Dictyostelium Cultivation, Transfection, Microscopy and Fractionation

The real time visualisation of fluorescently tagged proteins in live cells using ever more sophisticated microscopes has greatly increased our understanding of the dynamics of key proteins during fundamental physiological processes such as cell locomotion, chemotaxis, cell division and membrane trafficking. In addition the fractionation of cells and isolation of organelles or known compartments can often verify any subcellular localisation and the use of tagged proteins as bait for the immunoprecipitation of material from cell fractions can identify specific binding partners and multiprotein complexes thereby helping assign a function to the tagged protein. We have successfully applied these techniques to the Dictyostelium discoideum protein TSPOON that is part of an ancient heterohexamer membrane trafficking complex (Hirst et al ., 2013). TSPOON is the product of the tstD gene in Dictyostelium and is not required for growth or the developmental cycle in this organism. Dictyostelium amoebae will exist in a vegetative phase where growth is sustained by the phagocytosis of bacteria. When this food source is spent they enter a developmental phase where the amoebae aggregate, via chemotaxis to extracellular waves of cAMP, into multicellular structures that subsequently form a fruiting body containing viable spores (Muller-Taubenberger et al ., 2013). In the laboratory this cycle takes less than 24 h to complete and as a further aid to manipulation the requirement for a bacterial food source has been circumvented by the derivatisation of the wild type and isolation of axenic strains that can also grow in a nutrient rich broth. Axenic strains like Ax2 are the mainstay of laboratory research using Dictyostelium (Muller-Taubenberger et al ., 2013). A description of Dictyostelium cell cultivation, the generation of cell lines that overexpress TSPOON-GFP and TSPOON null cells, and subsequent analysis (Muller-Taubenberger and Ishikawa-Ankerhold,


3.
Alternatively, cells can be grown on tissue culture plates and harvested when confluent (each plate should yield 1-2 × 10 7 cells) ( Figure 1). This is useful when relatively few are required for confocal or TIRF microscopy. Pang et al., 1999) 1. Harvest mid log phase cells into 50 ml sterile plastic tubes and centrifuge at 300 × g for 2-3 min to pellet the cells. Aspirate supernatant and add 50 ml of ice cold E50 buffer and resuspend the cell pellet by gently tapping and shaking the tube.

2.
Determine the number of cells/ml using an automated cell counter or a haemocytometer.

3.
Centrifuge at 300 × g for 2-3 min to pellet the cells and then resuspend at 4 × 10 7 /ml in ice cold E50 buffer. Place the cell suspension on ice and incubate for 5 min (if the cells start to settle then gently mix by agitation).
a. For overexpression plasmids such as pJH101 (TSPOON-GFP fusion driven by the constitutively active actin15 promoter) or pDT58 and pDT61 (both contain the TSPOON-GFP fusion driven by the TSPOON promoter), transfer 0.1 ml of the cell suspension to a prechilled sterile electroporation cuvette (1 mm gap width) and add 10-30 μg of supercoiled plasmid DNA (preferably in ≤15 μl of Tris-HCl or TE buffer and prepared according to the manufacturer's instructions using the HiSpeed ® Plasmid Midi Kit) then mix by gently pipetting up and down avoiding introducing air bubbles to the cell suspension. Return the cuvette to ice ( Figure 2).
b. The most common and reliable gene disruption in Dictyostelium is achieved with blasticidin S as the selective agent using a plasmid containing the resistance cassette (such as pLPBLP) flanked on either side with DNA homologous to the targeted gene (Faix et al., 2004;Faix and Kimmel, 2006). This targeting/disruption cassette should be freed from the remainder of the construct by complete digestion with restriction enzymes. To knockout TSPOON, plasmid pDT70 (based on pLPBLP) was cut with ApaI and SacII (these enzymes generate 3′ overhangs in the cut DNA but enzymes that leave 5′ overahangs e. g. BamHI or blunt cutters like PvuII can be used in any combination to liberate the disruption cassette). The enzymes where removed by a single phenol extraction followed by three extractions with chloroform and the cut DNA precipitated with ethanol (Sambrook and Russel, 2001). Resuspend the cut DNA at 2-3 mg/ml in Tris-HCl or TE buffer. Add 15-20 μg of the disruption cassette DNA to each 0.1 ml of cell suspension, again in a chilled sterile electroporation cuvette (1 mm gap width) and mix as in step B4a.

