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An imaging flow cytometry method to assess ricin trafficking in A549 human lung epithelial cells
Methods xxx (2017) xxx–xxx
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An imaging flow cytometry method to assess ricin trafficking in A549 human lung epithelial cells Dominic Jenner a,⇑, Damien Chong b, Nicola Walker a, A. Christopher Green a a b
Defence Science and Technology Laboratory, CBR Division, Porton Down, Salisbury SP4 0JQ, United Kingdom Chemical & Toxin Medical Countermeasures, Land Division, Defence Science and Technology Group, 506 Lorimer St, Fishermans Bend, Victoria 3207, Australia
a r t i c l e
i n f o
Article history: Received 29 August 2017 Received in revised form 12 October 2017 Accepted 30 October 2017 Available online xxxx Keywords: Ricin Imaging flow cytometry ImageStream Golgi Toxin
a b s t r a c t The endocytosis and trafficking of ricin in mammalian cells is an important area of research for those producing ricin anti-toxins and other ricin therapeutics. Ricin trafficking is usually observed by fluorescence microscopy techniques. This gives good resolution and leads to a detailed understanding of the internal movement of ricin within cells. However, microscopy techniques are often hampered by complex analysis and quantification techniques, and the inability to look at ricin trafficking in large populations of cells. In these studies we have directly labelled ricin and assessed if its trafficking can be observed using Imaging Flow Cytometry (IFC) both to the cytoplasmic region of cells and specifically to the Golgi apparatus. Using IDEASÒ data analysis software the specific fluorescence location of the ricin within the cells was analysed. Then, using cytoplasmic masking techniques to quantify the number of cells with endocytosed cytoplasmic ricin or cells with Golgi-associated ricin, kinetic endocytosis curves were generated. Here we present, to the authors’ knowledge, the first example of using imaging flow cytometry for evaluating the subcellular transport of protein cargo, using the trafficking of ricin toxin in lung cells as a model. Ó 2017 Published by Elsevier Inc.
1. Introduction Ricin is a potent 66-kDa Type II ribosome inactivating protein produced by the seeds of the castor oil plant, Ricinus communis. Approximately one million tons of castor beans are processed annually in the production of castor oil worldwide leaving a ricin-rich pulp waste. Due to the ease of its extraction from castor seeds, its potentially lethal effects, ricin is recognised as a biological threat agent and is a Category B Agent on the Centres for Disease Control Select Agent List [1]. Ricin is a heterodimeric protein toxin comprising of A and B chains coupled by a disulphide linkage. The 34-kDa B chain is a lectin that facilitates toxin binding to terminal galactose residues on the target cell surface enabling ricin uptake by the cell. The holotoxin is internalised by endocytosis of which, a proportion of the ricin is transported via the Golgi apparatus to the endoplasmic reticulum, where the A and B chains are separated. The 32-kDa A chain is subsequently retrotranslocated into the cytosol and folded into a catalytically active RNA N-glycosidase. This active form of the ricin A chain then ⇑ Corresponding author at: Dstl Porton Down, CBR Division, Microbiology Bldg 7 Rm 201, Salisbury, Wiltshire SP1 3JQ, United Kingdom. E-mail address: [email protected] (D. Jenner).
depurinates base A4324 of 28S ribosomal RNA, thereby inhibiting protein synthesis and ultimately causing cell death [2,3]. The 50% lethal dose (LD50) depends on the route of exposure. In mice, the LD50 is less than 5 mg/kg ricin when administered intravenously, 4–6 mg/min/kg via inhalation and up to 30 mg/ kg via ingestion. In humans, the LD50 of ricin by ingestion is estimated to be 1–20 mg/kg [4–6]. Symptoms after ingestion may include fever, dilation of pupils, diarrhoea, nausea, vomiting, and abdominal pain and may progress to organ failure and hypotension. Inhalation of aerosolised ricin can result in dyspnoea, fever, cough, nausea, arthralgia, tightness in the chest and may progress to cyanosis with manifestations of heavy sweating and pulmonary oedema. Death may occur 24–72 h after exposure as a consequence of high permeability pulmonary oedema, acute respiratory distress syndrome or respiratory failure, either with or without hypotension and organ failure [5,7–14]. If death does not occur within 2–3 days after exposure, the patient will normally make a slow recovery, as there are no ricin treatments currently available other than palliative care. Understanding the mechanisms by which such toxins are transported through cells helps to determine how they may be exploited with novel inhibitors and how therapeutics can be used to disrupt their cytotoxicity [15,16].
https://doi.org/10.1016/j.ymeth.2017.10.012 1046-2023/Ó 2017 Published by Elsevier Inc.
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Conventionally, the mechanisms by which cells internalise proteins were often visualised by electron microscopy or measured using density gradient subcellular fractionation [17,18]. Although these approaches are still used, the development of fluorescence based technologies coupled with less-toxic fluorescent probes and user-friendly image analysis software means that measurement of a protein trafficking within cells is easily achievable. In the context of this paper the protein of interest is ricin, transport of which can be readily observed in relation to cellular sub-structure by quantitative multi-channel fluorescence microscopy using various reporter probes [15,16,19]. Although cutting edge fluorescence microscopy excels in image resolution and clarity, even when equipped with a motorised/ programmable stage one of its fundamental limitations is the time required to image cells and more importantly, to acquire data from sufficient numbers of cells to obtain a representative sample of the experiment. That is, it lacks the high throughput nature of other fluorescence technologies, such as flow cytometry. However, flow cytometry itself lacks the spatial data content provided by microscopy and only returns fluorescence intensity data for each particle interrogated. Imaging Flow Cytometry (IFC) is a cytometry technique that combines the high throughput nature of flow cytometry with the imaging and spatial capabilities of fluorescence microscopy [20]. IFC enables the capture of an optical slice image of individual cells in a flow in both brightfield and fluorescence channels, allowing for statistically robust, quantitative, image analysis in large populations of cells [20]. IFC is maturing quickly in the field of immunology, where the technology has been utilised to study many different aspects including, but not limited to, cell signalling [21], nuclear translocation [22,23] and host-pathogen interactions [24,25]. However, the study of how biological toxins interact with their target cells seems to be an as-yet unexplored application of IFC. Therefore, to demonstrate its utility, we have used IFC and Alexa FluorÒ 647 (AF647)-labelled ricin to show that it is possible to use this technology to generate kinetic data describing the uptake and endocytosis of ricin into the cytoplasm of human lung endothelial like A549 cells and we also demonstrate it is possible to visualise co-localisation of ricin with the Golgi apparatus of these cells. This methodology moves the analysis of toxin endocytosis away from making quantitative single cell measurements by confocal microscopy [15,16,19], and starts looking at toxin endocytosis on a cell population level with 1000s of cells, thus increasing the speed of data acquisition and analysis, along with the robustness that comes with looking at a population level.
2.2. Ricin production, fluorescent labelling and sodium dodecyl sulphate polyacrylamide gel electrophoresis of Ricin-AF647 Ricin was extracted and purified from Ricinus communis seeds as previously described [26] and was labelled using a microscale AF647 labelling kit (Invitrogen, UK). Briefly, ricin was mixed with AF647 succinimidyl ester at a molar ratio of 1:10 (ricin-AF647) for 15 min at room temperature. Unbound AF647 succinimidyl ester was removed using a NAP-5 column (GE Healthcare) according to the manufacturer’s instructions. Quantification of ricin concentration and degree of labelling was then calculated using A280 and A650 readings (using a Nano-drop 1000). To determine if there was any difference in AF647 binding between the A and B chains of ricin an SDS-PAGE gel was performed. Ricin-AF647 (25 ml) at a concentration of 15.6 ng/ml along with 20 ml of PageRulerTM prestained NIR protein ladder (Invitrogen, UK) were loaded onto a 1 mm NuPageTM NovexTM 10% Bis-Tris protein gel (Invitrogen, UK). Samples were run at 200 volts for 60 min in NuPageTM MOPS SDS running buffer (Invitrogen, UK). Gels were visualised using a BioRad Molecular ImagerÒ VersadocTM MP Imaging system using the 695BP55 filter and red illumination LED for a total exposure of 50 s with 4 gain applied. Images were inverted to increase band definition. 2.3. In vitro ricin toxicity assay To assess if ricin-AF647 had reduced toxicity a standard ricin toxicity assay was performed. Briefly, Vero cells were harvested by trypsinisation and seeded into a 96 well plate (Corning Costar) at 5000 cells/well. Cells were then incubated for 24 h at 37 °C in a 5% CO2 incubator with 95% humidity. Dilutions of ricin and ricin-AF647 were prepared at 1000, 100, 30, 10, 3, 1, 0.1, 0.03, 0.01, 0.003 pM. 100 ml of the ricin dilutions were added to the Vero cells in triplicate, cell only and media only controls were also included. Cells were then incubated at 37 °C in a 5% CO2 incubator with 95% humidity for 48 h. After which, 10 ml of WST-1 (a cell proliferation agent, Roche, UK) was added to the wells and cells were incubated for 3 h at 37 °C. Plates were agitated and the absorbance read on a plate reader at 450 nm. Analysis was performed in Graphpad Prism (Version 6.02), where OD values were plotted against log concentration values. The data were fitted to a sigmoidal, 4parameter logistic curve and the IC50 was used as a measure of toxicity. 2.4. In vitro ricin trafficking experiments
2. Materials and methods 2.1. Growth of A549 human lung epithelial-like cells and African green monkey kidney cells A549 human lung epithelial-like cells (ECACC, Salisbury, UK) were cultured in Dulbecco’s Modified Eagles Medium (DMEM, Gibco, Paisley, UK) supplemented with 10% fetal bovine serum (Gibco, Paisley, UK) and 2 mM glutamine (Gibco, UK) at 37 °C in a 5% CO2 incubator with 95% humidity. Passage of A549 cells was achieved by removal of attached cells by trypsinisation, and reseeding of fresh flasks with 3 104 cells/cm2. A549 cells are not used past passage 20. African green monkey kidney (Vero) cells (ECACC, Salisbury, UK) were routinely cultured in DMEM supplemented with 10% fetal bovine serum (Gibco, Paisley, UK), 100 mg/ml streptomycin and 100 Units penicillin (Sigma, Paisley, UK) and 2 mM glutamine (Sigma, Paisley, UK) at 37 °C in a 5% CO2 incubator with 95% humidity.
