A high-content screening microscopy approach to dissect the role of Rab proteins in Golgi-to-ER retrograde trafficking.

Here, we describe a high-content microscopy-based screen that allowed us to systematically assess and rank proteins involved in Golgi-to-endoplasmic reticulum (ER) retrograde transport in mammalian cells. Using a cell line stably expressing a GFP-tagged Golgi enzyme, we used brefeldin A treatment to stimulate the production of Golgi-to-ER carriers and then quantitatively analysed populations of cells for changes in this trafficking event. Systematic RNA interference (RNAi)-based depletion of 58 Rab GTPase proteins and 12 Rab accessory proteins of the PRAF, YIPF and YIF protein families revealed that nine of these were strong regulators. In addition to demonstrating roles for Rab1a, Rab1b, Rab2a, and Rab6a or Rab6a' in this transport step, we also identified Rab10 and Rab11a as playing a role and being physically present on a proportion of the Golgi-to-ER tubular intermediates. Combinatorial depletions of Rab proteins also revealed previously undescribed functional co-operation and physical co-occurrence between several Rab proteins. Our approach therefore provides a novel and robust strategy for a more complete investigation of the molecular components required to regulate Golgi-to-ER transport in mammalian cells.

Human diseases associated with form and function of the Golgi complex.

The Golgi complex lies at the heart of the secretory pathway and is responsible for modifying proteins and lipids, as well as sorting newly synthesized molecules to their correct destination. As a consequence of these important roles, any changes in its proteome can negatively affect its function and in turn lead to disease. Recently, a number of proteins have been identified, which when either depleted or mutated, result in diseases that affect various organ systems. Here we describe how these proteins have been linked to the Golgi complex, and specifically how they affect either the morphology, membrane traffic or glycosylation ability of this organelle.

Quantifying size-dependent interactions between fluorescently labeled polystyrene nanoparticles and mammalian cells.

BACKGROUND: Nanoparticles (NPs) are currently used in a wide variety of fields such as technology, medicine and industry. Due to the novelty of these applications and to ensure their success, a precise characterization of the interactions between NPs and cells is essential. FINDINGS: The current study explores the uptake of polystyrene NPs by 1321N1 human astrocytoma and A549 human lung carcinoma cell lines. In this work we show for the first time a comparison of the uptake rates of fluorescently labeled carboxylated polystyrene (PS) NPs of different sizes (20, 40 and 100 nm) in two different cell types, keeping the number of NPs per unit volume constant for all sizes. We propose a reliable methodology to control the dose of fluorescently labeled NPs, by counting individual NPs using automated particle detection from 3D confocal microscopy images. The possibility of detecting individual NPs also allowed us to calculate the size of each nanoparticle and compare the fluorescence of single NPs across different sizes, thereby providing a robust platform for normalization of NP internalization experiments as measured by flow cytometry. CONCLUSIONS: Our findings show that 40 nm NPs are internalized faster than 20 nm or 100 nm particles in both cell lines studied, suggesting that there is a privileged size gap in which the internalization of NPs is higher.

Genome-Wide siRNA Screening Using Forward Transfection: Identification of Modulators of Membrane Trafficking in Mammalian Cells.

RNA interference (RNAi) has become an essential tool for molecular and cellular biologists to dissect cell function. In recent years its application has been extended to genome-wide studies, enabling the systematic identification of new cell regulation mechanisms and drug targets. In this chapter, a protocol for a genome-wide RNAi screen coupled to high-content microscopy is presented. Specifically we describe key features of assay design, plate layout, and a protocol for forward transfection of small interfering RNAs (siRNAs) in a 384-well plate format. As an example of its application in identifying modulators of membrane trafficking, we also provide a protocol to measure the efficacy of intracellular delivery of the B subunit of Shiga-like toxin to the Golgi complex. Finally we show an automated image analysis routine that can be used to extract single cell data from the screen, thereby providing a quantitative ranking of how a large panel of siRNAs affects this biological process.

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

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.

RNAi-mediated inhibition of apoptosis fails to prevent cationic nanoparticle-induced cell death in cultured cells.

BACKGROUND: Nanoparticles are increasingly being considered as a novel and potent tool for drug delivery, and, therefore, concerns regarding the safety of their use in humans are pertinent. It has been shown that nanoparticles displaying unsaturated amines at their surface are toxic to cells, but the molecular and cellular mechanisms elicited in this response have yet to be described. AIMS: In this work we identify key proteins involved in the cytotoxicity of amine-modified polystyrene nanoparticles. We also demonstrate the suitability of RNAi to provide a molecular description of how nanoparticles and cells interact. MATERIALS & METHODS: We have used a focused RNAi strategy in 1321N1 cells to identify key proteins involved in the cytotoxicity induced by amine-modified polystyrene nanoparticles. RESULTS: We show that the apoptosome is central to the observed mechanism of toxicity and that, although the proapoptotic proteins BAX, BAK, BID, BIM and PUMA are critical modulators of the process, their cellular depletion is insufficient to protect cells from nanoparticle-induced cell death. CONCLUSION: We conclude that the apoptosome, together with proapoptotic proteins of the Bcl-2 family of proteins, is central to amine-modified polystyrene nanoparticle-induced cell death. We further demonstrate that RNAi is a powerful and suitable tool to study the effects of nanoparticles on cellular processes, in particular apoptosis.

