A systematic High-Content Screening microscopy approach reveals key roles for Rab33b, OATL1 and Myo6 in nanoparticle trafficking in HeLa cells.

Angela Panarella,Mariana G Bexiga,Kenneth A Dawson,George Galea,Jeremy C Simpson,Anna Salvati,Elaine D O’ Neill

doi:10.1038/srep28865

Abstract

Synthetic nanoparticles are promising tools for imaging and drug delivery; however the molecular details of cellular internalization and trafficking await full characterization. Current knowledge suggests that following endocytosis most nanoparticles pass from endosomes to lysosomes. In order to design effective drug delivery strategies that can use the endocytic pathway, or by-pass lysosomal accumulation, a comprehensive understanding of nanoparticle uptake and trafficking mechanisms is therefore fundamental. Here we describe and apply an RNA interference-based high-content screening microscopy strategy to assess the intracellular trafficking of fluorescently-labeled polystyrene nanoparticles in HeLa cells. We screened a total of 408 genes involved in cytoskeleton and membrane function, revealing roles for myosin VI, Rab33b and OATL1 in this process. This work provides the first systematic large-scale quantitative assessment of the proteins responsible for nanoparticle trafficking in cells, paving the way for subsequent genome-wide studies.

Highlights

Chemical inhibitors have traditionally been used to elucidate the molecular mechanism by which NPs enter and traffic through cells[8], but such tools are crude, providing relatively little specificity in terms of identifying the key molecules involved[9]
The ability to label them with fluorescent reporters means that they are an ideal model for imaging applications[21], potentially providing a first level of information about how NPs interact with cells
In order to demonstrate the suitability of our approach we tested the involvement of clathrin-mediated endocytosis machinery on NP uptake, as this pathway has been proposed as being significant for polystyrene NP uptake into cells[24]

Summary

Introduction

Chemical inhibitors have traditionally been used to elucidate the molecular mechanism by which NPs enter and traffic through cells[8], but such tools are crude, providing relatively little specificity in terms of identifying the key molecules involved[9]. The combination of RNAi with automated high-content screening (HCS) microscopy potentially allows for the molecular dissection of cellular events with spatial and temporal resolution from single cells and across a population[12], representing an unbiased approach for the discovery of new players in fundamental intracellular processes[13]. A small number of studies utilizing HCS microscopy to relate membrane trafficking to the efficiency of intracellular drug delivery have been reported, these were either based on the use of libraries of chemical inhibitors[14] or on a small number of individually selected siRNAs15. We describe and apply an RNAi-HCS approach utilizing two siRNA libraries to deplete families of proteins associated with either the cytoskeleton or the endomembrane system, in order to provide the first gene-based systematic overview of the mechanism by which synthetic NP internalization and delivery to acidic cellular organelles occurs

Methods

Results

Conclusion