A549 cells were harvested, enumerated and plated into a 24well plate (Corning Costar) at a density of 5 105 cells/well. Cells were incubated at 37 °C in a 5% CO2 incubator with 95% humidity for 18 h to allow cell adherence to the plate. Media was removed from the wells and replaced with 200 ml DMEM containing 1 mg/ml (the lowest limit of accurate detection using IFC for this assay) ricin-AF647. Exposed cells were then incubated for a range of times between 8 min and 6 h. After incubation the ricin-AF647 solution was removed and cells were washed once with phosphate buffered saline (PBS) before 200 ml detachin (AMS Biotechnology Limited, Abingdon, UK) was added to the cells. Cells were incubated at 37 °C in a 5% CO2 incubator with 95% humidity for 3 min to allow detachment from the plate surface. Cells were then harvested and centrifuged at 300g for 5 min and resuspended in 60 ml 4% paraformaldehyde. Cells were then counter stained with 3 mM 40 ,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, UK), a strong nuclear (DNA) stain, before data capture using IFC.
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2.5. Staining of Golgi by Golgi-ID Staining using Golgi-ID (Enzo Life Sciences) was achieved according to the manufacturer’s instructions. Briefly, A549 cell were plated as previously described and incubated overnight, washed with 100 ml of assay solution, before the addition of a 200 ml of Golgi-ID stain (1 in 100 dilution of provided stock solution) per well. Cells were then incubated at 4 °C for 30 min before removal of the Golgi-ID stain and washing of the cells with 100 ml assay buffer. A final 1 ml of DMEM was added to the cells and they were incubated for 30 min at 37 °C in a 5% CO2 incubator with 95% humidity before use in the ricin assays. 2.6. Staining of Golgi apparatus using anti-Golgin-97 antibody For staining Golgi apparatus with the anti golgin-97 antibody, 3 wells of a ricin trafficking experiment (described above) were pooled at each time point. Cells were harvested as previously described and resuspended in 250 ml BD fix/perm solution (BD Cytofix/CytopermTM Fixation/Permeabilization Solution Kit) and incubated on a roller at 23 °C for 20 min. Cells were then centrifuged at 300g for 5 min before being resuspended in 100 ml of 1 BD perm/wash solution, with the addition of 1.2 mg mouse anti-human Golgin-97 (ThermoFisher Scientific UK, clone: CDF4, Cat No: A-21270). Samples were then incubated on a roller for 1 h at 23 °C before the addition of 900 ml PBS and centrifugation at 300g for 5 min. After centrifugation cell pellets were resuspended in 100 ml of 1 BD perm/wash solution, with the addition of 1.2 mg goat anti-mouse IgG Alexa FluorÒ 405 secondary antibody (ThermoFisher Scientific UK Cat No: A-31553) and incubated on a roller at 23 °C for 1 h. 900 ml PBS was then added and the sample centrifuged at 300g for 5 min. Cell pellets were resuspended in 60 ml 4% paraformaldehyde and counter stained with 50 nM SytoxÒ green nucleic acid stain (ThermoFisher Scientific UK Cat No: S7020).
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from the images acquired; these are masks and features. Masks are used to define regions of interest within the cell or fluorescence image. Those masks are coupled with features (such as intensity and raw max pixel) that are used to calculate quantitative measurements from or within a masked region. 3.3. Creation of the cell cytoplasm mask A mask was used to identify the cytoplasmic region of cells (Fig. 1). Numbers in mask nomenclatures refer to the channel on which they are based (see Data capture above). The cytoplasmic mask was created by making an erode (M01, 8) mask (the default mask M01, eroded by 8 pixels around) and morphology (M07 or M02) mask of the nuclear channel. These two masks were then combined using Boolean logic (AND NOT) to make a mask that is specific for the cytoplasm. The full mask nomenclature for the cytoplasmic mask is: Erode (M01, 8) AND NOT Morphology (M07) for ricin only and Golgin-97 stained samples or Erode (M01, 8) AND NOT Morphology (M02) for samples where the Golgi apparatus was stained with Golgi-ID. A range of different cytoplasmic masks were assessed before selecting the most suitable mask for use in analysis these can be seen in Supplementary Fig. 2. 3.4. Creation of the Golgi mask When using Golgi-ID the default mask for the Golgi apparatus is not very specific (Fig. 1D), a more stringent mask was required to accurately reflect the Golgi location. To achieve this, a threshold mask was used; this masked a percentage of the brightest pixels within a given starting mask. Here we have masked the top 70% of pixels within the default M02 mask (Fig. 1D). The nomenclature for this mask is Threshold (M02, Ch02, 70), Fig. 1E. A range of different Golgi masks were assessed before selecting the most suitable mask for use in analysis these can be seen in Supplementary Fig. 2.
3. Imaging flow cytometry data collection and analysis
3.5. Data and statistical analysis
3.1. Data capture
All data shown is a percentage population using the in-focus single cells as the parent population. All modelling and statistical analysis was performed in GraphPad Prism version 6.02. All data has been fitted to a model that uses a one phase exponential association following a time delay with the following equation:
IFC data was acquired using an ImageStream X MkII (ISX, Amnis, Seattle, USA) equipped with dual cameras and 405, 488 and 642 nm excitation lasers. All samples were acquired at 60 magnification with a 0.9 numerical aperture (NA) objective, giving a 2.5 mm optical slice image of all cells. A minimum of 7000 infocus single cell events were collected for each sample (Supplementary Fig. 1). Only data from relevant channels were collected including Channel 01 (Ch01, brightfield camera 1), Channel 02 (Ch02, SytoxÒ Green 488 nm laser power: 100 mW) Channel 06 (Ch06, side scatter 785 nm laser power: 10 mW), Channel 7 (Ch07, DAPI fluorescence 405 nm laser power: 2 mW, Golgin-97Alexa Fluor 405, 405 nm laser power: 120 mW), Channel 09 (Ch09, brightfield camera 2) and Ch11 (AF647 fluorescence 642 nm laser power: 150 mW). Data from samples with only single stains were also captured to calculate the compensation matrix required to account for spectral overlap between the chosen fluorophores. All images shown are pseudo coloured the same regardless of the actual fluorescence wavelength to avoid confusion (nucleus = blue, Golgi = green, ricin = red). All images shown are displayed using the same image display parameters to avoid disparity between images in different figures. 3.2. Data analysis – IDEASÒ software Analysis of IFC data was achieved using IDEASÒ software (version 6.1). IDEASÒ utilizes two main principles to make calculations
Y ¼ IFðX < X0; Y0; Y0 þ ðPlateau Y0Þ ð1 expðK ðX X0ÞÞÞÞ ð1Þ where X = time (s), Y = the percentage of cells = Y0 until X = X0 when Y increases to the Plateau with a single phase association with a rate constant K (s1). All data shown are the result of a minimum three biological replicates. 4. Results 4.1. Ricin-AF647 analysis Optical density readings for AF647 labelled ricin, and subsequent calculations indicate that the ricin used for these experiments has a degree of labelling of 1.8 molecules of AF647 to 1 ricin molecule. SDS-Page analysis of ricin-AF647 (Supplementary Fig. 3A) indicates that both chains of the ricin are labelled with AF647 and therefore no judgement can be made regarding the point at which the A and B chain dissociate from each other. Ricin-AF647 was subjected to a standard Vero cell in vitro toxicity assay to assess if the labelling of the ricin had affected its function (Supplementary Fig. 3B). The toxicity assay indicated that the IC50
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Fig. 1. Creation of cytoplasmic mask & Golgi mask using IDEAS analysis software. The default masks (A) that IDEAS creates encompasses the whole cell. To identify cells that contain ricin in the cytoplasm a new mask was created using IDEAS. The default brightfield (BF) mask (A) was altered to be an erode (M01, 8) mask (the default mask eroded by 8 pixels around, B) and the default nuclear mask was altered to be a morphology mask (B). These two masks were then combined using AND NOT Boolean logic to create a cytoplasmic mask (C). The default mask for the Golgi apparatus (D) was also altered to be more specific to the Golgi staining. A threshold mask was used, which masks a percentage of the brightest pixels within a given starting mask. In this instance we have used a threshold (M07, Ch07, 70) mask (E) that masks the 70% brightest pixels contained within the default mask.