4.
The cell/DNA suspension should be electroporated quickly to minimise cell sedimentation in the cuvette.
a. Remove the cuvette from the ice and dry off any excess moisture on the electrodes with a tissue before placing in the electroporation pod.
b. The parameters should be set at 0.75-0.85 kV, 25 μF capacitance with resistance set at infinity (∞) for the Bio-Rad Xcell. The same parameters are used on the Bio-Rad GenePulser and GenePulserII however they may need to be optimised with electroporation devices from other manufacturers.
c. Deliver two pulses 5 sec apart to each cuvette and then quickly add 0.5 ml of sterile room temperature axenic medium, mix by gentle pipetting up and down and then return the cuvette to ice for 5 min.

1.
For each cuvette prepare a 6 well tissue culture plate with 3 ml of axenic medium in each well.

2.
Remove a cuvette from ice and resuspend the cells by gentle pipetting before adding 0.1 ml to each well of the tissue culture dish.

5.
Change the selection medium by aspiration to remove dead cells every 2-3 days and the wells should be confluent after 9-14 days. (Hygromycin appears to be less efficient at killing Dictyostelium amoebae than G418 and it is therefore important to replace the medium every 2-3 days as directed to ensure the removal of dead and dying cells that will become detached from the tissue culture plate).

6.
The wells are screened for optimum expression by dislodging the cells by pipetting up and down and removing 0.3 ml into a single well of a chambered coverslip (8 well).

7.
The cells are allowed to settle and attach for 20-30 min before the medium in each well is aspirated and replaced by 0.3 ml of LOFLO medium or KK 2 C.

8.
The cells are then examined by confocal or TIRF microscopy for the presence and distribution of the GFP fusion protein (Figures 3 and 4).

9.
To make a permanent stock, the well(s) with the optimum expression are harvested by pipetting the axenic medium up and down to dislodge the cells which are then used to initiate a larger culture (grown with the appropriate levels of G418 or hygromycin) either in shaken suspension or several 10 cm tissue culture dishes each with 12 ml of axenic medium.

10.
A minimum of 8 × 10 7 cells should be harvested and pelleted by centrifugation (300 × g for 2 min) then resuspended in 1.6 ml of freezing medium and processed as in step C23.

11.
To isolate knockout mutants the cell suspension should be diluted to appropriate densities to ensure clonality over a range of cell survival. This is particularly important when the targeted gene affects growth because wild type non-homologous recombinants are likely then to outgrow such a knockout, making the screen and its isolation more difficult.

12.
To ensure clonality, the cell suspension from a single cuvette is first added to a total of 15 ml of axenic medium in 50 ml plastic tube so that the cells are approximately 2.67 × 10 5 /ml.
14. Normally 4 × 96 well plates are prepared for the 100, 200 and 400 fold dilutions so 50 ml of diluted cells should be prepared for each of these dilutions.

15.
Incubate the plates in a moist atmosphere (such as in a large plastic cake storage box lined with wet tissues) at 22 °C overnight (normally 16-24 h) before adding 100 μl of axenic medium containing 20 μg/ml blasticidin S to each well (10 μg/ml final).

16.
Change the selection medium to remove dead cells every 2-3 days by forcefully 'throwing' the medium into a large tissue lined (stops splash back into wells) container. Add 200 μl of fresh axenic medium containing 10 μg/ml of blasticidin S to each well.
Note: Plating out the cells into the 96 well plates and replenishing the medium is best achieved with a 8 channel electronic pipette fitted with tips each with a capacity of 1,200 μl. The cell suspension or fresh media is dispensed from a sterile plastic reservoir. These reservoirs can be reused after autoclaving in a steribag pouch.

