Published: September 10th, 2018
Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.
Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.
Large-scale non-specific fluid uptake by macropinocytosis is important in several biological contexts1, including antigen sampling by immune cells2, pathogen entry into host cells3, cancer cell proliferation4 and the spreading of prion diseases5. In mammalian and Dictyostelium cells, actin6,7, PI(3,4,5)P38,9,10 (although the exact nature of the lipid differs between the two11), activated Ras12,13, and activated Rac14,15 are important for efficient fluid uptake by macropinocytosis, although there remain many unanswered questions about how the macropinocytic patch is formed, organized and eventually internalized. Discovering more proteins important for macropinocytosis, and subsequent determination of how they are important in the various biological contexts, will give a more comprehensive understanding of macropinocytosis and potentially allow development of targeted treatments for a range of conditions.
Dictyostelium is an ideal model system for studying macropinocytosis. The high level of constitutive macropinocytosis in standard laboratory strains means that fluid uptake is over 90% due to macropinocytosis6. This allows macropinocytosis to be measured solely by determining fluid uptake, unlike mammalian cells where the proportion of fluid uptake due to macropinocytosis is much lower. That macropinocytosis is so well defined and easily visualized12 in this system similarly offers distinct advantages for investigating core conserved components of the macropinosome over other systems where there may be multiple regulatory signals16,17.
The standard technique used to measure macropinocytosis by mammalian cells involves fixing cells after pulsing with dextran for a short period of time followed by microscopy to determine the area of a cell that is occupied by dextran-positive vesicles18. This technique does not however account for the possibility of macropinosomes shrinking upon entering the cell, which has been reported in Dictyostelium19, and only takes into account single planes of the cell, meaning the volume internalized is unclear. An alternative technique, of counting the number of macropinosomes internalized in a given time, has the same downsides20. Using Dictyostelium avoids these issues; however, existing techniques for measuring fluid uptake by Dictyostelium are relatively labor intensive, using a large amount of both cells and dextran21. Cells are shaken at high density in fluorescent dextran and samples removed at various time-points for determination of the internalized fluorescence using a fluorimeter. Cells prepared this way can be analyzed by flow cytometry to gain single cell, rather than population-level, resolution22, although this remains low-throughput.
Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment. Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. This method was designed to overcome the limitations of the above methods and allow simultaneous comparison of the fluid uptake of large numbers of strains/conditions while using fewer resources and reducing the labor involved.
1. Preparation of Cells and Materials
2. Converting Qualitative Measurements into Quantitative (Optional)
3. Measuring Fluid Uptake
Figure 1: Schematic of high-throughput measurement of macropinocytosis. (A) Grow Dictyostelium on an SM plate seeded with K. aerogenes bacteria (yellow). Harvest cells from the feeding front (orange), avoiding cells that are already developed (green), into 25 mL of KK2 buffer. Vortex to dissociate, pellet by 3 min centrifugation at 300 x g, then wash 3 times in 50 mL of KK2 buffer, discarding the supernatant each time. Resuspend to 1 x 105 cells/mL in HL5 growth medium and add 50 µL into three wells per sample of a flat bottom 96-well plate. Incubate at 22 °C for 24 h. (B) Dilute TRITC-dextran to 1 mg/mL in HL5 growth medium from a stock solution of 50 mg/mL. Add 50 µL to each sample (excluding the 0 min uptake control wells), and incubate at 22 °C for 1 h, after which add the dextran to the 0 min uptake control. (C) Immediately decant the media, pat the plate on a tissue to remove excess medium and submerge in a bath of ice-cold KK2 buffer, filling the wells to wash. Decant the buffer and pat dry again. Add 100 µL of 5 mM sodium azide dissolved in KK2MC to detach the cells. Take to flow cytometer for measurement of internalized fluorescence. Please click here to view a larger version of this figure.