for the unlabelled ricin was 1.09 pM and the ricin-AF647 was 2.64 pM. These concentrations are within the range of a normal ricin toxicity assay. The main concentration of ricin used for all the fluorescence studies in this assay was 1 mg/ml which equates to 15.15 nM, meaning an excess of ricin was -used when compared to the IC50 values. 4.2. Quantifying and modelling the endocytosis of ricin into the cell cytoplasm Using conventional flow technologies it is not possible to quantify fluorescence in a specific subcellular location. Here, using IFC and IDEASÒ software we have created a mask to identify the cytoplasmic region of the cell (Fig. 1). This cytoplasmic mask was used in conjunction with two IDEASÒ features namely raw max pixel
and intensity in the ricin channel (channel 11) to identify cells with cytoplasmic ricin. Raw max pixel returns the value of the brightest pixel within the input mask and intensity calculates the overall intensity of the fluorescence within the input mask. These two values were plotted on a dot plot and a polygon gate used to select cells with cytoplasmic ricin (Fig. 2) and this region was then used to calculate the percentage of cells with ricin in the cytoplasmic mask over time. When cells were incubated with ricin and subsequently assessed as described, the percentage of cells containing cytoplasmic ricin increased rapidly so that by about 45 min >90% of cells were positive (Fig. 3). The imagery generated indicated that the ricin-associated fluorescence was not distributed homogeneously throughout the cytoplasm but was present in punctate, high intensity areas particularly around the nucleus (Fig. 3B). Analysis of the kinetics of the ricin uptake
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Fig. 2. Identification of cells with cytoplasmic ricin. To identify those cells with cytoplasmic ricin two IDEAS features were calculated using the cytoplasmic mask as the region of interest. The features calculated were raw max pixel which returns the value of the brightest pixel within the cytoplasmic mask and intensity that calculates the overall intensity of the fluorescence within the cytoplasmic mask. The inset images show examples of the cytoplasmic mask (pink region) in the ricin channel (the red pseudo colour has been removed to increase the clarity of the cytoplasmic mask). The yellow text indicates the value associated with the feature used. On the Y axis the yellow text is the Raw Max Pixel value with in the cytoplasmic mask and on the X axis it is the intensity of the signal in the cytoplasmic mask. To select cells with cytoplasmic ricin a polygon gate was applied to the dot plot above to correspond to cells with cytoplasmic ricin. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
showed that the rate of increase in positive cells followed, very closely, a one phase exponential association but only if a short delay is included in the curve fit model (Eq. (1) and Fig. 3). In all cases Eq. (1) fitted the ricin uptake data significantly better than a standard one-phase association without a delay (p < .05, extra sum of squares F-test). Association of ricin with A549 cells had a t½ of 6.24 min, reaching a plateau of 91.12% at approximately 45 min. Extrapolation of the fitted curve indicated a delay of 6 min before the exponential association could adequately describe the data. The time points between 1 and 9 min could not be acquired in these studies and this extrapolation of the model (Eq. (1)) is shown as a dotted line (Fig. 3A inlay and Table 1). The R2 value for the curve is 0.9596 indicating a good fit of the model to the data. When counter staining the cells for Golgi apparatus using Golgin-97 the data was modelled using the same curve; however, the parameters of the curve fit changed. The t½ was 10.75 min and a plateau of 47.01% was reached after approximately 1 h, both parameters a significant difference from the ricin only curve, with an R2 value of 0.8494. The delay was similar to that seen when the analysis was conducted without the Golgi staining. When using the less invasive Golgi-ID stain the ricin kinetic curve was restored to the same level as when ricin was used in the absence of Golgi staining, however the parameters of the curve still remain significantly different. The Golgi-ID kinetic curve indicated a t½ of 3.69 min, reaching a plateau of 98.37 at between 30 and 60 min. The R2 value for the curve was 0.9854 and again the delay was similar to that seen with the other curve fits (Fig. 3 and Table 1). Thus Golgi staining had relatively little effect on the rate of association of ricin with A549 cells. 4.3. Quantifying and modelling of cells with ricin within the Golgi apparatus Quantification of ricin in the Golgi apparatus of cells was performed on cells stained with Golgi-ID only. Using the threshold
mask described previously (Fig. 1B), the intensity of ricin fluorescence was calculated within this region over the time course of the study (Fig. 4 and Fig. 5). The full feature and mask nomenclature for this is: Intensity_Threshold (M02, Ch02, 70)_Ch11. As with the uptake of ricin into the cytoplasm of A549 cells, the association of ricin fluorescence with the Golgi apparatus followed a singlephase exponential increase in the population of cells analysed (Fig. 5 and Table 2). Again, these data were best fit by including a delay preceding the one-phase association (p < .0001, extra sum of squares F test when compared to a standard one phase exponential association). The resulting percentage of cells returned as having Golgi-associated ricin fluorescence approached that seen when just cytoplasmic ricin was determined (Figs. 3 and 5 and Table 2). However, the rate at which the percentage of cells with Golgiassociated ricin increased was markedly slower than was seen for the cytoplasmic appearance of ricin having a t½ of 67.95 min (Table 2). 5. Discussion Determining the transport route of a biological toxin such as ricin into the cell enables further understanding of its mechanism of action and thus could be used to identify, design and assess therapeutics to interfere with this critical aspect of its mechanism of action. The imaging of ricin trafficking has long been achieved using conventional fluorescence and electron microscopy techniques [15,17,18]. These techniques excel in image resolution but lack the high throughput data capture of flow cytometry, which in itself lacks any kind of spatial resolution. IFC is a technology that combines flow cytometry with fluorescence microscopy to enable rapid imaging of cells in flow at the rate of up to thousands of cells per second [20]. This allows for high throughput data capture of a large cell population from an experiment as opposed to a typical fluorescence/confocal microscope, through which, a relatively small sample of cells are imaged to represent the experiment.
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Fig. 3. Ricin uptake into A549 cells. Kinetic curves were generated by fitting a one phase association to all the three ricin curves following a delay (A). The inlay in panel A is the same data shown on a log scale to indicate the delay (dotted lines extrapolate to the delay time given in Table 1. Data are shown as means ± sem from at least 3 independent experiments. Examples of the staining observed between each curve can be observed in panel B (ricin only), panel C (ricin with Golgin-97 staining), panel D (ricin with Golgi-ID staining). BF = Brightfield, Composite = Ricin, Golgi (C&D) and Nucleus.
Table 1 Kinetic parameters for ricin uptake into the cytoplasmic region of interest under the different Golgi staining conditions. Staining condition
Plateau (%)
t½ (min)
Delay (min)
No Golgi stain
91.1 (89.0–93.3) 98.37* (96.4–100.4) 48.6* (42.9–54.4)
6.26 (5.41–7.44) 3.69* (3.13–4.50) 10.1* (6.95–18.6)
6.17 (5.26–7.08) 6.84 (6.23–7.46) 7.24 (4.32–10.16)
Golgi-ID Golgin 97 *
Significantly different to no Golgi stain ricin curve p < .05.
Hence, IFC platforms greatly increase the statistical robustness of any downstream image analyses. In this study we have exposed A549 human lung epithelial cells to AF647-labelled ricin and examined the cells at different times to ascertain if IFC is a technology that can be used to observe ricin movement into sub-cellular compartments, specifically the cytoplasm and Golgi apparatus. It should be noted however that in these studies it is not possible to distinguish between fluorescent ricin that is free in the cytosol from that which is membranebound within the endocytic pathway. We have developed image analyses protocols to demonstrate that it is possible to track AF647-ricin endocytosis and movement
to the Golgi apparatus using IFC; an application of IFC that to the authors’ knowledge has not previously been reported in the literature. All data that were produced indicate a large proportion of cells contain internalised ricin within the first 15–20 min of exposure with the percentage of cells showing internalised ricin plateauing between 30 and 60 min. This agrees with previous work published using confocal microscopy of ricin uptake by HeLa cells [15] and earlier studies using Vero cells with 125I-labelled ricin [27], thus lending validity to this method for studying ricin trafficking. It is well established that ricin is trafficked through a retrograde transport pathway via the Golgi apparatus [28]. To investigate if it was possible to observe co-localisation of ricin and the Golgi apparatus within A549 cells, the Golgi apparatus was counterstained using either a mouse anti-human Golgin-97 antibody and a secondary anti-mouse Alexa Fluor 405 antibody or with the proprietary Golgi-specific fluorescent probe (Golgi-ID). Comparing the datasets for cells with unstained Golgi and cells co-stained for Golgi apparatus using the two different approaches, indicated a difference between the plateaus for the percentage of cells with internalised ricin between the Golgin-97 stained samples and the Golgi-ID labelled or unlabelled cells. It is apparent when immunolabelling the Golgi apparatus that the plateau drops from >90% when no staining is performed or when Golgi-ID is used to 47%
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Fig. 4. Quantification of ricin in the Golgi apparatus of cell when stained with Golgi-ID. Cells stained with Golgi-ID had Golgi associated ricin quantified using the feature and mask with the following nomenclature: Intensity_Threshold(M02, Ch02, 70)_Ch11. A line gate was used to select cells that were positive for a Golgi-associated ricin signal. Percentage of cells as a proportion of single cells was used as the output parameter. BF = Brightfield. Composite = Nucleus, Golgi and Ricin.
Table 2 Kinetic parameters for ricin uptake into the Golgi in Golgi-ID stained cells.