17.
The 96 well plate containing the least diluted cells should have confluent wells after 9 days whereas the plates containing the 100, 200 and 400 fold dilutions may take up to 21 days (times are average based on the use of many different knockout vectors but deletion of a gene that impairs growth in axenic medium is likely to increase these times). Dilutions are usually screened from plates where <30 wells/plate are occupied, as these are likely to be clonal.

18.
The cells from each confluent well are harvested by pipetting up and down, then transferred to 1.5 ml microcentrifuge tube containing 0.75 ml of sterile KK 2 (this is to dilute residual axenic medium after the cells are pelleted).

19.
Refill the well with 200 μl of fresh axenic medium containing 10 μg/ml of blasticidin S. The cells are pelleted in a benchtop microfuge at full speed for 20 sec and the supernatant removed by aspiration.

20.
Genomic DNA from the cell pellet can then be isolated using a variety of commercial kits but we prefer the Quick-gDNA MiniPrep kit from Zymo Reasearch as it is quick, has few steps and reproducibly yields good quality genomic DNA for screening by PCR using a variety of polymerases [Expand 20 kb plus polymerase (Roche) was found to be optimal for the TSPOON screen but we have also used Q5, Pwo and KOD polymerases].

21.
In the case of the TSPOON knockout, isolation and screening took 17 days from the start of selection until confluent wells where observed in the plates containing the 200 and 400 fold dilutions.
22. The PCR screen used TSPOON specific primer (TCP) oligonucleotides TCP15: (5′-GATGAAATTTATCAGATATTGATTTCATGAATGTTTCACC-3′) and d. The insertion of blasticidin resistance cassette and flanking DNA introduces restriction sites into the knockout PCR product that are absent in the wild type. This is a useful further proof of correct targeting as digestion of these products with a restriction enzyme such as SmaI for TSPOON, leaves the wild type product intact but cuts the knockout product into 3 fragments.

e.
A total of 46 wells where screened by this method, 37 were TSPOON knockouts, 6 were wild type and 3 were not clonal as they contained both PCR products. Therefore, the targeting frequency for this the tstD gene was 80% and though high was in keeping with other genes targeted by this procedure in our laboratory such as 76% for iplA, 67% mscS, 57% mclN, 50% phdA and 12% dagA.

23.
Four TSPOON knockouts, isolated from different 96 well plates, were processed further (2 for study and 2 as backup).
a. The wells containing these cells were harvested as before, the cells diluted in KK2 to 0.6-1.2 cells/μl (a confluent well contains ~10 5 cells).
b. SM agar plates were prepared each with a 0.4 ml drop of a dense suspension of Klebsiella aerogenes bacteria ( Figure 5).
c. Add 10 μl of the diluted cell suspension to this drop and spread evenly over the plate with a sterile plastic spreader.
d. Discrete colonies appear within 4-5 days and one per knockout was harvested when they had reached ~1.5 cm in diameter.
e. The flat end of a sterilised small metal spatula was used to transfer the entire colony directly into 1.6 ml of freezing medium within a CryoTube on ice. The CryoTubes were intermittently vortexed to ensure an even suspension and then transferred to cryo preservation module chilled to 4 °C.
f. The module is then transferred to a −80 °C freezer overnight and then the tubes can remain at −80 °C (cells remain viable for >2 years) or be transferred to a liquid nitrogen storage tank (indefinite viability).

1.
Fixed and live cells were analysed my microscopy at the vegetative or the aggregation competent stage of development.

a.
Simply harvest growing cells from axenic culture and transfer an appropriate number of cells so that final density is 0.5-1.5 × 10 5 /cm 2 into a glass bottom dish or chambered coverslip containing LOFLO medium (this has been formulated to minimise background and cellular autofluorescence compared to standard axenic medium and it will keep the cells vegetative for a while) or KK 2 C (they will start to develop in this buffer but autofluorescence and photosensitivity will be minimised).

b.
Incubate at 22 °C for 10 min to allow the cells to attach then aspirate the medium (do not allow the cells to dry) and replace with fresh LOFLO or KK 2 C. They can then be used for live cell imaging or fixed.

