Molecular architecture of lymphocyte cortex

淋巴细胞皮层的分子结构

• 批准号: 7965913
• 负责人: James Shaw
• 金额: $ 25.68万
• 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/7965913
• 关键词: Actins; Antibodies; Architecture; Back; Bacteria; Base Sequence; Binding; Biochemical; Bioinformatics; Biological; Biological Process; Cell Line; Cell membrane; Cells; Chimeric Proteins; Collaborations; Deletion Mutation; Eukaryota; Genetic; Goals; Human; Immune system; Immunologics; Knock-out; Knockout Mice; Ligand Binding; Lipid Binding; Lipids; Lymphocyte; Lymphocyte Function; Malignant Neoplasms; Membrane; Membrane Lipids; Modeling; Molecular; Mouse Strains; Mutate; Myosin Type I; PH Domain; Process; Property; Protein Isoforms; Proteomics; Recombinant Proteins; Regulation; Rest; Role; Sequence Alignment; Signal Transduction; Specificity; Structure; T-Lymphocyte; Tail; Testing; Time; base; cell type; cellular microvillus; improved; interest; migration; overexpression

We have initiated studies of Myo1G in lymphocytes, because of our finding that: 1) Myo1G is highly enriched in lymphocyte membrane / microvilli; 2) Myo1G is previously uncharacterized; and 3) Myo1G is expected to be involved in regulating cortical organization (based on other Myo isoforms in eukaryotes). We have generated a structure based sequence alignment, and generated antibodies and GFP fusion proteins to show that Myo1G is present exclusively at the plasma membrane in lymphocytes. The PH domain in class I myosins is divergent and may represent an ancestral PH domain, since our analysis identified for the first time a homologous PH domain in bacteria. Overexpression studies and mutational analysis of conserved basic residues implicated in ligand binding showed that Myo1G membrane localization is in part due to the PH domain that, which we find is present in the tail of all class I myosins (Myo1A-H). Using a set of deletion and mutation constructs we demonstrated that the PH domain is necessary but not sufficient for membrane localization of Myo1G and have characterized the additional requirements. We have modeled the PH domain and mutated multiple basic residues to identify the critical residues for membrane localization. Analysis of lipid binding will be undertaken upon completion of appropriate constructs and recombinant proteins. Perturbations of membrane lipids in intact cells reveals different requirements for Myo1G from the previously described requirements for Myo1C. . In summary, these studies shown that there is a conserved PH domain in all human class I myosins, and this domain is important for normal localization, however the specificity for interaction with lipids at the plasma membrane differs between them. In order to understand the broader biological function of Myo1G, we have generated a Myo1G conditional knockout mouse and are characterizing functional alterations. We have also initiated studies of NHERF1, a PDZ-containing adaptor molecule important in regulating molecular localization and function in multiple cells types. An additional rationale for study is recent evidence implicating NHERF1 in cancer. But the function of NHERF1 in the immune system is largely unstudied. The importance of ERM binding to NHERF1 is regulating localization at the membrane has been described but is not well understood. We have therefore undertaken structure function analysis to better characterize this process, especially with regard to the role of regulation by membrane lipid and autoinhibition. In addition, 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.

我们已经在淋巴细胞中对MyO1g进行了研究，因为我们的发现：1）Myo1g高度富集在淋巴细胞膜 /微绒毛中； 2）Myo1g以前未经表征； 3）预计Myo1g将参与调节皮质组织（基于真核生物中的其他Myo同工型）。我们已经产生了基于结构的序列比对，并生成了抗体和GFP融合蛋白，以表明肌肉膜仅存在于淋巴细胞中的质膜。 I类肌动物中的pH结构域是不同的，可能代表祖先的pH结构域，因为我们的分析首次确定了细菌中同源的pH结构域。对配体结合涉及的保守基本残基的过表达研究和突变分析表明，Myo1g膜定位部分是由于pH结构域的一部分是由于我们发现的所有I级肌动物（MyO1A-H）的尾巴都存在。使用一组删除和突变构建体我们证明了pH结构域是必要的，但不足以用于Myo1g的膜定位，并表征了其他要求。我们已经对pH结构域进行了建模并突变了多个基本残基，以识别膜定位的关键残基。完成适当的构建体和重组蛋白后，将进行脂质结合的分析。 完整细胞中膜脂质的扰动揭示了对MyO1c的先前描述的要求，对Myo1g的需求不同。 。总而言之，这些研究表明，所有人类I类肌球蛋白中都有一个保守的pH结构域，并且该结构域对于正常定位很重要，但是与质膜在质膜上相互作用的特异性在它们之间有所不同。为了了解Myo1g的更广泛的生物学功能，我们已经产生了MyO1g有条件的基因敲除小鼠，并且正在表征功能变化。我们还启动了NHERF1的研究，NHERF1是一种含PDZ的衔接子分子，对调节多种细胞中的分子定位和功能很重要。额外的研究基本原理是最近的证据，暗示了NHERF1癌症。但是NHERF1在免疫系统中的功能在很大程度上没有研究。 ERM与NHERF1结合的重要性是调节在膜上的定位，但尚不清楚。因此，我们进行了结构函数分析以更好地表征这一过程，尤其是在膜脂质和自身抑制的调节作用方面。此外，我们已经启动了一项协作来研究敲除小鼠，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。