Monitoring Submicron and Micron-Size Membrane Compartments using Quantum Dots Monovalently Conjugated to Tracer Molecules

Abstract

The current paper describes the application of highly-photostable quantum dot (QD)-conjugated lipids and membrane proteins to explore membrane compartmentalization in model and plasma membranes over a wide range of length and time scales. A rigorous screening protocol is described that assures the bioinertness of QD coatings and the monovalent binding of QDs to tracer molecules. Here, the quality of several bioinert surface coatings is tested by determining their impact on the colloidal stability of CdSe/ZnS QDs in aqueous solution using confocal fluorescence correlation spectroscopy. The monovalent binding of QDs to tracer molecules is verified using a sensitive single molecule tracking assay, which is based on QD-conjugated lipids in a solid-supported lipid bilayer. Three different examples are discussed, in which QD-tracking probes are successfully employed to elucidate the membrane organization in model and plasma membranes. Tracking experiments on compartmentalized polymer-tethered lipid bilayers illustrate that, unlike organic fluorescence dyes, photostable QD-based membrane probes are well-suited to detect micron-size compartments with partially permeable diffusion barriers. Results from wide-field single molecule fluorescence microscopy experiments with frame rates of up to 1000 fps on several cell lines show that QD-conjugated tracer molecules are well-suited for fluorescence-based, long-term, high-speed tracking experiments in plasma membranes. Finally, we discuss changes in membrane compartmentalization induced by insulin as shown through tracking results from QD-conjugated transferrin receptors in healthy and insulin-resistant adipocytes and the impact of chromium picolinate on receptor mobility.