Activation of the Drosophila transmembrane receptor protein Notch is induced by association with its transmembrane ligands, Delta and Serrate. The ability to assay binding between Notch and its ligands has been essential for characterizing the influence of posttranslational modifications, such as glycosylation, as well as for characterizing structural motifs involved in receptor-ligand interactions. We describe here a simple, widely used method for assaying receptor-ligand binding. This method involves expression of soluble forms of either Notch or its ligands, comprising the extracellular domains fused to an easily assayed tag, the enzyme alkaline phosphatase. These soluble proteins are then incubated with their binding partners, either as transmembrane proteins expressed on the surface of cultured cells or as extracellular protein domains attached to agarose beads. After washing, the amount of bound protein can be readily assayed by measuring alkaline phosphatase activity.
Regeneration is a process by which organisms replace damaged or amputated organs to restore normal body parts. Regeneration of many tissues or organs requires proliferation of stem cells or stem cell-like blastema cells. This regenerative growth is often initiated by cell death pathways induced by damage. The executors of regenerative growth are a group of growth-promoting signaling pathways, including JAK/STAT, EGFR, Hippo/YAP, and Wnt/β-catenin. These pathways are also essential to developmental growth, but in regeneration, they are activated in distinct ways and often at higher strengths, under the regulation by certain stress-responsive signaling pathways, including JNK signaling. Growth suppressors are important in termination of regeneration to prevent unlimited growth and also contribute to the loss of regenerative capacity in nonregenerative organs. Here, we review cellular and molecular growth regulation mechanisms induced by organ damage in several models with different regenerative capacities.