Molecular architecture of lymphocyte cortex

淋巴细胞皮层的分子结构

• 批准号: 8157563
• 负责人: James Shaw
• 金额: $ 26.18万
• 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/8157563
• 关键词: Actins; Architecture; Binding; Bioinformatics; F-Actin; Lipid Binding; Lymphocyte; Lymphocyte Function; Molecular; Motor; Mutate; PH Domain; in vivo

Class I myosins, which link F-actin to membrane, are largely undefined in lymphocytes. Mass spectrometric analysis of lymphocytes identified two short tail forms: (Myo1G and Myo1C) and one long tail (Myo1F). We investigated Myo1G, the most abundant in T-lymphocytes, and compared key findings with Myo1C and Myo1F. Myo1G localizes to the plasma membrane and associates in an ATP-releasable manner to the actin-containing insoluble pellet. The IQ+tail region of Myo1G (Myo1C and Myo1F) is sufficient for membrane localization, but membrane localization is augmented by the motor domain. The minimal region lacks IQ motifs but includes: 1) a PH-like domain; 2) a "Pre-PH" region; and 3) a "Post-PH" region. The Pre-PH predicted alpha helices may contribute electrostatically, because two conserved basic residues on one face are required for optimal membrane localization. Our sequence analysis characterizes the divergent PH domain family, Myo1PH, present also in long tail myosins, in eukaryotic proteins unrelated to myosins, and in a probable ancestral protein in prokaryotes. The Myo1G Myo1PH domain utilizes the classic lipid binding site for membrane association, because mutating either of two basic residues in the "signature motif" destroys membrane localization. Mutation of each basic residue of the Myo1G Myo1PH domain reveals another critical basic residue in the beta3 strand, which is shared only by Myo1D. Myo1G differs from Myo1C in its phosphatidylinositol 4,5-bisphosphate dependence for membrane association, because membrane localization of phosphoinositide 5-phosphatase releases Myo1C from the membrane but not Myo1G. Thus Myo1PH domains likely play universal roles in myosin I membrane association, but different isoforms have diverged in their binding specificity. We predict that Myo1G will be functionally important in lymphocytes. Therefore we have generated a conditional Myo1G knockout mouse and are investigating immune system development and hematopoietic cell function. We find that immune system development is generally normal. However, knockout T-lymphocytes have altered migratory properties in vitro and in vivo, which we are analyzing in detail.

将F-肌动蛋白与膜联系起来的I类肌球蛋白在淋巴细胞中基本上不确定。淋巴细胞的质谱分析确定了两种短尾巴形式：（MyO1g和MyO1c）和一个长尾巴（MyO1F）。我们研究了Myo1g，这是T淋巴细胞中最丰富的，并将关键发现与MyO1C和Myo1F进行了比较。 Myo1g将质膜定位于质膜，并以ATP可释放的方式与含有肌动蛋白的无溶性颗粒相关。 Myo1g（MyO1C和MyO1F）的IQ+尾部区域足以用于膜定位，但是膜定位被运动域增强。最小区域缺乏智商基序，但包括：1）pH状域； 2）一个“ pH前”区域； 3）一个“后”区域。 pH前预测的α螺旋可能会产生静电性，因为最佳膜定位需要两个脸上的两个保守基本残基。我们的序列分析表征了发散的pH结构域家族，肌蛋白，在与肌醇无关的真核蛋白中，以及原核生物中可能的祖先蛋白质中。 Myo1g Myo1ph结构域利用经典的脂质结合位点作为膜关联，因为“签名基序”中的两个基本残基中的任何一个都破坏了膜定位。 Myo1g myo1ph结构域的每个基本残基的突变揭示了beta3链中的另一个关键基本残基，仅由myo1d共享。 Myo1g与MyO1c在其磷脂酰肌醇4,5-双磷酸依赖性的膜关联中有所不同，因为磷酸肌醇5-磷酸酶的膜定位从膜上释放了MyO1c，而不是肌肉。因此，Myo1ph结构域可能在肌球蛋白I膜关联中起普遍的作用，但是不同的同工型在其结合特异性方面有所不同。我们预测，MyO1g在淋巴细胞中在功能上很重要。因此，我们已经产生了有条件的Myo1g敲除小鼠，并正在研究免疫系统发育和造血细胞功能。我们发现免疫系统开发通常是正常的。但是，敲除晶状体细胞在体外和体内改变了迁移特性，我们正在详细分析。