4. Performing Fluid Uptake Time-courses
5. Dose Response Curves
6. Phagocytosis and Membrane Uptake
Once the technique has been performed and cells are loaded with dextran and ready for analysis (Figure 1), ensure the flow cytometer is not blocked and adjust the forward scatter/side scatter profile to look like the cells shown in Figure 2A. If the machine is blocked, it will look more like that shown in Figure 2B and must be unblocked before continuing. Ensure the parameters show control axenic cells have high internalized dextran fluorescence at longer time points and low internalized fluorescence at shorter ones (Figure 2C).
When looking for differences between mutants, it is likely that there will be one of three phenotypes. The mutants could have normal fluid uptake, they could have a partial defect, or fluid uptake could be completely abolished. Figure 2D shows a strain with normal fluid uptake, in this case the standard laboratory strain Ax2, a mutant with a ~50% decrease in fluid uptake (Ax2 rasG-14) and one with abolished fluid uptake (Ax3 gefB-28). Obtain the average median fluid uptake (section 3.5) and use it to either calculate the volume of fluid internalized (as in Figure 3B) or compare the data to a control (as in Figure 4B and 4C).
When performing a fluid uptake time course, as in Figure 3A, the internalized fluorescence should increase for 60–90 min, after which the dextran begins to be exocytosed and a plateau is reached (Figure 3B). Using 60 min as the standard time point when comparing macropinocytosis in different mutants/conditions therefore allows a good signal to be achieved, and no signal is lost due to exocytosis. Mutants where exocytosis is severely obstructed may take longer to reach a plateau29.
When treating cells with inhibitors that are effective against macropinocytosis in Dictyostelium (set up as in Figure 4A), the dextran internalized in 1 h will go down to almost nothing at higher inhibitor concentrations in the majority of cases (Figure 4B). Some inhibitors may not be 100% effective, however, e.g. nocodazole only inhibits up to 50% of fluid uptake by macropinocytosis when added acutely (Figure 4C). If the inhibitors are not effective, the cells will internalize a similar amount of dextran as the control, and a decrease in fluorescence will not be seen. This technique allows a large range of different inhibitors and inhibitor concentrations to be screened for effects on fluid uptake by macropinocytosis very quickly, reducing the time spent optimizing the inhibitor treatment.
Figure 2: Set up of flow cytometer and representative data. (A) The forward scatter (FSC) and side scatter (SSC) profiles of cells should be set so the cells can be easily distinguished. An example of how Ax2 cells should look is shown. (B) If the flow cytometer is blocked, as in this example, the cells have very low side scatter. The laser will not excite the fluorophores properly and the machine should be unblocked before continuing. Any data obtained while the machine was blocked should be discarded. (C) The fluorescence should be set so that a 0 min uptake sample has low fluorescence, which increases when cells have been incubated for longer in the fluorescent medium, as shown in this example taken from Williams & Kay 201824. (D) Examples of cells that have been incubated with TRITC dextran for 1 h with normal macropinocytosis (Ax2, green), reduced macropinocytosis (Ax2 rasG-, HM172614, orange) and abolished macropinocytosis (Ax3 gefB-, HM177628, blue). Please click here to view a larger version of this figure.
Figure 3: Performing fluid uptake time-courses in 96-well plates. (A) Dextran should be added to each set of samples sequentially, with the same finish time. Then wash the wells, detach and measure the internalized fluorescence by flow cytometry. Example times to add the dextran are shown here. (B) Fluid uptake time-course of Ax2 cells performed in 96-well plates. Taken from Williams & Kay 201824, error bars show the standard error of three independent experiments. Please click here to view a larger version of this figure.
Figure 4: Fluid uptake dose response curves. (A) Add the compound of interest, in this case the PI3K inhibitor LY294002, to HL5 containing 1 mg/mL TRITC-dextran at double the desired final maximum concentration. Mix with HL5 growth medium + 1 mg/mL dextran containing vehicle alone in various proportions to generate a dilution series of 200 µL medium per condition. Add to wells as normal for 1 h before washing and measuring internalized fluorescence. (B) Fluid uptake dose response curve for Ax2 cells incubated with the LY294002-containing medium from A. Adapted from Williams & Kay 201824. Fluid uptake is normalized to an untreated control. Error bars show the standard error of three independent experiments. (C) Fluid uptake dose response curve for Ax2 cells incubated with the nocodazole. Adapted from Williams & Kay 201824. Fluid uptake is normalized to an untreated control. Error bars show the standard error of three independent experiments. Please click here to view a larger version of this figure.
