High-Speed Imaging of Rab Family Small GTPases Reveals Rare Events in Nanoparticle Trafficking in Living Cells

Despite the increased application of nanomaterials in diagnostics and therapeutics, methods to study the interactions of nanoparticles with subcellular structures in living cells remain relatively undeveloped. Here we describe a robust and quantitative method that allows for the precise tracking of all cell-associated nanoparticles as they pass through endocytic compartments in a living cell. Using rapid multicolor 3D live cell confocal fluorescence microscopy, combined with transient overexpression of small GTPases marking various endocytic membranes, our studies reveal the kinetics of nanoparticle trafficking through early endosomes to late endosomes and lysosomes. We show that, following internalization, 40 nm polystyrene nanoparticles first pass through an early endosome intermediate decorated with Rab5, but that these nanoparticles rapidly transfer to late endosomes and ultimately lysosomes labeled with Rab9 and Rab7, respectively. Larger nanoparticles of 100 nm diameter also reach acidic Rab9- and Rab7-positive compartments although at a slower rate compared to the smaller 40 nm nanoparticles. Our work also reveals that relatively few nanoparticles are able to access endocytic recycling pathways, as judged by lack of significant colocalization with Rab11. Finally, we demonstrate that this quantitative approach is sufficiently sensitive to be able to detect rare events in nanoparticle trafficking, specifically the presence of nanoparticles in Rab1A-labeled structures, thereby revealing the wide range of intracellular interactions between nanoparticles and the intracellular environment

High-Speed Imaging of Rab Family Small GTPases Reveals Rare Events in Nanoparticle Trafficking in Living Cells

Despite the increased application of nanomaterials in diagnostics and therapeutics, methods to study the interactions of nanoparticles with subcellular structures in living cells remain relatively undeveloped. Here we describe a robust and quantitative method that allows for the precise tracking of all cell-associated nanoparticles as they pass through endocytic compartments in a living cell. Using rapid multicolor 3D live cell confocal fluorescence microscopy, combined with transient overexpression of small GTPases marking various endocytic membranes, our studies reveal the kinetics of nanoparticle trafficking through early endosomes to late endosomes and lysosomes. We show that, following internalization, 40 nm polystyrene nanoparticles first pass through an early endosome intermediate decorated with Rab5, but that these nanoparticles rapidly transfer to late endosomes and ultimately lysosomes labeled with Rab9 and Rab7, respectively. Larger nanoparticles of 100 nm diameter also reach acidic Rab9- and Rab7-positive compartments although at a slower rate compared to the smaller 40 nm nanoparticles. Our work also reveals that relatively few nanoparticles are able to access endocytic recycling pathways, as judged by lack of significant colocalization with Rab11. Finally, we demonstrate that this quantitative approach is sufficiently sensitive to be able to detect rare events in nanoparticle trafficking, specifically the presence of nanoparticles in Rab1A-labeled structures, thereby revealing the wide range of intracellular interactions between nanoparticles and the intracellular environment

DataSheet1_An imaging-based RNA interference screen for modulators of the Rab6-mediated Golgi-to-ER pathway in mammalian cells.docx

In mammalian cells, membrane traffic pathways play a critical role in connecting the various compartments of the endomembrane system. Each of these pathways is highly regulated, requiring specific machinery to ensure their fidelity. In the early secretory pathway, transport between the endoplasmic reticulum (ER) and Golgi apparatus is largely regulated via cytoplasmic coat protein complexes that play a role in identifying cargo and forming the transport carriers. The secretory pathway is counterbalanced by the retrograde pathway, which is essential for the recycling of molecules from the Golgi back to the ER. It is believed that there are at least two mechanisms to achieve this - one using the cytoplasmic COPI coat complex, and another, poorly characterised pathway, regulated by the small GTPase Rab6. In this work, we describe a systematic RNA interference screen targeting proteins associated with membrane fusion, in order to identify the machinery responsible for the fusion of Golgi-derived Rab6 carriers at the ER. We not only assess the delivery of Rab6 to the ER, but also one of its cargo molecules, the Shiga-like toxin B-chain. These screens reveal that three proteins, VAMP4, STX5, and SCFD1/SLY1, are all important for the fusion of Rab6 carriers at the ER. Live cell imaging experiments also show that the depletion of SCFD1/SLY1 prevents the membrane fusion event, suggesting that this molecule is an essential regulator of this pathway.</p

Time-dependent Cell Response Profile (TCRP) of infected macrophage monolayers.

