Using hierarchical clustering and dendrograms to quantify the clustering of membrane proteins.

Cell biologists have developed methods to label membrane proteins with gold nanoparticles and then extract spatial point patterns of the gold particles from transmission electron microscopy images using image processing software. Previously, the resulting patterns were analyzed using the Hopkins statistic, which distinguishes nonclustered from modestly and highly clustered distributions, but is not designed to quantify the number or sizes of the clusters. Clusters were defined by the partitional clustering approach which required the choice of a distance. Two points from a pattern were put in the same cluster if they were closer than this distance. In this study, we present a new methodology based on hierarchical clustering to quantify clustering. An intrinsic distance is computed, which is the distance that produces the maximum number of clusters in the biological data, eliminating the need to choose a distance. To quantify the extent of clustering, we compare the clustering distance between the experimental data being analyzed with that from simulated random data. Results are then expressed as a dimensionless number, the clustering ratio that facilitates the comparison of clustering between experiments. Replacing the chosen cluster distance by the intrinsic clustering distance emphasizes densely packed clusters that are likely more important to downstream signaling events.We test our new clustering analysis approach against electron microscopy images from an experiment in which mast cells were exposed for 1 or 2 minutes to increasing concentrations of antigen that crosslink IgE bound to its high affinity receptor, FcϵRI, then fixed and the FcϵRI β subunit labeled with 5 nm gold particles. The clustering ratio analysis confirms the increase in clustering with increasing antigen dose predicted from visual analysis and from the Hopkins statistic. Access to a robust and sensitive tool to both observe and quantify clustering is a key step toward understanding the detailed fine scale structure of the membrane, and ultimately to determining the role of spatial organization in the regulation of transmembrane signaling.