Cofilin和Myosin1g myo1g）。 MMV中的其他微丝蛋白，例如塔林，凝胶蛋白，肌动蛋白II和叶胶蛋白，中间丝和微管相关蛋白也显着降低。在MMV制备中富含异三聚体G蛋白和一些小的G蛋白（尤其是RAS和RAP1）。还出现了两个值得注意的一般观察。在跨膜蛋白中，两个细胞之间的重叠率少于其他类别的蛋白质，这与质膜蛋白在分化中的特殊作用一致。 其次，未刺激的原代T淋巴细胞具有异常高的肌动蛋白和其他与微丝相关的蛋白质，与肌动蛋白介导的运动性在这些原代细胞执行的免疫监测中的奇异作用一致。我们选择了对上述研究中强烈富集的两个更丰富的分子进行密集研究：myo1g和nherf1。之所以选择Myo1g，是因为已经证明了I类在其他细胞中的非常规的肌球蛋白在调节微绒毛形成，内吞作用，囊泡交通和其他关键细胞事件中起重要作用，但是它们在造血细胞功能中的作用在很大程度上尚未研究。尽管Myo1g在T细胞中很明显，但尤其是完全没有研究。因此，我们已经对Myo1g进行了系统的研究。已经生成了关键试剂，包括特定的多克隆抗体，研究结构功能关系的结构以及有条件的基因敲除小鼠这些工具用于研究细胞和小鼠中的MyO1g表达，定位和功能。同样，NHERF1是一种含PDZ的衔接子分子，对于调节多种细胞中的分子定位和功能很重要，但其功能在免疫系统中未被研究。我们已经启动了一种研究淘汰小鼠的协作，并将小鼠菌株反向BL6进行了反向，以提高其对免疫学分析的有用性。最初的结果表明，基因敲除淋巴细胞在细胞扩散和迁移方面发生了变化。系统分析正在进行中。