The Molecular Basis of Allorecognition in Social Amoeba

描述（由申请人提供）：异体识别是生物体区分自我与非自我的能力，是许多生物体的共同主题，也是多细胞生物进化的核心组成部分。在哺乳动物中，同种异体识别是由主要组织相容性复合体（MHC）介导的，它是免疫系统的一部分。MHC有助于识别感染过程中的寄生虫和移植医学中的移植排斥反应。有证据表明其他蛋白质参与移植排斥反应，但很少有模型系统可用于研究这些蛋白质。在非哺乳动物系统中有几种异体识别系统，特别是在海洋生物中，但它们中的大多数不是遗传上可处理的。在之前的资助期内，我们在社会土壤中发现了一种异体识别系统。该系统基于两种蛋白质，TgrB1和TgrC1，它们与哺乳动物MHC蛋白有许多相同的特性，但不是它们的氨基酸序列。我们发现这些跨膜蛋白在天然盘基骨菌群体中具有高度多态性，是异体识别的必要和充分条件。当食物充足时，盘基骨柱细胞以自由变形虫的形式生活在土壤中，但当饥饿时，它们聚集成多细胞生物。聚集涉及细胞外cAMP的趋化性，盘基骨柱是研究趋化性的最佳模型系统之一，趋化性是胚胎发生和先天免疫的核心过程。当盘基骨柱细胞在聚集过程中遇到携带不相容的TgrB1和TgrC1的细胞时，它们会彼此分离，形成独立的多细胞生物。我们提出，一个细胞表面的TgrB1与相邻细胞表面的TgrC1结合，这种结合启动了改变细胞行为的信号转导级联。我们打算通过使用两个基因的不同多态性等位基因进行等位基因替换和蛋白结构域交换实验来验证这一假设。我们最近的工作也表明，同种异体识别对趋化性有意想不到的影响。许多趋化性模型认为这一过程是细胞自主的，但我们的研究结果表明，一种合作效应需要相同同种异型细胞之间的相互作用。为了进一步研究这一革命性的发现，我们将测试同种异体识别对趋化性特定方面的影响，并确定哪些经过充分研究的趋化性模块被同种异体识别改变。我们还建议使用转录组分析和基因抑制来识别整合异体识别的信号转导基因。我们已经发现了几个候选基因，包括染色质重塑成分和蛋白激酶，它们可能是这个过程中的候选基因。我们将在异体识别和趋化性的背景下研究这些基因，并寻找其他基因。最后，我们建议测试同种异体识别在盘基骨柱聚集期后发育中的作用。我们发现tgrB1和tgrC1在茎前细胞中富集，因此我们建议使用条件表达和嵌合聚集体来测试这些基因在细胞类型分化和组织分离中的可能作用。

DESCRIPTION (provided by applicant): Allorecognition is the ability of organisms to distinguish self from non-self, a common theme in many organisms and a central component in the evolution of multicellularity. In mammals, allorecognition is mediated by the Major Histocompatibility Complex (MHC), which is part of the immune system. The MHC facilitates identification of parasites in infection processes, and graft rejection in transplantation medicine There is evidence for the involvement of other proteins in graft rejection but there are very few model systems available to study these proteins. There are several allorecognition systems in non-mammalian systems, notably in marine organisms, but most of them are not genetically tractable. In the previous grant period we found an allorecognition system in the social soil amoeba Dictyostelium discoideum. The system is based on two proteins, TgrB1 and TgrC1 that share many properties with mammalian MHC proteins, but not their amino acid sequences. We found these trans-membrane proteins to be highly polymorphic in natural Dictyostelium populations and necessary and sufficient for allorecognition. Dictyostelium cells live as free amoebae in the soil when food is abundant but they aggregate into multicellular organisms when starved. Aggregation involves chemotaxis to extracellular cAMP and Dictyostelium is one of the best model systems for the study of chemotaxis, which is a central process in embryogenesis and in innate immunity. When Dictyostelium cells encounter cells that carry incompatible TgrB1 and TgrC1 during aggregation, they segregate from one another and form separate multicellular organisms. We propose that TgrB1 on the surface of one cell binds TgrC1 on the surface of an adjacent cell and that binding initiates a signal transduction cascade that alters cell behavior. We intend to test this hypothesis by performing allele replacement and protein-domain swapping experiments using different polymorphic alleles of the two genes. Our recent work also showed that allorecognition has an unexpected effect on chemotaxis. Many models of chemotaxis consider the process to be cell-autonomous, but our findings suggest a cooperative effect that requires interactions between cells of the same allotype. To study this revolutionary finding further, we will test the effect of allorecognition on specific aspects of chemotaxis and identify which of the well-studied chemotaxis modules is altered by allorecognition. We also propose to use transcriptome analysis and genetic suppressors to identify signal-transduction genes that integrate allorecognition. We already found several candidate genes, including chromatin remodeling components and protein kinases that are likely candidates in the process. We will study these genes in the context of allorecognition and chemotaxis and search for additional ones as well. Lastly, we propose to test the role of allorecognition in Dictyostelium development past the aggregation stage. We found that the tgrB1 and tgrC1 are enriched in prestalk cells so we propose to use conditional expression and chimeric aggregates to test the possible role of these genes in cell-type differentiation and tissue segregation.