Figure S1: Structural details of model components. (A) nephrin, (B) Nck, (C) NWASP. Diameter of the sites are shown in brown below the corresponding site. Center-to-center distance between sites are shown in blue. All distance units are nm.

Dynamic molecular clusters are assembled through weak multivalent interactions and are platforms for cellular functions, especially receptor-mediated signaling. Clustering is also a prerequisite for liquidliquid phase separation. But it is not well understood how molecular structure and cellular organization control clustering. Using coarse-grain kinetic Langevin dynamics, we performed computational experiments on a prototypical ternary system modeled after membrane-bound nephrin, the adaptor Nck1 and the actin nucleation promoting factor NWASP. Steady state cluster size distributions favored stoichiometries that optimized binding (stoichiometry matching), but still were quite broad. At high concentrations, the system can be driven beyond the saturation boundary such that cluster size is limited only by the number of available molecules. This behavior would be predictive of phase separation. Domains close to binding sites sterically inhibited clustering much less than terminal domains because the latter effectively restrict access to the cluster interior. Increased flexibility of interacting molecules diminished clustering by shielding binding sites within compact conformations. Membrane association of nephrin increased the cluster size distribution in a density-dependent manner. These properties provide insights into how molecular ensembles function to localize and amplify cell signaling.

Extracellular signal-Regulated Kinase 2) and STAT3 (Signal Transducer and Activator of Transcription 3) was inhibited in same cell line. And then, in order to examine whether this DNA Nano-Foeces can modulate the internalization by EGFR activation in this cell, we monitored the internalization level of this protein after treatment of EGF in the DNA Nano-Forceps treated cell through both epi-fluorescence and TIRF (total internal reflection fluorescence microscopy) microscopy. Interestingly, the treatment of EGFR antibody conjugated DNA Nano-Forceps inhibited the internalization of EGFR even if EGF was treated. Based on these results, we conclude that EGFR antibody conjugated DNA Nano-Forceps can modulate EGFR and related signaling pathways through inhibition of dimerization by dysregulated lateral diffusion and internalization of EGFR simultaneously.