For developed cells c.
Transfer 0.5-1.5 × 10 5 /cm 2 into KK 2 C and replace the KK 2 C once the cells have attached.

d.
Incubate at 22 °C for 8-10 h to allow the cells to become aggregation competent.

e.
Alternatively transfer 1 × 10 6 cells into a 35 mm tissue culture dish containing 2 ml of KK 2 C, allow the cells to attach and then replace the KK 2 C and incubate at 22 °C for 1 h.

f.
Transfer the dish to 15 °C overnight (15-17 h) and then return the dish to 22 °C for 1 h before transfer (at 0.5-1.5 × 10 5 /cm 2 ) to a glass bottom dish (the cells should aggregate within 2-3 h at 22 °C).

g.
If larger quantities of developed cells are needed then resuspend vegetative cells at 2 × 10 7 /ml in KK 2 C and transfer them into a conical flask with a volume of at least 5 times that of the cell suspension to ensure optimum aeration. The cells are shaken at 180 rpm at 22 °C for 1 h then a 100 μl droplet of KK 2 C, containing enough cAMP (diluted from a master stock) so that the final concentration of the entire suspension is 100 nM, is dispensed into the suspension every six minutes for 3.5-5.0 h via a programmable peristaltic pump. This procedure mimics the pulsatile waves of cAMP that are emitted through a developing population of amoebae and is useful for mutant strains defective in early development and cAMP signalling.

h.
Harvest the cells and pellet by centrifugation 300 × g for 2 min, aspirate off the supernatant and resuspend the cells at 1 × 10 7 /ml in KK 2 C and plate as before at 0.5-1.5 × 10 5 /cm 2 .

i.
There are numerous ways to fix Dictyostelium amoebae for immunocytochemistry (ICC) and they can be found elsewhere

a.
Live cell imaging is normally a balance between detecting the tagged fusion protein (signal to noise ratio) and damaging the cells with the laser due to free radicals produced by the illumination interacting with cellular constituents or the fluorophore tag. With a 10× or 20× lens this is never normally a problem but at these magnifications there is little or no spatial resolution within a cell to be gained given that Dictyostelium cells are ~10-15 μm in diameter.

b.
Normally a 63× or 100× lens is needed to resolve the subcellular localisation of a tagged protein within a Dictyostelium amoeba.

c.
Vegetative amoebae are particularly sensitive to damage especially from shorter wavelengths such as the 405 nm laser. An Argon laser (25-35 mW) is fitted to most confocal miscroscopes for imaging YFP, GFP and CFP fusion proteins and excessive power will result in phototoxicity and photobleaching so the laser strength should be kept to a minimum (normally 2-10% for the 488 nm laser line).

d.
Longer wavelength laser lines such as 561 mn used for RFP tagged proteins present less of a problem and can be used at higher power (>15%) signal can be increased by opening the pinhole.

e.
Avoid line averaging, slow scans and real time deconvolution.

f.
Developed cells are usually less sensitive to laser damage but it can be fusion protein dependent. For instance a GFP tagged protein that resides in the plasma membrane may render the cells more sensitive (due to lipid oxidation in the membrane) than a cytoplasmic protein. L-Ascorbic acid (50-100 μm final) to scavenge free radicals can be added to minimise this problem and the vitamin E analogue Trolox C has also been used to this end (add 1 mM to the growth medium and incubate overnight before imaging).

g.
If phototoxicity cannot be overcome then consider switching to a spinning disc confocal microscope (SDCM) where illumination is restricted to thousands of small confocal volumes during image acquisition rather than through the whole sample thus reducing photobleaching and phototoxicity.

h.
To follow the movement of vegetative cells frames should be captured every 2-30 sec and for developed cells every 1-20 sec. The fastest realistic frame rate is 1/sec. It may be necessary to reduce the area scanned from the default 512 pixels × 512 pixels to 512 × 300 as this allows slower scans (better images) and maximises the frame rate (Frigault et al., 2009; Muller-Taubenberger and Ishikawa-Ankerhold, 2011).

i.
For cells expressing the promoter_TSPOON-GFP construct typical settings used had the 488 nm laser power set at 5% and the pinhole opened up to 2.5 Airy units with a 512 pixels × 300 pixels frame collected every second. For faster temporal resolution switch to a SDCM.

j.
With fixed cells and ICC photobleaching can be a problem but the use of an antifade mounting medium (typically Fluoromount-G ® ) can minimise this and allows slower scan speeds and averaging to give better images.