Time resolved study of cell death mechanisms induced by amine-modified polystyrene nanoparticles.

Positively charged polymers and nanoparticles (NPs) can be toxic to cells in various systems. Using human astrocytoma cells, we have previously shown that 50 nm amine-modified polystyrene NPs damage mitochondria and induce cell death by apoptosis. Here we provide comprehensive details of the cellular events occurring after exposure to the NPs in a time-resolved manner. We demonstrate that the accumulation of NPs in lysosomes plays a central role in the observed cell death, leading to swelling of the lysosomes and release of cathepsins into the cytosol, which ultimately propagates the damage to the mitochondria with subsequent activation of apoptosis. This is accompanied and sustained by other events, such as increasing ROS levels and autophagy. Using various inhibitors, we also show the interplay between apoptosis and autophagy as a response to NP accumulation in lysosomes.

Sorafenib-mediated targeting of the AAA⁺ ATPase p97/VCP leads to disruption of the secretory pathway, endoplasmic reticulum stress, and hepatocellular cancer cell death.

The molecular mechanisms and cellular targets of sorafenib, a multikinase inhibitor used for the treatment of hepatocellular carcinoma (HCC), remain to be fully characterized. Recent studies have shown that sorafenib induces tumor cell death through the activation of endoplasmic reticulum stress signaling and/or autophagy in various cellular models. Using liver cancer-derived cell lines, we specifically show that the IRE1 and phosphorylated extracellular signal-regulated kinase arms of the unfolded protein response (UPR) become activated upon sorafenib treatment, whereas the ATF6 arm is inhibited. Our results also reveal that sorafenib treatment causes disruption to the secretory pathway, as witnessed by the fragmentation of the Golgi apparatus and the induction of autophagy. On the basis of these observations, we tested the relevance of the AAA⁺ ATPase p97/VCP as a potential functional target of sorafenib. Our results show that p97/VCP tyrosine phosphorylation is prevented upon sorafenib treatment, and that this can be correlated with enhanced membrane association. Moreover, we show that DBeQ, a recently discovered inhibitor of p97/VCP, enhances sorafenib-mediated toxicity in cultured cells. Our data show a novel mechanism for sorafenib-mediated cell death in HCC, which depends on the integrity of the secretory pathway; and we identify p97/VCP phosphorylation as a potential target for improved sorafenib treatment efficacy in patients.

Genome-wide RNAi screening identifies human proteins with a regulatory function in the early secretory pathway.

The secretory pathway in mammalian cells has evolved to facilitate the transfer of cargo molecules to internal and cell surface membranes. Use of automated microscopy-based genome-wide RNA interference screens in cultured human cells allowed us to identify 554 proteins influencing secretion. Cloning, fluorescent-tagging and subcellular localization analysis of 179 of these proteins revealed that more than two-thirds localize to either the cytoplasm or membranes of the secretory and endocytic pathways. The depletion of 143 of them resulted in perturbations in the organization of the COPII and/or COPI vesicular coat complexes of the early secretory pathway, or the morphology of the Golgi complex. Network analyses revealed a so far unappreciated link between early secretory pathway function, small GTP-binding protein regulation, actin cytoskeleton organization and EGF-receptor-mediated signalling. This work provides an important resource for an integrative understanding of global cellular organization and regulation of the secretory pathway in mammalian cells.

Cationic nanoparticles induce caspase 3-, 7- and 9-mediated cytotoxicity in a human astrocytoma cell line.

On a daily basis we are exposed to cationic nanoparticulates in many different ways. They are known to distribute to many organs of the body, and while some evidence suggests that these nanoparticles are toxic to cells, the mechanism of their toxicity is not clear. Here we apply a combination of biochemical and imaging techniques to study the mechanism by which amine-modified polystyrene nanoparticles induce cell death in a human brain astrocytoma cell line. Flow cytometry analysis of cells exposed to cationic nanoparticles revealed an increase in cell membrane permeability of the dyes YoPro-1 and propidium iodide, indicating onset of an apoptotic followed by a secondary necrotic response. Activation of caspases 3/7 and 9 and cleavage of poly(ADP-ribose) polymerase (PARP)-1 was also detected, providing clear molecular evidence of the apoptotic pathway induced by the nanoparticles. Transmission electron microscopy also revealed that these nanoparticles induce morphological changes in lysosomes and mitochondria, consistent with our observation of a rapid increase in the formation of reactive oxygen species in these cells. Together these results suggest that amine-modified polystyrene nanoparticles can mediate cell death through an apoptotic mechanism mediated by damage to the mitochondria.