Fig. 5. Ricin trafficking to the Golgi of A549 cells. Cells identified as having ricin located in the Golgi were fitted to The kinetic model as described in the methods; 2 Kinetic parameters derived from the fit are shown in Table 2. Data are shown as mean ± sem from at least 3 independent experiments.
when Golgi are immunolabelled with Golgin-97. The reason for this difference is not clearly defined but it is likely that during the fix/permeabilise step and additional wash steps of the
Staining condition
Plateau (%)
t½ (min)
Delay (min)
Golgi-ID
98.5 (90.8–106)
68.0 (57.5–83.1)
13.1 (10.6–15.7)
Golgin-97 staining method that a proportion of the cytoplasmic ricin is being washed out of the cells before they are fully fixed causing a decrease in the percentage of cells with cytoplasmic ricin. When, the fixation and permeabilization steps are removed by pre-staining the cells with Golgi-ID the plateau is restored to the same as when no Golgi staining is applied, adding weight to the argument that the additional processing involved with the Golgin-97 staining is washing ricin from the cells. Although the Golgi-ID curve is restored there is a significant difference observed between it and the no Golgi stained ricin curve. The reason for this is unknown but the cells in this curve have had other staining steps prior to ricin intoxication. Although cells stained for Golgi-ID are cooled they are also then re-equilibrated to 37 °C before use in the assay, there is a small possibility this could have an effect on the cells. Furthermore, the proprietary formulation of Golgi-ID is not freely available which may also inherently affect cellular uptake of ricin.
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In addition to examining the kinetics of the endocytosis of ricin into the cytoplasm of the cells, we have also monitored the movement of ricin into the Golgi apparatus. This was achieved using the data set generated when cells were stained with Golgi-ID. Previous studies have indicated that ricin associates with cellular compartments including the Golgi within one hour of exposure [15,29,30]. Our data are consistent with these observations but additionally demonstrate that, in a small proportion of cells, ricin can be detected in the Golgi as early as 15 min after exposure thus demonstrating the ability of IFC to identify early consequences of ricin trafficking in a small proportion of cells within the exposed population. Ricin trafficking to the Golgi is, however, slower than uptake into the cytoplasm with 50% of cells having ricin within the Golgi region of interest by 80 min. The data derived from these studies are generated at a cell population level rather than an individual cell level. Therefore, it is not possible to ascertain what is happening at an individual cell level at any one time; however it is possible to derive what is happening to the ricin movement within the whole cell population. Ricin is endocytosed into the cytoplasm within the population quickly and reaches plateau at around 30–45 min, at this time some 25% of cells have ricin within the Golgi apparatus and this percentage increases relatively slowly until most cells with cytoplasmic ricin also have Golgi-associated ricin too. Ricin is transported from the Golgi to the ER, where the A and B chains dissociate and move independently within the cell [2]. It is important to note that it is not possible to discriminate between the A and B chains of ricin with the labelled ricin used in these studies. Therefore once a fluorescence signal is observed in the Golgi apparatus, and the ricin chains dissociate in the ER, it is currently not possible to follow the fate of the individual chains. In our model of Golgi-associated ricin, an estimated plateau is not reached until after the final time point at 4 h. This means that, as a population, there is a lag between ricin entering the cytoplasmic compartment of the cell population, and being trafficked to the Golgi environment of approximately 3–3.5 h. This lag to plateau is expected due to the nature of the movement of ricin from the endosome though the retrograde transport system to the Golgi apparatus. The rapid cellular uptake and trafficking of ricin noted in these studies is consistent with previous in vitro studies demonstrating that only short duration exposures are sufficient to cause cytotoxicity at later time points [31]. These observations seem at odds with in vivo protection studies using anti-ricin antibody treatments where protection can be observed for many hours after the initial ricin exposure. This has been demonstrated for intraperitoneal [32], inhalation [33] and intranasal [34–36] challenges with ricin. For the latter route, partial protection was achieved by antibody administrations as late as 72 h after ricin challenge in mice. It has been generally assumed that the anti-toxin mediated neutralisation of toxins occurs extracellularly whilst the toxin is extracellular (free or cell surface attached), however, given the rapid kinetics of ricin uptake it is difficult to reconcile this mode of neutralisation with the in vivo protection observed many hours after intoxication. This suggests that either the kinetics of ricin cellular trafficking observed in vitro are not reflected in vivo, or that the assumption of a purely extracellular mode of neutralisation is an incomplete explanation of the mechanism of post-exposure antitoxin. Indeed it has been observed that both A and B-chain directed anti-ricin antibodies have the ability to neutralise ricin activity intracellularly and can disrupt the intracellular trafficking of ricin [15,16,37,38]. Clearly, understanding the impact of antitoxinbased therapies on intracellular ricin trafficking is important. IFC technology could therefore be applied to provide quantitative cell population data to support mechanistic studies of antitoxins or small molecule-mediated strategies [39,40] that block or redirect
normal intracellular trafficking of ricin towards proteolytic or other non-toxic routes. Methods for studying ricin trafficking have, over the years, used many different methodologies including confocal microscopy [15], radio-labelling [27], electron microscopy [29] and more recently, GFP reporter assays [41]. Here we have shown that it is possible to examine the endocytosis of ricin using imaging flow cytometry. Although this technique does not have as high resolution as confocal/electron microscopy, IFC benefits from the collection of high throughput data and large populations of cellular images. This provides robust image analysis and data sets that better represent the cell population of interest. The assay presented in this work is an example of using imaging flow cytometry for evaluating the trafficking of toxins. This approach can be used as a tool for the early evaluation of toxin therapies to help establish their mechanisms of action, or more broadly, to compare the endocytosis kinetics of toxins in different cell types and tissues. Acknowledgements The authors would like to thank Fiona Stahl for her help and assistance running the ricin toxicity assays. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.ymeth.2017.10.012. References [1] CDC, Biological and chemical terrorism: strategic plan for preparedness and response: recommendations of the CDC Strategic Planning Workgroup, Morbidity and Mortality Weekly Report. Recommendations and Reports, 49 (RR-4), 2000, 1–14. [2] R.A. Spooner, J.M. Lord, Ricin trafficking in cells, Toxins 7 (1) (2015) 49–65. [3] W. Montfort, J.E. Villafranca, A.F. Monzingo, S.R. Ernst, B. Katzin, E. Rutenber, N. H. Xuong, R. Hamlin, J.D. Robertus, The three-dimensional structure of ricin at 2.8 A, J. Biol. Chem. 262 (11) (1987) 5398–5403. [4] G. Griffiths, P. Rice, A. Allenby, S. Bailey, D. Upshall, Inhalation toxicology and histopathology of ricin and abrin toxins, Inhalation Toxicol. 7 (2) (1995) 269– 288. [5] D. Franz, N. Jaax, Ricin toxin, in: F.R. Sidell, E.T. Takafuji, D.R. Franz (Eds.), Medical Aspects of Chemical and Biological Warfare, Office of the Surgeon General, Falls Church, Virginia, 1997, pp. 631–642. [6] J. Audi, M. Belson, M. Patel, J. Schier, J. Osterloh, Ricin poisoning: a comprehensive review, JAMA 294 (a18) (2005) 2342–2351. [7] K.R. Challoner, M.M. McCarron, Castor bean intoxication, Ann. Emerg. Med. 19 (10) (1990) 1177–1183. [8] B. Furbee, M. Wermuth, Life-threatening plant poisoning, Crit. Care Clin. 13 (4) (1997) 849–888. [9] P.A. Kinamore, R.W. Jaeger, F.J. de Castro, Abrus and ricinus ingestion: management of three cases, Clin. Toxicol. 17 (3) (1980) 401–405. [10] W. Palatnick, M. Tenenbein, Hepatotoxicity from castor bean ingestion in a child, J. Toxicol. Clin. Toxicol. 38 (1) (2000) 67–69. [11] R.P. Reed, Castor oil seed poisoning: a concern for children, Med. J. Aust. 168 (8) (1998) 423–424. [12] G.P. Wedin, J.S. Neal, G.W. Everson, E.P. Krenzelok, Castor bean poisoning, Am. J. Emerg. Med. 4 (3) (1986) 259–261. [13] H. Bigalke, A. Rummel, Medical aspects of toxin weapons, Toxicology 214 (3) (2005) 210–220. [14] M. Kortepeter, G. Christopher, T. Cieslak, USAM-RIID’s Medical Management of Biological Casulaties Handbook, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, 2001. [15] K. Song, R.R. Mize, L. Marrero, M. Corti, J.M. Kirk, S.H. Pincus, Antibody to ricin A chain hinders intracellular routing of toxin and protects cells even after toxin has been internalized, PLoS One 8 (4) (2013) e62417. [16] A. Yermakova, T.I. Klokk, R. Cole, K. Sandvig, N.J. Mantis, Antibody-mediated inhibition of ricin toxin retrograde transport, mBio 5 (2) (2014). e00995. [17] K. Sandvig, K. Prydz, S.H. Hansen, B. van Deurs, Ricin transport in brefeldin Atreated cells: correlation between Golgi structure and toxic effect, J. Cell Biol. 115 (4) (1991) 971–981. [18] J.P. Frenoy, E. Turpin, M. Janicot, F. Gehin-Fouque, B. Desbuquois, Uptake of injected 125I-ricin by rat liver in vivo Subcellular distribution and characterization of the internalized ligand, Biochem. J 284 (Pt 1) (1992) 249–257. [19] P.Z. Chia, P.A. Gleeson, Imaging and quantitation techniques for tracking cargo along Endosome-to-Golgi transport pathways, Cells 2 (1) (2013) 105–123.