<p>Monolayers were infected with a virulent (<i>R. equi</i> 103+; top panel) or with an avirulent (<i>R. equi</i> 103−; bottom panel) strain of <i>R. equi</i> at increasing multiplicities (MOI) of infection. The TCRP was normalized against the TCRP of a non-infected macrophage monolayer.</p

Intracellular growth of virulent and attenuated <i>R. equi</i>.

<p>J774A.1 monolayers were infected with virulent (♦, <i>R. equi</i> 103+; ∇, <i>R. equi</i> Δ35000) and attenuated (□, <i>R. equi</i> 103−; •, <i>R. equi</i> Δ<i>vapA</i>) <i>R. equi</i> strains. Following a 1 hour incubation to allow phagocytosis, monolayers were washed and treated with vancomycin to kill remaining extra cellular bacteria. Intracellular bacteria were enumerated via real time qPCR of the <i>R. equi</i> 16S rRNA gene. Results are expressed as fold change in bacterial numbers relative to t = 0 (h) values. Error bars denote the standard deviation of the mean.</p

Macrophages in infected monolayers are not homogeneous with respect to the number of intracellular <i>R. equi</i> they harbour.

<p>Macrophage monolayers were infected with ATTO 488 labeled virulent (<i>R. equi</i> 103+; top panel) or attenuated (<i>R. equi</i> 103−; bottom panel) strains of <i>R. equi</i> at increasing multiplicities (MOI) of infection. Monolayers were fixed and labelled 24 h post-infection. High content image analysis using Columbus software was employed to enumerate infected macrophages and to determine the number of intracellular <i>R. equi</i>. Shown are the number of macrophages that contained no intracellular bacteria (grey), between 1 and 5 bacteria (white) or more than 5 bacteria (black).</p

The morphology of macrophages changes following infection with <i>R. equi</i>.

<p>J774A.1 monolayers were infected with ATTO 488 labelled virulent <i>R. equi</i> 103+ (green). Monolayers were fixed 24 h post-infection; the actin cytoskeleton and cell nuclei were stained with Texas Red Phalloidin (red) and Hoechst 33258 (blue), respectively. Panel A: non-infected monolayers, panel B: monolayers infected with <i>R. equi</i> 103+. Arrows indicate the change in cell shape following infection with <i>R. equi</i>. The length of the white bar is 40 µm.</p

Bacterial strains, plasmids and oligonucleotides used in this study.

<p>Bacterial strains, plasmids and oligonucleotides used in this study.</p

Infection of macrophages with <i>R. equi</i> affects host cell size and morphology.

<p>Macrophage monolayers were infected with ATTO 488 labeled virulent <i>R. equi</i> 103+ or attenuated <i>R. equi</i> 103− strains at increasing multiplicities (MOI) of infection. Monolayers were fixed and labelled 24 h post-infection. The cell size (panel A) and cell roundness (panel B) of infected cells were compared to control cells (not infected, indicated as MOI = 0). Green bars: no intracellular bacteria; blue bars: 1 to 5 intracellular bacteria; red bars: more than 5 intracellular bacteria. +: <i>R. equi</i> 103+; −: <i>R. equi</i> 103−. Error bars denote the standard deviation of the mean.</p

Key TCRP parameters of macrophage monolayers infected with virulent or attenuated <i>R. equi</i> strains.

<p>Shown are the maximum cell indices (Ci) values (panel A) and the the C_i inflection times (panel B) following infection of J774A.1 macrophage monolayers with virulent (♦, <i>R. equi</i> 103+; ∇, <i>R. equi</i> Δ35000) and attenuated (□, <i>R. equi</i> 103−; •, <i>R. equi</i> Δ<i>vapA</i>) <i>R. equi</i> strains at increasing multiplicity of infection. Values are the means ± SD.</p