Fig. 5
From: The LRR receptor Islr2 is required for retinal axon routing at the vertebrate optic chiasm
Vasna and Vasnb specifically and directly bind Islr2 with low affinity. a. Identification of the Vasnb-Islr2 interaction using the AVEXIS assay. Vasnb interacted with Islr2 and no other protein when presented either as bait against 194 prey ectodomains (upper panel) or as prey against 149 baits (lower panel). Statistical threshold (dashed lines) is calculated as 3 standard deviations over the mean. b. Quantification of the interaction between Vasna and Islr2 using surface plasmon resonance. Upper panel: Serial dilutions of purified monomeric Islr2-Cd4d3+4-6xHis were sequentially injected until binding equilibrium was reached over flow cells in which biotinylated Vasna ectodomains were immobilized on a streptavidin-coated sensor chip at high (solid line) and low (dashed line) levels. An equilibrium binding constant (K D) of 12.4 μM was calculated by non-linear curve fitting to both binding curves. Lower panel: kinetic analysis of the Vasna-Islr2 interaction. A dissociation rate constant (k off) was calculated by globally fitting a first order decay curve to the dissociation phase of three concentrations (7 μM, 14 μM, 28 μM) of Islr2-Cd4d3+4-6xHis; the interaction half-life (t1/2) was calculated as t1/2 = ln2/k off. Each data point represents average values from normalized dissociation data (error bars = ± 1 standard deviation, n = 3). Control dissociation measurements (dashed line) are from 28 μM Islr2-Cd4d3+4-6xHis injected over a flow cell containing the Cd4d3+4 tag alone. c. AVEXIS screening to identify the extracellular domains mediating the interaction between Vasna and Islr2. We observed nitrocefin hydrolysis whenever the EGF domain of Vasna could bind to the LRR domain of Islr2. Conversely, the truncated LRR domain encoded by islr2 sa82 did not show activity, indicating that the zebrafish mutant allele is functionally null. d. Schematic model for the interaction between Vasorin and Islr2, where the proteins bind in trans, a property suggested by the non-overlapping expression patterns of the two genes