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Methods journal homepage: www.elsevier.com/locate/ymeth
An imaging flow cytometry method to assess ricin trafficking in A549 human lung epithelial cells Dominic Jenner a,⇑, Damien Chong b, Nicola Walker a, A. Christopher Green a a b
Defence Science and Technology Laboratory, CBR Division, Porton Down, Salisbury SP4 0JQ, United Kingdom Chemical & Toxin Medical Countermeasures, Land Division, Defence Science and Technology Group, 506 Lorimer St, Fishermans Bend, Victoria 3207, Australia
a r t i c l e
i n f o
Article history: Received 29 August 2017 Received in revised form 12 October 2017 Accepted 30 October 2017 Available online xxxx Keywords: Ricin Imaging flow cytometry ImageStream Golgi Toxin
a b s t r a c t The endocytosis and trafficking of ricin in mammalian cells is an important area of research for those producing ricin anti-toxins and other ricin therapeutics. Ricin trafficking is usually observed by fluorescence microscopy techniques. This gives good resolution and leads to a detailed understanding of the internal movement of ricin within cells. However, microscopy techniques are often hampered by complex analysis and quantification techniques, and the inability to look at ricin trafficking in large populations of cells. In these studies we have directly labelled ricin and assessed if its trafficking can be observed using Imaging Flow Cytometry (IFC) both to the cytoplasmic region of cells and specifically to the Golgi apparatus. Using IDEASÒ data analysis software the specific fluorescence location of the ricin within the cells was analysed. Then, using cytoplasmic masking techniques to quantify the number of cells with endocytosed cytoplasmic ricin or cells with Golgi-associated ricin, kinetic endocytosis curves were generated. Here we present, to the authors’ knowledge, the first example of using imaging flow cytometry for evaluating the subcellular transport of protein cargo, using the trafficking of ricin toxin in lung cells as a model. Ó 2017 Published by Elsevier Inc.
1. Introduction Ricin is a potent 66-kDa Type II ribosome inactivating protein produced by the seeds of the castor oil plant, Ricinus communis. Approximately one million tons of castor beans are processed annually in the production of castor oil worldwide leaving a ricin-rich pulp waste. Due to the ease of its extraction from castor seeds, its potentially lethal effects, ricin is recognised as a biological threat agent and is a Category B Agent on the Centres for Disease Control Select Agent List [1]. Ricin is a heterodimeric protein toxin comprising of A and B chains coupled by a disulphide linkage. The 34-kDa B chain is a lectin that facilitates toxin binding to terminal galactose residues on the target cell surface enabling ricin uptake by the cell. The holotoxin is internalised by endocytosis of which, a proportion of the ricin is transported via the Golgi apparatus to the endoplasmic reticulum, where the A and B chains are separated. The 32-kDa A chain is subsequently retrotranslocated into the cytosol and folded into a catalytically active RNA N-glycosidase. This active form of the ricin A chain then ⇑ Corresponding author at: Dstl Porton Down, CBR Division, Microbiology Bldg 7 Rm 201, Salisbury, Wiltshire SP1 3JQ, United Kingdom. E-mail address: [email protected] (D. Jenner).
depurinates base A4324 of 28S ribosomal RNA, thereby inhibiting protein synthesis and ultimately causing cell death [2,3]. The 50% lethal dose (LD50) depends on the route of exposure. In mice, the LD50 is less than 5 mg/kg ricin when administered intravenously, 4–6 mg/min/kg via inhalation and up to 30 mg/ kg via ingestion. In humans, the LD50 of ricin by ingestion is estimated to be 1–20 mg/kg [4–6]. Symptoms after ingestion may include fever, dilation of pupils, diarrhoea, nausea, vomiting, and abdominal pain and may progress to organ failure and hypotension. Inhalation of aerosolised ricin can result in dyspnoea, fever, cough, nausea, arthralgia, tightness in the chest and may progress to cyanosis with manifestations of heavy sweating and pulmonary oedema. Death may occur 24–72 h after exposure as a consequence of high permeability pulmonary oedema, acute respiratory distress syndrome or respiratory failure, either with or without hypotension and organ failure [5,7–14]. If death does not occur within 2–3 days after exposure, the patient will normally make a slow recovery, as there are no ricin treatments currently available other than palliative care. Understanding the mechanisms by which such toxins are transported through cells helps to determine how they may be exploited with novel inhibitors and how therapeutics can be used to disrupt their cytotoxicity [15,16].
https://doi.org/10.1016/j.ymeth.2017.10.012 1046-2023/Ó 2017 Published by Elsevier Inc.
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Conventionally, the mechanisms by which cells internalise proteins were often visualised by electron microscopy or measured using density gradient subcellular fractionation [17,18]. Although these approaches are still used, the development of fluorescence based technologies coupled with less-toxic fluorescent probes and user-friendly image analysis software means that measurement of a protein trafficking within cells is easily achievable. In the context of this paper the protein of interest is ricin, transport of which can be readily observed in relation to cellular sub-structure by quantitative multi-channel fluorescence microscopy using various reporter probes [15,16,19]. Although cutting edge fluorescence microscopy excels in image resolution and clarity, even when equipped with a motorised/ programmable stage one of its fundamental limitations is the time required to image cells and more importantly, to acquire data from sufficient numbers of cells to obtain a representative sample of the experiment. That is, it lacks the high throughput nature of other fluorescence technologies, such as flow cytometry. However, flow cytometry itself lacks the spatial data content provided by microscopy and only returns fluorescence intensity data for each particle interrogated. Imaging Flow Cytometry (IFC) is a cytometry technique that combines the high throughput nature of flow cytometry with the imaging and spatial capabilities of fluorescence microscopy [20]. IFC enables the capture of an optical slice image of individual cells in a flow in both brightfield and fluorescence channels, allowing for statistically robust, quantitative, image analysis in large populations of cells [20]. IFC is maturing quickly in the field of immunology, where the technology has been utilised to study many different aspects including, but not limited to, cell signalling [21], nuclear translocation [22,23] and host-pathogen interactions [24,25]. However, the study of how biological toxins interact with their target cells seems to be an as-yet unexplored application of IFC. Therefore, to demonstrate its utility, we have used IFC and Alexa FluorÒ 647 (AF647)-labelled ricin to show that it is possible to use this technology to generate kinetic data describing the uptake and endocytosis of ricin into the cytoplasm of human lung endothelial like A549 cells and we also demonstrate it is possible to visualise co-localisation of ricin with the Golgi apparatus of these cells. This methodology moves the analysis of toxin endocytosis away from making quantitative single cell measurements by confocal microscopy [15,16,19], and starts looking at toxin endocytosis on a cell population level with 1000s of cells, thus increasing the speed of data acquisition and analysis, along with the robustness that comes with looking at a population level.
2.2. Ricin production, fluorescent labelling and sodium dodecyl sulphate polyacrylamide gel electrophoresis of Ricin-AF647 Ricin was extracted and purified from Ricinus communis seeds as previously described [26] and was labelled using a microscale AF647 labelling kit (Invitrogen, UK). Briefly, ricin was mixed with AF647 succinimidyl ester at a molar ratio of 1:10 (ricin-AF647) for 15 min at room temperature. Unbound AF647 succinimidyl ester was removed using a NAP-5 column (GE Healthcare) according to the manufacturer’s instructions. Quantification of ricin concentration and degree of labelling was then calculated using A280 and A650 readings (using a Nano-drop 1000). To determine if there was any difference in AF647 binding between the A and B chains of ricin an SDS-PAGE gel was performed. Ricin-AF647 (25 ml) at a concentration of 15.6 ng/ml along with 20 ml of PageRulerTM prestained NIR protein ladder (Invitrogen, UK) were loaded onto a 1 mm NuPageTM NovexTM 10% Bis-Tris protein gel (Invitrogen, UK). Samples were run at 200 volts for 60 min in NuPageTM MOPS SDS running buffer (Invitrogen, UK). Gels were visualised using a BioRad Molecular ImagerÒ VersadocTM MP Imaging system using the 695BP55 filter and red illumination LED for a total exposure of 50 s with 4 gain applied. Images were inverted to increase band definition. 2.3. In vitro ricin toxicity assay To assess if ricin-AF647 had reduced toxicity a standard ricin toxicity assay was performed. Briefly, Vero cells were harvested by trypsinisation and seeded into a 96 well plate (Corning Costar) at 5000 cells/well. Cells were then incubated for 24 h at 37 °C in a 5% CO2 incubator with 95% humidity. Dilutions of ricin and ricin-AF647 were prepared at 1000, 100, 30, 10, 3, 1, 0.1, 0.03, 0.01, 0.003 pM. 100 ml of the ricin dilutions were added to the Vero cells in triplicate, cell only and media only controls were also included. Cells were then incubated at 37 °C in a 5% CO2 incubator with 95% humidity for 48 h. After which, 10 ml of WST-1 (a cell proliferation agent, Roche, UK) was added to the wells and cells were incubated for 3 h at 37 °C. Plates were agitated and the absorbance read on a plate reader at 450 nm. Analysis was performed in Graphpad Prism (Version 6.02), where OD values were plotted against log concentration values. The data were fitted to a sigmoidal, 4parameter logistic curve and the IC50 was used as a measure of toxicity. 2.4. In vitro ricin trafficking experiments
2. Materials and methods 2.1. Growth of A549 human lung epithelial-like cells and African green monkey kidney cells A549 human lung epithelial-like cells (ECACC, Salisbury, UK) were cultured in Dulbecco’s Modified Eagles Medium (DMEM, Gibco, Paisley, UK) supplemented with 10% fetal bovine serum (Gibco, Paisley, UK) and 2 mM glutamine (Gibco, UK) at 37 °C in a 5% CO2 incubator with 95% humidity. Passage of A549 cells was achieved by removal of attached cells by trypsinisation, and reseeding of fresh flasks with 3 104 cells/cm2. A549 cells are not used past passage 20. African green monkey kidney (Vero) cells (ECACC, Salisbury, UK) were routinely cultured in DMEM supplemented with 10% fetal bovine serum (Gibco, Paisley, UK), 100 mg/ml streptomycin and 100 Units penicillin (Sigma, Paisley, UK) and 2 mM glutamine (Sigma, Paisley, UK) at 37 °C in a 5% CO2 incubator with 95% humidity.
