Figure 1.
Protein microarrays identify Netrin-1 and CBLN4 as specific DCC binders.
The intersection plots show data from two replicate screens (Array 1 and Array 2) of DCC-Fc against two extracellular protein-microarray libraries, representing 686 genes in Library 1 (A) and 562 genes in Library 2 (B). Each point represents a protein sample and the oval represents the cutoff for selected hits. CBLN4-Fc, CBLN4-His, Netrin-1-His and gD-Netrin-1 proteins were the top scoring hits in each library.
Figure 2.
CBLN4 binds specifically with DCC on cells.
A) Representative images of CBLN4-AP protein binding to COS-7 cells transfected with DCC, but not NgR expression constructs (top panels). Nogo 66-AP protein bound to NgR but not to DCC transfected cells (middle panels). AP did not interact with either DCC or NgR (lower panels). B) Flow cytometric analysis confirmed cell-surface expression of transiently transfected Flag-tagged DCC on HEK239T cells (top panels). The CBLN4-Fc and gD-Netrin-1 proteins bound to DCC expressing cells as demonstrated by the shift in fluorescent intensity compared to untransfected cells (bottom panels).
Figure 3.
CBLN family binding and cell-based DCC interaction affinity measurements.
A) Sensograms are shown from SPR analysis of immobilized DCC-Fc (left; 15,082 response units) and negative control Robo3-Fc (right; 9,833 response units) with CBLN1, CBLN2, CBLN3 and CBLN4 injected as analytes at 10 µg/ml. Robust binding was detected to CBLN4 (black trace), while no binding signal was detected for the other Cerebellin family members (gray traces). B) A radio-lignad assay was used to determine the affinity of CBLN4-Fc (left) and gD-Netrin-1 (right) to DCC transiently transfected into HEK293T cells. 125I-Labeled ligand was allowed to bind transfected cells in the presence of increasing amounts of unlabeled ligand. Calculated KD values are indicated on the graphs and are representative of 3 independent experiments.
Figure 4.
Characterizing CBLN4 binding partners.
A) quantification of CBLN4-Fc binding to COS-7 cells transfected with the indicated axon guidance proteins. Data represent three independent replicate plates, each containing triplicate wells and error bars represent standard error of the mean. B) Flag-DCC and Flag-NEO1 were transiently expressed at the surface of COS-7 cell as detected by anti-Flag staining (top panel). The lower panel shows a titration series of CBLN4-Fc binding to DCC or NEO1. Triplicate independent experiments were performed and error bars represent the standard error of the mean.
Figure 5.
CBLN4 binds to the FN4-6 region of DCC.
A) a schematic representation of DCC is shown with the Ig domains in black and the FN domains in green. Representative fluorescent images are shown for CBLN4-Fc binding to COS-7 cells expressing full-length DCC, DCC Ig1-4, DCC FN1-6, DCC FN4-6, and negative control APP. Detection of CBLN4-Fc binding was through anti-human Alexa Fluor-488 (green). Detection of cell surface expression of all flag-tagged DCC deletion mutants was confirmed with an Alex Fluor-647 labeled anti-mouse IgG (red). B) quantification of CBLN4-Fc binding to the DCC domain deletion constructs. Triplicate independent experiments were performed and error bars represent the standard error of the mean.
Figure 6.
Netrin-1 competes CBLN4 binding to DCC.
A) quantification of the amount of 125I-radiolabeled CBLN4-Fc or gD-Netrin-1 bound to DCC expressing HEK293T cells, or untransfected control cells, preincubated with unlabeled gD-Netrin-1 or CBLN4-Fc, respectively. Data from four independent experiments are shown. Each bar represents 3 technical replicates expressed as means ± SD. One-tailed Student’s t test were applied: *, P<0.05; **, P<0.01; ***, P<0.001. B) displacement plot, representative from triplicate independent experiments, is shown for gD-Netrin-1 competing with binding of 125I-radiolabeled CBLN4-Fc to DCC expressing HEK293T cells.
Figure 7.
Distinct expression pattern of CBLN4 during embryonic development and subtle effects on axon pathfinding in CBLN4−/− mice.
A) Lac-Z reporter expression of CBLN4 in E11.5 CBLN4+/− mice (top panels) showing distinctive expression in limbs including dorsal limb expression. Lac-Z reporter staining of CBLN4 in E13.5 CBLN4+/− mice (lower panels) showing expression in the spinal cord motor column. B) commissural axons of E11.5 spinal cord visualized with TAG-1 immunostaining in combinations of CBLN4 and Netrin-1 genotypes. C) Representative images and quantification of wandering axons (white arrows) in the brachial plexus of E11.5 CBLN4−/− mice. Tukey contrasts; **, P<0.01; ***, P<0.001; n is left and right forelimb nested in mouse; n = 4 CBLN4+/+; n = 5 CBLN4+/−; n = 6 CBLN4−/−.