Clustered protocadherin cis-interactions are required for combinatorial cell-cell recognition underlying neuronal self-avoidance

Abstract

In the developing human brain, only 53 stochastically expressed clustered protocadherin (cPcdh) isoforms enable neurites from individual neurons to recognize and self-avoid while simultaneously maintaining contact with neurites from other neurons. Cell assays have demonstrated that self-recognition occurs only when all cPcdh isoforms perfectly match across the cell boundary, with a single mismatch in the cPcdh expression profile interfering with recognition. It remains unclear, however, how a single mismatched isoform between neighboring cells is sufficient to block erroneous recognitions. Using systematic cell aggregation experiments, we show that abolishing cPcdh interactions on the same membrane (cis) results in a complete loss of specific combinatorial binding between cells (trans). Our computer simulations demonstrate that the organization of cPcdh in linear array oligomers, composed of cis and trans interactions, enhances self-recognition by increasing the concentration and stability of cPcdh trans complexes between the homotypic membranes. Importantly, we show that the presence of mismatched isoforms between cells drastically diminishes the concentration and stability of the trans complexes. Overall, we provide an explanation for the role of the cPcdh assembly arrangements in neuronal self/non-self-discrimination underlying neuronal self-avoidance.

Keywords: adhesion; cadherins; cell–cell recognition specificity; clustered protocadherins; neuronal self-avoidance.

Fig. 1.

Abrogating cis interactions results in smaller aggregates. The figure depicts typical aggregates of cells transfected with a single cPcdh isoform, either WT or lacking the EC6 ectodomains are shown. The Bottom row displays the sizes of aggregates for each isoform, and the bar plot on the Right provides a summary of the overall aggregate size for each isoform. (Scale bar: 100 µm.)

Fig. 2.

Mismatch coaggregation assays demonstrate the essential role of cis interactions in cell–cell combinatorial homophilic binding specificity. (A and B) Red and green cells coexpressing matched and mismatched WT cPcdhs did not bind to each other and formed separate homotypic aggregates (Top). In contrast, eliminating cis binding resulted in indiscriminate adhesion and mixed red and green coaggregates between cells expressing mismatched isoforms (Bottom). The mean mixing score values are shown in the Upper Right corner of each representative image (SI Appendix, Fig. S5). (C) Illustration of the cell aggregation experiment. (Scale bar: 100 µm.)

Fig. 3.

Computer simulations reveal how cis interactions and the zipper-like assemblies impact adhesion strength. (A) Illustrates the lattice simulations, designed as two stacked grids representing both membranes. Squares on the grid occupied by cPcdhs are colored based on membrane affiliation, with orange representing membrane A and blue representing membrane B. Trans dimers are indicated by black squares (i.e., zipper-like arrays are represented as black lines). (B) Simulation snapshots of the 2D lattice model when both cis and trans interactions are present (Left) and when cis interaction is absent (Right). Trans interactions can only occur in the diffusion trap area shown as a square in the middle and magnified. Only with cis interactions, 2D stacking of zipper-like structures are found at the diffusion trap area. (C) Boxplot of 60 independent simulations with or in absence of cis interactions demonstrates a reduction in trans dimers when cis interactions are abolished.

Fig. 4.

Computer simulations reveal the role of cis interactions and the zipper-like assemblies in adhesion combinatorial specificity. Simulation snapshots of the 2D lattice model for WT (A) and ΔEC6 (B) cPcdhs. Colors represent cPcdhs membrane origin, with orange squares representing cPcdhs from the Top and blue from the Bottom membranes. Black squares represent trans-dimers. The Top images represent the results of simulations with two isoforms (i and ii), and the Bottom images represent the results of simulations with three isoforms (iii and iv). Only the diffusion trap areas are shown. (A) The simulation of WT matching cPcdhs generated clusters of long zippers-like arrays and significantly more trans dimers than simulations of cells expressing a mismatched isoform. (B) Once cis interactions are eliminated, a mismatched isoform only slightly reduces the number of trans dimers compared to simulations of all matched isoforms. (C) Boxplots demonstrating the number of trans dimers observed from simulations of the different match-to-mismatch ratios with and without cis interactions (30 independent simulation runs for each condition).

Fig. 5.

cis interactions provide tolerance to a higher level of matching isoform expression. (A) Illustration of the varying ratios of matched to mismatched isoforms across five experimental conditions. Starting from the Top, two distinct cell populations (depicted in red and green) express two different isoforms, resulting in a 0% match. Next, both populations express two isoforms each, including one matching isoform. The proportion of the matching isoform relative to all isoforms increases gradually from 25 to 100%. (B) Summary of eleven simulation sets (50 repeats per simulation), modeling protein–protein interactions between cells with ratios of matching isoforms ranging from 0 to 100%. Without cis interactions, the extent of mixing is predicted to increase gradually as the concentration of the matching isoform rises (light gray). In contrast, with cis interactions, the curve resembles a step function, indicating a binary threshold for tolerating matching isoform presence (black). Below, cells transfected with increasing levels of a matching isoform were tested for coaggregation (see SI Appendix, Fig. S7 for more data).

Fig. 6.

A model of cPcdhs combinatorial cell–cell recognition. WT cPcdh isoforms generate strong adhesion in homotypic contacts by forming 2D zipper stacks (zippers are highlighted in different colors). In contrast, heterotypic contacts with mismatching isoforms produce shorter zippers that cannot stack into larger complexes. These differences in the number of trans complexes lead to different adhesion strengths and a preference for homotypic binding in WT cPcdhs (separate aggregates, Fig. 2). In neurons, this binding can trigger self-avoidance. In contrast, mutated cPcdh isoforms incapable of cis interactions cannot form zippers as they require both cis and trans interactions, resulting in weak and comparable adhesion in both homotypic and heterotypic contacts via trans dimers only (shown in gray) leading to no binding preference (mixed aggregates, Fig. 2).