Abstract
Quantum dots (QDs) are extremely bright, photostable, nanometer particles broadly used to investigate single molecule dynamics in vitro. However, the use of QDs in vivo to investigate single molecule dynamics is impaired by the absence of an efficient way to chemically deliver them into the cytosol of cells. Indeed, current methods (using cell-penetrating peptides for instance) provide very low yields: QDs stay at the plasma membrane or are trapped in endosomes. Here, we introduce a technology based on cell-penetrating poly(disulfide)s that solves this problem: we deliver about 70 QDs per cell, and 90% appear to freely diffuse in the cytosol. Furthermore, these QDs can be functionalized, carrying GFP or anti-GFP nanobodies for instance. Our technology thus paves the way toward single molecule imaging in cells and living animals, allowing to probe biophysical properties of the cytosol.
Highlights
U nderstanding how tissue and cellular behavior emerges from single molecule dynamics is a major endeavor of modern cell biology
This faces a unique challenge because single molecule dynamics occurs at very short time scales, whereas cell and tissue dynamics occur at time scales of minutes or even hours
Though numerous studies have elegantly used quantum dots (QDs) to investigate transmembrane protein dynamics at the plasma membrane,[2,3] their use to investigate single molecule dynamics in the cytoplasm has suffered from the lack of an efficient cytosolic delivery technique
Summary
CPDs in the presence of excess biotin (Figure S12) did efficiently enter into cells, but rather remained trapped in endosomal compartments This suggests that cytosolic delivery requires specific interactions with CPDs, at least for streptavidin-coated QDs. We checked that QDs were delivered into the cytosol using three independent methods (see below): (i) delivered QDs have a diffusive motion with a diffusion coefficient too high for endosomes (mean square displacement (MSD) analysis), (ii) QDs have low colocalization with endosomal markers and (iii) most QDs are found beyond the plasma membrane imaged by TIRFM (total internal reflection fluorescence microscopy).
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