Quantitatively Mapping the Assembly Pattern of EpCAM on Cell Membranes with Peptide Probes

Epithelial cell adhesion molecule (EpCAM) is an important type I transmembrane protein that is overexpressed on the surfaces of most cancer cells and involved in various biological processes such as cell adhesion and cell signaling. Although it plays crucial roles in cell functions and tumorigenesis, questions concerning the detailed morphology, molecular stoichiometry and the assembly mechanisms of EpCAM on cell membranes have not been fully elucidated. Here we used direct stochastic optical reconstruction microscopy (dSTORM) and relied on fluorophore-conjugated peptides to quantitatively analyze the assembly pattern of EpCAM with single-molecule precision. EpCAM was found to organize heterogeneous clusters with different sizes, of which contains dif-ferent number of EpCAM molecules on MCF-7 cell membranes. Moreover, dual-color dSTORM imaging revealed a significant correlation between EpCAM and tetraspanin CD9, and part of EpCAM clusters could be disrupted by knockdown of CD9, which indicated that EpCAM ...