TIRF microscopy.
a. The problem of phototoxicity is usually more acute with vegetative Dictyostelium cells, in part because although the evanescent field is limited to the initial ~100 nm of the sample from the coverslip, the plasma membrane is strongly illuminated.
b. Image cells that have been in LOFLO or KK 2 C for at least one hour. The inclusion of L-Ascorbic acid may be necessary.
c. Keep the laser power and the illumination time to a minimum.

d.
Since there is no scanning the frame rate is higher. A 100× lens (high NA) should be used with an additional 1.5× zoom lens if present as this allows ameobae to be imaged in great detail.
f. Typical settings for cells expressing promoter_TSPOON-GFP construct had the 488 nm laser power set at 7% with an exposure time of 0.08 sec collecting 512 × 512 frames at up to 12 per sec.

1.
Cells expressing A15_GFP (control) or promoter_TSPOON-GFP were grown until they reached a density of 2-4 × 10 6 /ml in selective media, and by microscopy >50% of cells were expressing GFP.

2.
Starting with a maximum of 8 × 10 8 cells, the cells were washed in KK 2 buffer and then pelleted at 300 × g for 3 min. All subsequent steps were performed at 4 °C.

3.
The cells were resuspended in PBS with a protease inhibitor cocktail, lysed by 8 strokes of a motorized Potter-Elvehjem homogenizer (grinding chamber clearance 0.1-0.15 mm) followed by 5 strokes through a 21-g needle to ensure full lysis.

4.
Alternatively, the cells can be resuspended at 1-5 × 10 8 /ml and placed in a syringe fitted with a 25 mm filter holder containing a prefilter and a Nucleopore filter (3.0 μm). The cell suspension is then passed through this filter assembly twice.

5.
The lysate was then centrifuged at 4,100 × g for 32 min to pellet nuclei and unbroken cells, and the postnuclear supernatant further centrifuged at 50,000 rpm (135,700 × g RCF max ) for 30 min in a TLA-110 rotor to recover the membrane pellet and cytosolic supernatant.

6.
A standard protein assay was used to assess protein recovery in the 2 fractions, and volumes adjusted for equal protein.

7.
Alternatively, to equalise volumes, the cytosolic supernatant was concentrated by precipitation with 10% tricholoroacetic acid at 4 °C for 30 min and recovered by centrifugation 14,000 × g for 10 min. Samples precipitated with trichloroacetic acid were washed with ice cold acetone (−20 °C), air dried for 2 min and then resuspended in the same volume as the pellet fractions.

F. Protein pulldowns
Immunoprecipitations were performed using amoebae stably expressing TSPOON-GFP under a constitutive (A15_ TSPOON-GFP) and its own promoter (prom_TSPOON-GFP), and non-transformed cells were used as a control. The amounts given here are recommended for a large scale immunoprecipitation sufficient for proteomic identification of interacting proteins under native conditions. The size of the starting culture, and all subsequent volumes can be reduced accordingly for smaller scale immunoprecipitations, for instance where the identification of proteins are made by Western blotting. The protocol can also be amended for denatured (by heat and/or the presence of SDS but are not detailed here) immunoprecipitations.

1.
Cells were grown until they reached a density of 2-4 × 10 6 /ml in selective media, and by microscopy >50% of cells were expressing GFP.

2.
Up to 8 × 10 8 cells were pelleted by centrifugation at 300 × g for 2 min, washed twice in 50 ml of KK 2 buffer before being resuspended at 2 × 10 7 cells/ml in KK 2 buffer and starved for 4-6 h at 22 °C whilst shaking at 180 rpm.

3.
The cells were then pelleted at 300 × g for 3 min, lysed in 4 ml PBS-T plus protease inhibitor cocktail tablet, extracted for 20 min with rotation at 8 rpm in a 15 ml tube at 4 °C, and then spun 20,000 × g for 15 min to remove debris and insoluble material.

