Many organisms across the tree of life can distinguish between themselves and other members of their species via cell-cell contact. In vertebrates, this process controls graft rejection and auto-immune responses. In colonial invertebrates -- animals like marine sponges, corals, and sea squirts -- this ability is called allorecognition. Allorecognition controls whether colonies aggressively compete for space or peacefully coexist when they encounter one another as they grow. Despite the biological significance of this phenomenon, the genes and proteins that underlie it remain poorly understood. In this project, the investigators will study the molecular basis of allorecognition in a cnidarian, Hydractinia symbiolongicarpus. Recently, two genes controlling allorecognition in Hydractinia have been identified. The investigators will determine how the proteins encoded by these genes allow Hydractinia to properly distinguish self from non-self. To do this, the investigators will combine biochemical experiments with computer modeling and x-ray crystallography to build a structural model of the proteins, which will reveal how they enable Hydractinia to discriminate between self and non-self. This knowledge will fill a significant gap in our understanding of invertebrate allorecognition and form the basis of future work to investigate how these phenomena evolve. As part of their work, the investigators will train two undergraduates, two graduate students, and one postdoc in protein biochemistry and structure determination. They will provide opportunities for three high school students to conduct independent research projects in their laboratories. They will also develop and implement an educational program about Hydractinia for elementary school students in southwestern Pennsylvania.
In Hydractinia, two allorecognition proteins, Alr1 and Alr2, discriminate between self and non-self. Preliminary studies indicate these proteins engage in isoform-specific binding across cell membranes. This binding is hypothesized to be a general feature of both proteins and to be mediated by trans interactions between immunoglobulin superfamily-like (IgSF-like) domains in their extracellular regions and the formation of disulphide-linked cis multimers. Aim 1 will identify segments in the extracellular regions of each protein that control binding specificity and test whether the proteins form cis multimers via disulphide bonds. Aim 2 will identify amino acid positions within these segments that determine their binding profiles and use computational modeling to explore hypothesized binding mechanisms. Together, Aims 1 and 2 will be used to create a structural model of isoform-specific binding. Aim 3 will validate this model by determining the crystal structures of the extracellular domains of Alr1 and Alr2. Our team combines three PIs with expertise in invertebrate allorecognition, computational biology, and x-ray crystallography. The expected outcome of this project will be the first mechanistic understanding of how allorecognition proteins discriminate between self and non-self in any invertebrate. Comparison of this structural mechanism with those in other self-recognition systems will reveal which aspects of molecular recognition are unique to Hydractinia and which are shared with other species. The data generated in this project will guide future projects investigating how sequence evolution at allorecognition genes leads to novel allorecognition specificities and how signaling through the allorecognition proteins regulates allorecognition responses.
