Figure 6. δ-Pcdhs possess differences in apparent adhesive affinity.

(A) Representative images of cell aggregates at select speeds. Pcdh7 cells possessed small aggregates even at 220 RPM while Pcdh1 and Pcdh17 cells dissociated. Scale bar, 100 μm. (B) Mean aggregate size at each speed. Pcdh1 and Pcdh17 were significantly different from Pcdh7 by ANOVA, p=1×10⁻¹⁵. Error bars indicate ±SEM. Results for each assay were determined from four independent electroporations. (C) Representative images of a mismatch coaggregation assay with Pcdh1+Pcdh7 cells. At higher speeds, Pcdh1 cells change from interfacing to segregating (middle column), while the other two populations remain intermixed. Scale bar, 100 μm. (D) Mean CoAg values of (C). Error bars indicate ±SEM, * indicates p≤0.05, ** indicates p≤0.01. Results for each assay were determined from three independent electroporations. (E) Monte Carlo simulations incorporating affinity and relative expression level capture most, but not all, mismatch assay results. We modeled the behavior of a given mismatch assay (e.g. row 1, Pcdh1+Pcdh7). The Y-axis represents the CoAg Index (simulated (solid black and red lines) and observed (thick dashed line with standard error represented by thin dashed lines). Solid lines represent simulations where the relative expression level of the two δ-Pcdhs has been varied (from 1:1 to 20:1). The X-axis represents increasing differences in apparent adhesive affinity (e.g. the left most point on the X-axis represents conditions where both δ-Pcdhs are of equal apparent adhesive affinity). In all three simulated coaggregation assays, the model predicted intermixing conditions (e.g. CoAg index above 0.2), but was not able to precisely model segregation or interfacing behaviors (compare right most graph in each row against the other two).

Figure 6—figure supplement 1. δ-Pcdhs possess differences in apparent adhesive affinity, which appears to be mediated by EC domains 1–4.

(A) Western blot showing similar surface level expression for Pcdh1 and Pcdh19 cells. A second, high-molecular weight band is frequently observed for Pcdh19. (B) Representative images of cell aggregates taken at various speeds. Scale bar, 100 µm. (C) Quantification of aggregate size shows Pcdh1 cells maintained larger aggregates than Pcdh19 cells at all speeds. Pcdh1 and Pcdh19 cell behaviors were significantly different by ANOVA, p=1×10⁻¹⁵. Error bars indicate ±SEM. Results for each assay were determined from four independent electroporations. (D–G) Cells expressing high, medium, and low levels of Pcdh7 (D) and Pcdh17 (F) were subject to increasing rotational speeds. Mean aggregate sizes for Pcdh7 (E) and Pcdh17 (G). Increased surface expression led to larger aggregates at each speed tested. Error bars indicate ±SEM. Results for each assay were determined from three independent electroporations. (H) MUSCLE protein alignments show low overall sequence identities for EC1-4. (I) Pcdh1^Δ1-4, Pcdh7^Δ1-4 and Pcdh17^Δ1-4 constructs fail to mediate adhesion. Cartoon illustrates EC1-4 deletion for Pcdh1 and Pcdh7. Pcdh17 construct has only two EC domains following deletion of EC1-4. Scale bar, 100 µm. (J) Western blot of biotinylated surface show proteins with deletions in EC1-4 are transported to the surface.

Figure 6—figure supplement 2. EC1-4 mediate adhesive interactions among δ-Pcdhs.

(A) Representative images of coaggregation assay with Pcdh1+Pcdh7^Δ1-4 cells.

Pcdh1 cells now intermix with this population while Pcdh7 cells segregate. Scale bar, 100 µm. (B) Mean CoAg values, error bars indicate ±SEM, * indicates p≤0.05, ** indicates p≤0.01. Results for each assay were determined from three independent electroporations. (C) Representative images of coaggregation assay where EC1-4 of Pcdh7 has been swapped with that from Pcdh1. Results are best interpreted by comparing images within individual columns. Columns 3 and 4 shows the swap construct Pcdh7^EC1-4:Pcdh1 enables these cells to now intermix with Pcdh1 cells, and cause Pcdh7 cells to now segregate. Scale bar, 100 µm. (D) Mean CoAg values. Values from the top half of each bar should be compared against those in the bottom half to visualize differences in coaggregation behavior. Error bars indicate ±SEM, * indicates p≤0.05, ** indicates p≤0.01. Results for each assay were determined from three independent electroporations.