4.
By BCA protein assay the resulting lysate contained 10-15 mg total protein.

5.
The lysates were precleared by adding 100 μl of PA sepharose bead slurry (50% v/v in PBS) and incubated for 30 min with rotation at 8 rpm, followed by centrifugation at 2,200 × g for 3 min to pellet beads.

6.
The supernatant was transferred (~ 5 ml) to a fresh tube, and this is the starting material for the immunoprecipitation (protein concentration should be 2-4 mg/ml).

7.
For maximum recovery the lysates were immunoprecipitated using an inhouse antibody against GFP overnight with rotation at 4 °C, though it may be sufficient to incubate for as little as 90 min.

8.
The appropriate antibody concentration requires individual optimization, though the recommended starting point is generally 2 to 5-fold higher than used for Western blotting.

9.
Following incubation with anti-GFP, 50 μl PA sepharose bead slurry (50% v/v in PBS) was added for 90 min at 4 °C with rotation at 8 rpm.
10. An alternative would be to use a commercial source of polyclonal anti-GFP, or an anti-GFP that is already coupled to sepharose, for example GFP-TRAP that has the benefit of the antibody remaining coupled to the PA sepharose when immunoprecipitated proteins are eluted from the beads. In this case the addition of PA sepharose at this step is omitted.

11.
The antibody complexes recovered by centrifugation 2,200 × g for 3 min, and then washed twice with 10 ml PBS-T, resuspended in 1 ml of PBS, and transferred into a 1.5 ml microfuge tube.

12.
The antibody complexes were washed a further two times with PBS, pelleting the beads at 8,000 × g for 20 sec, and then eluted from the beads with 200 μl elution buffer warmed to 60 °C for 10 min.

13.
The beads are then pelleted at 8,000 × g for 20 sec, and the supernatant carefully removed; this can be facilitated by using a fine gel loading tip to remove the last 50 μl as the beads do not enter the tip, or by the use a micro bio-spin chromatography column.

15.
The supernatant is removed and the pellet air dried for 2 min and resuspended in a buffer of choice (e.g. 1× LDS sample buffer for SDS-PAGE).

16.
For Western blotting of samples SDS-PAGE gels were run and transferred according to a standard protocol (Sambrook and Russel, 2001).

17.
For proteomics, the samples were run on pre-cast NuPAGE 4-12% Bis-Tris gels, stained with Coomassie G-250 SimplyBlue SafeStain and then cut into 8 gel slices. Each gel slice was processed by filter-aided sample preparation solution digest, and the sample was analyzed by liquid chromatographytandem mass spectrometry in an Orbitrap mass spectrometer.

18.
Proteins that came down in the non-transformed control were eliminated, as were any proteins with less than 5 identified peptides, proteins that did not consistently coimmunoprecipitate in three independent experiments, or proteins of very low abundance compared with the bait (i.e., molar ratios of <0.002). The remaining proteins were considered to be specifically immunoprecipitated.    In the background the BioRad Xcell is shown with the pod open and loaded with an electroporation cuvette (1cm gap width). In the ice bucket is two more cuvettes awaiting electroporation. In the foreground a tube (15 ml) containing the axenic medium that will be added (0.5 ml) after each cuvette is pulsed twice to electroporate the cell suspension within. A tissue culture dish with 6 wells each of which contains 3 ml of axenic medium is also shown and after recovery on ice for 5 min, 100 μls of the electroporated cell suspension will be added to each well of this dish.  The translucent periphery of a colony consists of vegetative amoebae while at the centre fruiting bodies and other multicellular structures have formed. Colonies suitable for harvesting and freezing should be ~1 to 1.5 cm in diameter and clearly separated from neighbours. Suitable colonies in this example are indicated by the black arrowheads. Approximately 6 amoebae in 10 μl of KK 2 were directly added a 400 μl drop of a dense suspension of Klebsiella aerogenes already on the plate and then evenly spread. Colonies were visible 4 days after plating and this photograph was taken after 6 days. The scale is shown in the bottom right.