A549 cells were harvested, enumerated and plated into a 24well plate (Corning Costar) at a density of 5 105 cells/well. Cells were incubated at 37 °C in a 5% CO2 incubator with 95% humidity for 18 h to allow cell adherence to the plate. Media was removed from the wells and replaced with 200 ml DMEM containing 1 mg/ml (the lowest limit of accurate detection using IFC for this assay) ricin-AF647. Exposed cells were then incubated for a range of times between 8 min and 6 h. After incubation the ricin-AF647 solution was removed and cells were washed once with phosphate buffered saline (PBS) before 200 ml detachin (AMS Biotechnology Limited, Abingdon, UK) was added to the cells. Cells were incubated at 37 °C in a 5% CO2 incubator with 95% humidity for 3 min to allow detachment from the plate surface. Cells were then harvested and centrifuged at 300g for 5 min and resuspended in 60 ml 4% paraformaldehyde. Cells were then counter stained with 3 mM 40 ,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, UK), a strong nuclear (DNA) stain, before data capture using IFC.
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2.5. Staining of Golgi by Golgi-ID Staining using Golgi-ID (Enzo Life Sciences) was achieved according to the manufacturer’s instructions. Briefly, A549 cell were plated as previously described and incubated overnight, washed with 100 ml of assay solution, before the addition of a 200 ml of Golgi-ID stain (1 in 100 dilution of provided stock solution) per well. Cells were then incubated at 4 °C for 30 min before removal of the Golgi-ID stain and washing of the cells with 100 ml assay buffer. A final 1 ml of DMEM was added to the cells and they were incubated for 30 min at 37 °C in a 5% CO2 incubator with 95% humidity before use in the ricin assays. 2.6. Staining of Golgi apparatus using anti-Golgin-97 antibody For staining Golgi apparatus with the anti golgin-97 antibody, 3 wells of a ricin trafficking experiment (described above) were pooled at each time point. Cells were harvested as previously described and resuspended in 250 ml BD fix/perm solution (BD Cytofix/CytopermTM Fixation/Permeabilization Solution Kit) and incubated on a roller at 23 °C for 20 min. Cells were then centrifuged at 300g for 5 min before being resuspended in 100 ml of 1 BD perm/wash solution, with the addition of 1.2 mg mouse anti-human Golgin-97 (ThermoFisher Scientific UK, clone: CDF4, Cat No: A-21270). Samples were then incubated on a roller for 1 h at 23 °C before the addition of 900 ml PBS and centrifugation at 300g for 5 min. After centrifugation cell pellets were resuspended in 100 ml of 1 BD perm/wash solution, with the addition of 1.2 mg goat anti-mouse IgG Alexa FluorÒ 405 secondary antibody (ThermoFisher Scientific UK Cat No: A-31553) and incubated on a roller at 23 °C for 1 h. 900 ml PBS was then added and the sample centrifuged at 300g for 5 min. Cell pellets were resuspended in 60 ml 4% paraformaldehyde and counter stained with 50 nM SytoxÒ green nucleic acid stain (ThermoFisher Scientific UK Cat No: S7020).
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from the images acquired; these are masks and features. Masks are used to define regions of interest within the cell or fluorescence image. Those masks are coupled with features (such as intensity and raw max pixel) that are used to calculate quantitative measurements from or within a masked region. 3.3. Creation of the cell cytoplasm mask A mask was used to identify the cytoplasmic region of cells (Fig. 1). Numbers in mask nomenclatures refer to the channel on which they are based (see Data capture above). The cytoplasmic mask was created by making an erode (M01, 8) mask (the default mask M01, eroded by 8 pixels around) and morphology (M07 or M02) mask of the nuclear channel. These two masks were then combined using Boolean logic (AND NOT) to make a mask that is specific for the cytoplasm. The full mask nomenclature for the cytoplasmic mask is: Erode (M01, 8) AND NOT Morphology (M07) for ricin only and Golgin-97 stained samples or Erode (M01, 8) AND NOT Morphology (M02) for samples where the Golgi apparatus was stained with Golgi-ID. A range of different cytoplasmic masks were assessed before selecting the most suitable mask for use in analysis these can be seen in Supplementary Fig. 2. 3.4. Creation of the Golgi mask When using Golgi-ID the default mask for the Golgi apparatus is not very specific (Fig. 1D), a more stringent mask was required to accurately reflect the Golgi location. To achieve this, a threshold mask was used; this masked a percentage of the brightest pixels within a given starting mask. Here we have masked the top 70% of pixels within the default M02 mask (Fig. 1D). The nomenclature for this mask is Threshold (M02, Ch02, 70), Fig. 1E. A range of different Golgi masks were assessed before selecting the most suitable mask for use in analysis these can be seen in Supplementary Fig. 2.
3. Imaging flow cytometry data collection and analysis
3.5. Data and statistical analysis
3.1. Data capture
All data shown is a percentage population using the in-focus single cells as the parent population. All modelling and statistical analysis was performed in GraphPad Prism version 6.02. All data has been fitted to a model that uses a one phase exponential association following a time delay with the following equation:
IFC data was acquired using an ImageStream X MkII (ISX, Amnis, Seattle, USA) equipped with dual cameras and 405, 488 and 642 nm excitation lasers. All samples were acquired at 60 magnification with a 0.9 numerical aperture (NA) objective, giving a 2.5 mm optical slice image of all cells. A minimum of 7000 infocus single cell events were collected for each sample (Supplementary Fig. 1). Only data from relevant channels were collected including Channel 01 (Ch01, brightfield camera 1), Channel 02 (Ch02, SytoxÒ Green 488 nm laser power: 100 mW) Channel 06 (Ch06, side scatter 785 nm laser power: 10 mW), Channel 7 (Ch07, DAPI fluorescence 405 nm laser power: 2 mW, Golgin-97Alexa Fluor 405, 405 nm laser power: 120 mW), Channel 09 (Ch09, brightfield camera 2) and Ch11 (AF647 fluorescence 642 nm laser power: 150 mW). Data from samples with only single stains were also captured to calculate the compensation matrix required to account for spectral overlap between the chosen fluorophores. All images shown are pseudo coloured the same regardless of the actual fluorescence wavelength to avoid confusion (nucleus = blue, Golgi = green, ricin = red). All images shown are displayed using the same image display parameters to avoid disparity between images in different figures. 3.2. Data analysis – IDEASÒ software Analysis of IFC data was achieved using IDEASÒ software (version 6.1). IDEASÒ utilizes two main principles to make calculations
Y ¼ IFðX < X0; Y0; Y0 þ ðPlateau Y0Þ ð1 expðK ðX X0ÞÞÞÞ ð1Þ where X = time (s), Y = the percentage of cells = Y0 until X = X0 when Y increases to the Plateau with a single phase association with a rate constant K (s1). All data shown are the result of a minimum three biological replicates. 4. Results 4.1. Ricin-AF647 analysis Optical density readings for AF647 labelled ricin, and subsequent calculations indicate that the ricin used for these experiments has a degree of labelling of 1.8 molecules of AF647 to 1 ricin molecule. SDS-Page analysis of ricin-AF647 (Supplementary Fig. 3A) indicates that both chains of the ricin are labelled with AF647 and therefore no judgement can be made regarding the point at which the A and B chain dissociate from each other. Ricin-AF647 was subjected to a standard Vero cell in vitro toxicity assay to assess if the labelling of the ricin had affected its function (Supplementary Fig. 3B). The toxicity assay indicated that the IC50
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Fig. 1. Creation of cytoplasmic mask & Golgi mask using IDEAS analysis software. The default masks (A) that IDEAS creates encompasses the whole cell. To identify cells that contain ricin in the cytoplasm a new mask was created using IDEAS. The default brightfield (BF) mask (A) was altered to be an erode (M01, 8) mask (the default mask eroded by 8 pixels around, B) and the default nuclear mask was altered to be a morphology mask (B). These two masks were then combined using AND NOT Boolean logic to create a cytoplasmic mask (C). The default mask for the Golgi apparatus (D) was also altered to be more specific to the Golgi staining. A threshold mask was used, which masks a percentage of the brightest pixels within a given starting mask. In this instance we have used a threshold (M07, Ch07, 70) mask (E) that masks the 70% brightest pixels contained within the default mask.