Whereas other methods to assess fluid uptake are low throughput, washing the cells in situ and the use of sodium azide to detach cells are the critical steps in this method, which allow high-throughput measurement of macropinocytosis, membrane uptake, or phagocytosis by Dictyostelium. As the cells are attached to a surface and the medium is not, they can be left attached while the medium around them is first thrown off and then changed by immersion in buffer and thrown off again. Sodium azide, which depletes cellular ATP and depolarizes the membrane30, is then used to detach the cells, and also prevents exocytosis without affecting cell viability24.
While using flow cytometry to measure macropinocytosis by Dictyostelium gives a very accurate measurement of fluid uptake very quickly, to establish the reason why a particular strain or condition has altered fluid uptake, further investigation using microscopy is required24. It should also be noted that previously published results have, in some cases, shown a difference in fluid uptake by mutant strains grown either on a surface (as in this case), or in shaking suspension (as in the standard protocol)31. Using this method may mean that, in rare cases, apparent fluid uptake defects are missed. Additionally, when measuring phagocytosis, only low concentrations of particles can be used. The maximum rate of phagocytosis that can be determined with this technique is far below the real maximum, although it is still possible to measure relevant differences in phagocytosis between strains and conditions24. To determine the maximum rate of phagocytosis, uptake must be measured in shaking suspension by an alternative protocol27. Cells that have phagocytosed beads have increased side scatter, so this should be corrected for accordingly when setting up the flow cytometer.
Flow cytometry can be used to measure fluid uptake in mammalian cells32, however the higher proportion of fluid phase uptake by other endocytic pathways than seen in Dictyostelium is a concern. In addition, cells are typically detached using trypsin at 37 °C, allowing further endocytic progression of internalized dextran. Ice-cold sodium azide does not cause macrophages to detach from a surface (Williams, unpublished observation), making this technique not applicable to mammalian cells without further optimization.
High throughput measurement of macropinocytosis has the potential to be used to screen quickly and cheaply for the effects of inhibitors, genetic mutation or gene knockdown on Dictyostelium cells. Mutants should always be compared to their direct parent only. If the reader has no prior preference for Dictyostelium strain, non-axenic strains such as DdB or NC4 are more "wild-type" than axenic ones and can be manipulated as effectively as axenic strains33. Otherwise, Ax2 strains are the axenic strains with the fewest genome duplications34, while many strains of Ax4 are Talin A knockouts and should be avoided if possible23. Most previously published strains can be ordered from the Dicty Stock Center35.
This technique allows greater investigative possibilities than was previously possible into the effects of different conditions, inhibitors and mutations on macropinocytosis by Dictyostelium.
The authors have no conflicts of interest to disclose.
We thank the Medical Research Council UK for core funding (U105115237) to RRK.
|LSR_II flow cytometer
|Other Flow cytometers can also do this role, e.g. the LSRFortessa by BD
|TRITC-dextran (155 kDa)
|Other non-quenchable dextrans, and other sizes are also fine
|96-well tissue culture plate
|Any flat-bottom tissue culture treated 96-well plate will work
|Magnesium sulfate hydrate
|Calcium chloride dihydrate
|Potassium dihydrogen phopshate
|Di-potassium hydrogen phosphate
|LS 50 B
|Fluoresbrite YG-carboxy microspheres 1.00 µm
|Fluoresbrite YG-carboxy microspheres 1.50 µm
|Fluoresbrite YG-carboxy microspheres 1.75 µm
|Fluoresbrite YG-carboxy microspheres 2.00 µm
|Texas Red E. coli bioparticles
|0.22 µm syringe filter
|Elkay Laboratory Products
|10 mL Syringe
|Round-bottom polystyrene tubes
|Use a tube that will fit onto your flow cytometer.
|70 µm cell strainer
|50 mL centrifuge tube
|5 mL repeating pipette tips
|Cayman Chemical Company
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