Super Resolution Mapping of Adhesion Molecules in Confined Cellular Environments using Monomeric Streptavidin Ligands

Abstract

High resolution tracking of individual cell surface proteins is limited by several factors, including probe size that can impair protein trafficking and restrict their access to crowded cellular environments, ligand multivalency that induces protein cross-linking, and potential loss of function of recombinant proteins fused to large tags. To overcome these issues, we developed a labeling method that uses monomeric streptavidin (mStrav) conjugated to highly photo-robust organic Atto dyes. mStrav is a 3-nm-molecule, stable and easy to produce, that binds biotinylated proteins with high affinity (Lim et al., Biochemistry 2011). Atto-conjugated mStrav is then used as a probe to label cells co-transfected with a membrane molecule of interest carrying a 15 amino acid extra-cellular acceptor peptide (AP), and the biotin ligase BirA that covalently adds biotin to the tag during protein maturation. We used this technique to study membrane diffusion and nanoscale organization of the synaptic adhesion molecules neurexin-1β and neuroligin-1 in hippocampal neurons and β3-integrins in fibroblasts. We show that the monovalency of mStrav prevents protein cross-linking, allowing more accurate diffusion measurements. The small sizes of the AP tag (<1nm) and mStrav (∼3nm) do not perturb protein function and allow access to single molecule dynamics and high resolution mapping in confined regions such as synapses or cell-matrix contacts. We also used mStrav along with nanobodies (∼3nm) for dual-color single molecule tracking. Moreover, the binding of mStrav can be reversed with excess biotin, a property that can be used to study protein endocytosis and recycling. Finally, the photostability of Atto dyes conjugated to mStrav allows for long trajectory recordings and live STED imaging. Taken together, monomeric streptavidin is a simple and versatile tool that can be used to track an arbitrary membrane protein in living cells.