for the unlabelled ricin was 1.09 pM and the ricin-AF647 was 2.64 pM. These concentrations are within the range of a normal ricin toxicity assay. The main concentration of ricin used for all the fluorescence studies in this assay was 1 mg/ml which equates to 15.15 nM, meaning an excess of ricin was -used when compared to the IC50 values. 4.2. Quantifying and modelling the endocytosis of ricin into the cell cytoplasm Using conventional flow technologies it is not possible to quantify fluorescence in a specific subcellular location. Here, using IFC and IDEASÒ software we have created a mask to identify the cytoplasmic region of the cell (Fig. 1). This cytoplasmic mask was used in conjunction with two IDEASÒ features namely raw max pixel
and intensity in the ricin channel (channel 11) to identify cells with cytoplasmic ricin. Raw max pixel returns the value of the brightest pixel within the input mask and intensity calculates the overall intensity of the fluorescence within the input mask. These two values were plotted on a dot plot and a polygon gate used to select cells with cytoplasmic ricin (Fig. 2) and this region was then used to calculate the percentage of cells with ricin in the cytoplasmic mask over time. When cells were incubated with ricin and subsequently assessed as described, the percentage of cells containing cytoplasmic ricin increased rapidly so that by about 45 min >90% of cells were positive (Fig. 3). The imagery generated indicated that the ricin-associated fluorescence was not distributed homogeneously throughout the cytoplasm but was present in punctate, high intensity areas particularly around the nucleus (Fig. 3B). Analysis of the kinetics of the ricin uptake
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Fig. 2. Identification of cells with cytoplasmic ricin. To identify those cells with cytoplasmic ricin two IDEAS features were calculated using the cytoplasmic mask as the region of interest. The features calculated were raw max pixel which returns the value of the brightest pixel within the cytoplasmic mask and intensity that calculates the overall intensity of the fluorescence within the cytoplasmic mask. The inset images show examples of the cytoplasmic mask (pink region) in the ricin channel (the red pseudo colour has been removed to increase the clarity of the cytoplasmic mask). The yellow text indicates the value associated with the feature used. On the Y axis the yellow text is the Raw Max Pixel value with in the cytoplasmic mask and on the X axis it is the intensity of the signal in the cytoplasmic mask. To select cells with cytoplasmic ricin a polygon gate was applied to the dot plot above to correspond to cells with cytoplasmic ricin. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
showed that the rate of increase in positive cells followed, very closely, a one phase exponential association but only if a short delay is included in the curve fit model (Eq. (1) and Fig. 3). In all cases Eq. (1) fitted the ricin uptake data significantly better than a standard one-phase association without a delay (p < .05, extra sum of squares F-test). Association of ricin with A549 cells had a t½ of 6.24 min, reaching a plateau of 91.12% at approximately 45 min. Extrapolation of the fitted curve indicated a delay of 6 min before the exponential association could adequately describe the data. The time points between 1 and 9 min could not be acquired in these studies and this extrapolation of the model (Eq. (1)) is shown as a dotted line (Fig. 3A inlay and Table 1). The R2 value for the curve is 0.9596 indicating a good fit of the model to the data. When counter staining the cells for Golgi apparatus using Golgin-97 the data was modelled using the same curve; however, the parameters of the curve fit changed. The t½ was 10.75 min and a plateau of 47.01% was reached after approximately 1 h, both parameters a significant difference from the ricin only curve, with an R2 value of 0.8494. The delay was similar to that seen when the analysis was conducted without the Golgi staining. When using the less invasive Golgi-ID stain the ricin kinetic curve was restored to the same level as when ricin was used in the absence of Golgi staining, however the parameters of the curve still remain significantly different. The Golgi-ID kinetic curve indicated a t½ of 3.69 min, reaching a plateau of 98.37 at between 30 and 60 min. The R2 value for the curve was 0.9854 and again the delay was similar to that seen with the other curve fits (Fig. 3 and Table 1). Thus Golgi staining had relatively little effect on the rate of association of ricin with A549 cells. 4.3. Quantifying and modelling of cells with ricin within the Golgi apparatus Quantification of ricin in the Golgi apparatus of cells was performed on cells stained with Golgi-ID only. Using the threshold
mask described previously (Fig. 1B), the intensity of ricin fluorescence was calculated within this region over the time course of the study (Fig. 4 and Fig. 5). The full feature and mask nomenclature for this is: Intensity_Threshold (M02, Ch02, 70)_Ch11. As with the uptake of ricin into the cytoplasm of A549 cells, the association of ricin fluorescence with the Golgi apparatus followed a singlephase exponential increase in the population of cells analysed (Fig. 5 and Table 2). Again, these data were best fit by including a delay preceding the one-phase association (p < .0001, extra sum of squares F test when compared to a standard one phase exponential association). The resulting percentage of cells returned as having Golgi-associated ricin fluorescence approached that seen when just cytoplasmic ricin was determined (Figs. 3 and 5 and Table 2). However, the rate at which the percentage of cells with Golgiassociated ricin increased was markedly slower than was seen for the cytoplasmic appearance of ricin having a t½ of 67.95 min (Table 2). 5. Discussion Determining the transport route of a biological toxin such as ricin into the cell enables further understanding of its mechanism of action and thus could be used to identify, design and assess therapeutics to interfere with this critical aspect of its mechanism of action. The imaging of ricin trafficking has long been achieved using conventional fluorescence and electron microscopy techniques [15,17,18]. These techniques excel in image resolution but lack the high throughput data capture of flow cytometry, which in itself lacks any kind of spatial resolution. IFC is a technology that combines flow cytometry with fluorescence microscopy to enable rapid imaging of cells in flow at the rate of up to thousands of cells per second [20]. This allows for high throughput data capture of a large cell population from an experiment as opposed to a typical fluorescence/confocal microscope, through which, a relatively small sample of cells are imaged to represent the experiment.
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Fig. 3. Ricin uptake into A549 cells. Kinetic curves were generated by fitting a one phase association to all the three ricin curves following a delay (A). The inlay in panel A is the same data shown on a log scale to indicate the delay (dotted lines extrapolate to the delay time given in Table 1. Data are shown as means ± sem from at least 3 independent experiments. Examples of the staining observed between each curve can be observed in panel B (ricin only), panel C (ricin with Golgin-97 staining), panel D (ricin with Golgi-ID staining). BF = Brightfield, Composite = Ricin, Golgi (C&D) and Nucleus.
Table 1 Kinetic parameters for ricin uptake into the cytoplasmic region of interest under the different Golgi staining conditions. Staining condition
Plateau (%)
t½ (min)
Delay (min)
No Golgi stain
91.1 (89.0–93.3) 98.37* (96.4–100.4) 48.6* (42.9–54.4)
6.26 (5.41–7.44) 3.69* (3.13–4.50) 10.1* (6.95–18.6)
6.17 (5.26–7.08) 6.84 (6.23–7.46) 7.24 (4.32–10.16)
Golgi-ID Golgin 97 *
Significantly different to no Golgi stain ricin curve p < .05.
Hence, IFC platforms greatly increase the statistical robustness of any downstream image analyses. In this study we have exposed A549 human lung epithelial cells to AF647-labelled ricin and examined the cells at different times to ascertain if IFC is a technology that can be used to observe ricin movement into sub-cellular compartments, specifically the cytoplasm and Golgi apparatus. It should be noted however that in these studies it is not possible to distinguish between fluorescent ricin that is free in the cytosol from that which is membranebound within the endocytic pathway. We have developed image analyses protocols to demonstrate that it is possible to track AF647-ricin endocytosis and movement
to the Golgi apparatus using IFC; an application of IFC that to the authors’ knowledge has not previously been reported in the literature. All data that were produced indicate a large proportion of cells contain internalised ricin within the first 15–20 min of exposure with the percentage of cells showing internalised ricin plateauing between 30 and 60 min. This agrees with previous work published using confocal microscopy of ricin uptake by HeLa cells [15] and earlier studies using Vero cells with 125I-labelled ricin [27], thus lending validity to this method for studying ricin trafficking. It is well established that ricin is trafficked through a retrograde transport pathway via the Golgi apparatus [28]. To investigate if it was possible to observe co-localisation of ricin and the Golgi apparatus within A549 cells, the Golgi apparatus was counterstained using either a mouse anti-human Golgin-97 antibody and a secondary anti-mouse Alexa Fluor 405 antibody or with the proprietary Golgi-specific fluorescent probe (Golgi-ID). Comparing the datasets for cells with unstained Golgi and cells co-stained for Golgi apparatus using the two different approaches, indicated a difference between the plateaus for the percentage of cells with internalised ricin between the Golgin-97 stained samples and the Golgi-ID labelled or unlabelled cells. It is apparent when immunolabelling the Golgi apparatus that the plateau drops from >90% when no staining is performed or when Golgi-ID is used to 47%
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Fig. 4. Quantification of ricin in the Golgi apparatus of cell when stained with Golgi-ID. Cells stained with Golgi-ID had Golgi associated ricin quantified using the feature and mask with the following nomenclature: Intensity_Threshold(M02, Ch02, 70)_Ch11. A line gate was used to select cells that were positive for a Golgi-associated ricin signal. Percentage of cells as a proportion of single cells was used as the output parameter. BF = Brightfield. Composite = Nucleus, Golgi and Ricin.
Table 2 Kinetic parameters for ricin uptake into the Golgi in Golgi-ID stained cells.
Fig. 5. Ricin trafficking to the Golgi of A549 cells. Cells identified as having ricin located in the Golgi were fitted to The kinetic model as described in the methods; 2 Kinetic parameters derived from the fit are shown in Table 2. Data are shown as mean ± sem from at least 3 independent experiments.
when Golgi are immunolabelled with Golgin-97. The reason for this difference is not clearly defined but it is likely that during the fix/permeabilise step and additional wash steps of the
Staining condition
Plateau (%)
t½ (min)
Delay (min)
Golgi-ID
98.5 (90.8–106)
68.0 (57.5–83.1)
13.1 (10.6–15.7)
Golgin-97 staining method that a proportion of the cytoplasmic ricin is being washed out of the cells before they are fully fixed causing a decrease in the percentage of cells with cytoplasmic ricin. When, the fixation and permeabilization steps are removed by pre-staining the cells with Golgi-ID the plateau is restored to the same as when no Golgi staining is applied, adding weight to the argument that the additional processing involved with the Golgin-97 staining is washing ricin from the cells. Although the Golgi-ID curve is restored there is a significant difference observed between it and the no Golgi stained ricin curve. The reason for this is unknown but the cells in this curve have had other staining steps prior to ricin intoxication. Although cells stained for Golgi-ID are cooled they are also then re-equilibrated to 37 °C before use in the assay, there is a small possibility this could have an effect on the cells. Furthermore, the proprietary formulation of Golgi-ID is not freely available which may also inherently affect cellular uptake of ricin.
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In addition to examining the kinetics of the endocytosis of ricin into the cytoplasm of the cells, we have also monitored the movement of ricin into the Golgi apparatus. This was achieved using the data set generated when cells were stained with Golgi-ID. Previous studies have indicated that ricin associates with cellular compartments including the Golgi within one hour of exposure [15,29,30]. Our data are consistent with these observations but additionally demonstrate that, in a small proportion of cells, ricin can be detected in the Golgi as early as 15 min after exposure thus demonstrating the ability of IFC to identify early consequences of ricin trafficking in a small proportion of cells within the exposed population. Ricin trafficking to the Golgi is, however, slower than uptake into the cytoplasm with 50% of cells having ricin within the Golgi region of interest by 80 min. The data derived from these studies are generated at a cell population level rather than an individual cell level. Therefore, it is not possible to ascertain what is happening at an individual cell level at any one time; however it is possible to derive what is happening to the ricin movement within the whole cell population. Ricin is endocytosed into the cytoplasm within the population quickly and reaches plateau at around 30–45 min, at this time some 25% of cells have ricin within the Golgi apparatus and this percentage increases relatively slowly until most cells with cytoplasmic ricin also have Golgi-associated ricin too. Ricin is transported from the Golgi to the ER, where the A and B chains dissociate and move independently within the cell [2]. It is important to note that it is not possible to discriminate between the A and B chains of ricin with the labelled ricin used in these studies. Therefore once a fluorescence signal is observed in the Golgi apparatus, and the ricin chains dissociate in the ER, it is currently not possible to follow the fate of the individual chains. In our model of Golgi-associated ricin, an estimated plateau is not reached until after the final time point at 4 h. This means that, as a population, there is a lag between ricin entering the cytoplasmic compartment of the cell population, and being trafficked to the Golgi environment of approximately 3–3.5 h. This lag to plateau is expected due to the nature of the movement of ricin from the endosome though the retrograde transport system to the Golgi apparatus. The rapid cellular uptake and trafficking of ricin noted in these studies is consistent with previous in vitro studies demonstrating that only short duration exposures are sufficient to cause cytotoxicity at later time points [31]. These observations seem at odds with in vivo protection studies using anti-ricin antibody treatments where protection can be observed for many hours after the initial ricin exposure. This has been demonstrated for intraperitoneal [32], inhalation [33] and intranasal [34–36] challenges with ricin. For the latter route, partial protection was achieved by antibody administrations as late as 72 h after ricin challenge in mice. It has been generally assumed that the anti-toxin mediated neutralisation of toxins occurs extracellularly whilst the toxin is extracellular (free or cell surface attached), however, given the rapid kinetics of ricin uptake it is difficult to reconcile this mode of neutralisation with the in vivo protection observed many hours after intoxication. This suggests that either the kinetics of ricin cellular trafficking observed in vitro are not reflected in vivo, or that the assumption of a purely extracellular mode of neutralisation is an incomplete explanation of the mechanism of post-exposure antitoxin. Indeed it has been observed that both A and B-chain directed anti-ricin antibodies have the ability to neutralise ricin activity intracellularly and can disrupt the intracellular trafficking of ricin [15,16,37,38]. Clearly, understanding the impact of antitoxinbased therapies on intracellular ricin trafficking is important. IFC technology could therefore be applied to provide quantitative cell population data to support mechanistic studies of antitoxins or small molecule-mediated strategies [39,40] that block or redirect
normal intracellular trafficking of ricin towards proteolytic or other non-toxic routes. Methods for studying ricin trafficking have, over the years, used many different methodologies including confocal microscopy [15], radio-labelling [27], electron microscopy [29] and more recently, GFP reporter assays [41]. Here we have shown that it is possible to examine the endocytosis of ricin using imaging flow cytometry. Although this technique does not have as high resolution as confocal/electron microscopy, IFC benefits from the collection of high throughput data and large populations of cellular images. This provides robust image analysis and data sets that better represent the cell population of interest. The assay presented in this work is an example of using imaging flow cytometry for evaluating the trafficking of toxins. This approach can be used as a tool for the early evaluation of toxin therapies to help establish their mechanisms of action, or more broadly, to compare the endocytosis kinetics of toxins in different cell types and tissues. Acknowledgements The authors would like to thank Fiona Stahl for her help and assistance running the ricin toxicity assays. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.ymeth.2017.10.012. References [1] CDC, Biological and chemical terrorism: strategic plan for preparedness and response: recommendations of the CDC Strategic Planning Workgroup, Morbidity and Mortality Weekly Report. Recommendations and Reports, 49 (RR-4), 2000, 1–14. [2] R.A. Spooner, J.M. Lord, Ricin trafficking in cells, Toxins 7 (1) (2015) 49–65. [3] W. Montfort, J.E. Villafranca, A.F. Monzingo, S.R. Ernst, B. Katzin, E. Rutenber, N. H. Xuong, R. Hamlin, J.D. Robertus, The three-dimensional structure of ricin at 2.8 A, J. Biol. Chem. 262 (11) (1987) 5398–5403. [4] G. Griffiths, P. Rice, A. Allenby, S. Bailey, D. Upshall, Inhalation toxicology and histopathology of ricin and abrin toxins, Inhalation Toxicol. 7 (2) (1995) 269– 288. [5] D. Franz, N. Jaax, Ricin toxin, in: F.R. Sidell, E.T. Takafuji, D.R. Franz (Eds.), Medical Aspects of Chemical and Biological Warfare, Office of the Surgeon General, Falls Church, Virginia, 1997, pp. 631–642. [6] J. Audi, M. Belson, M. Patel, J. Schier, J. Osterloh, Ricin poisoning: a comprehensive review, JAMA 294 (a18) (2005) 2342–2351. [7] K.R. Challoner, M.M. McCarron, Castor bean intoxication, Ann. Emerg. Med. 19 (10) (1990) 1177–1183. [8] B. Furbee, M. Wermuth, Life-threatening plant poisoning, Crit. Care Clin. 13 (4) (1997) 849–888. [9] P.A. Kinamore, R.W. Jaeger, F.J. de Castro, Abrus and ricinus ingestion: management of three cases, Clin. Toxicol. 17 (3) (1980) 401–405. [10] W. Palatnick, M. Tenenbein, Hepatotoxicity from castor bean ingestion in a child, J. Toxicol. Clin. Toxicol. 38 (1) (2000) 67–69. [11] R.P. Reed, Castor oil seed poisoning: a concern for children, Med. J. Aust. 168 (8) (1998) 423–424. [12] G.P. Wedin, J.S. Neal, G.W. Everson, E.P. Krenzelok, Castor bean poisoning, Am. J. Emerg. Med. 4 (3) (1986) 259–261. [13] H. Bigalke, A. Rummel, Medical aspects of toxin weapons, Toxicology 214 (3) (2005) 210–220. [14] M. Kortepeter, G. Christopher, T. Cieslak, USAM-RIID’s Medical Management of Biological Casulaties Handbook, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, 2001. [15] K. Song, R.R. Mize, L. Marrero, M. Corti, J.M. Kirk, S.H. Pincus, Antibody to ricin A chain hinders intracellular routing of toxin and protects cells even after toxin has been internalized, PLoS One 8 (4) (2013) e62417. [16] A. Yermakova, T.I. Klokk, R. Cole, K. Sandvig, N.J. Mantis, Antibody-mediated inhibition of ricin toxin retrograde transport, mBio 5 (2) (2014). e00995. [17] K. Sandvig, K. Prydz, S.H. Hansen, B. van Deurs, Ricin transport in brefeldin Atreated cells: correlation between Golgi structure and toxic effect, J. Cell Biol. 115 (4) (1991) 971–981. [18] J.P. Frenoy, E. Turpin, M. Janicot, F. Gehin-Fouque, B. Desbuquois, Uptake of injected 125I-ricin by rat liver in vivo Subcellular distribution and characterization of the internalized ligand, Biochem. J 284 (Pt 1) (1992) 249–257. [19] P.Z. Chia, P.A. Gleeson, Imaging and quantitation techniques for tracking cargo along Endosome-to-Golgi transport pathways, Cells 2 (1) (2013) 105–123.
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