Introduction: Naegleria gruberi is an amebo-flagellate. Amebo-flagellates are unusual organisms that can exist as amebae and also as swimming flagellates. The ameboid stage feeds by phagocytosis of bacteria and divides by binary fission. The flagellated stage does not feed or divide. Flagellates are thought to provide a method of dispersal when food supplies become low.

Amebae are induced to become flagellates by removal of their food supply, bacteria. This is done by gently washing the amebae with ice-cold buffer in the centrifuge. The differentiation of amebae into flagellates is started by suspending washed amebae in a warm-dilute buffer, 2 mM Tris-HCl, pH 7.6 at 25 ^oC in our experiments. This is defined as 0 min. of the differentiation.

The differentiation of Naegleria provides a useful system for examining a number of interesting problems in Cell and Developmental Biology. Some of the advantages of the system stem from the ease and speed with which cells can be grown and induced to differentiate. Another important advantage is that the differentiation of amebae into flagellates is relatively synchronous. This means that following the population of amebae as they differentiate into flagellates is similar to following the changes in a single cell. In this way we can ask about the molecular basis for the changes that take place.

These changes include a disassembly of the actin-myosin cytoskeleton that provides movement in amebae. This is reflected in the first morphological change in the cells when they change from an ameboid shape to a spherical shape at about 60 min. after initiation.

The next visible change is the appearance of short flagella on the surface of the spherical cells. The time when 50% of the population has developed visible flagella is defined as the T50 for flagella formation. One of the great advantages of the Naegleria system is the reproducibility of the differentiation. The T₅₀ for flagellum formation (the time for 50% of the cells to form visible flagella) is 68 +/- 2 min.

About the time flagella have reached three quarters of their full-length, the cells change shape again. This time the spherical cells become flattened ovals referred to as the flagellate shape. The T50 for flagellate shape formation is 85 min. Formation of the flagellate shape is correlated with the formation of an extensive cytoskeleton composed of microtubules. The T50 for cytoskeletal microtubule (CSMT) formation is 75 min. This is particularly dramatic because amebae lack any kind of microtubules except in the mitotic spindle.

Although not visible without staining for tubulin, the de novo formation of basal bodies precedes the formation of flagella by about 10 min. The de novo formation of basal bodies is a nearly unique feature of the Naegleria differentiation and it provides the potential for investigating the control of these poorly understood organelles.

Our lab today will involve the differentiation of amebae into flagellates. We will follow this process by fixing cells in Lugol's iodine and scoring for the presence of flagella. We will fix cells in a mixture of formaldehyde and nonionic detergent for staining of the cytoskeleton which will be done in two weeks. We will also collect samples for the analysis of tubulin by Western blotting next week.

At the end of the differentiation we will harvest flagellates and remove the flagella using a pH shock. We will prepare the isolated flagella for examination by negative staining in the election microscope in two weeks.

Materials:

• plates of Naegleria gruberi grown on Klebseiella pneumoniae. (see below).
• ice-cold 2 mM Tris-HCl, pH 7.6, 1 liter.
• Lugol's iodine (see below)
• 0.5% PMSF in n-propanol, 100X stock soln. (CAUTION - TOXIC)
• 2 M leupeptin, 100X stock soln.
• 100 mM EGTA, pH adjusted to 8.25 with NaOH, = 100X or 1000X stock.
• 100 mM dithiothreitol (DTT), = 1000X stock.
• 0.1 M Tris-HCl, pH 7.6, = 50X stock.
• 1 M MgCl₂ stock.
• 100 mM sodium acetate, pH 3.7, prepared by adding 0.575 ml glacial acetic acid to about 85 ml DIW(deionized water) . The pH is adjusted to 3.7 with 10 N NaOH and then the volume is brought to 100 ml with DIW. This is a 10X stock.
• MgCF,

50 mM sodium phosphate, pH 7.2, 125 mM sucrose, 5 mM MgCl₂, 1 mM EGTA, 0.1% w/v NP-40 (dilute from 10% w/v stock), 0.9% formaldehyde (dilute from 36% soln = formalin).
• ice-cold Detailing Medium, 10 mM sodium acetate, pH 3.7 [from 10X stock], 2 mM MgCl₂, 75 mM sucrose, 0.1 mM EGTA [from 1000X stock] and 0.005% PMSF and 20 mM leupeptin added just before use from 100X stocks.
• ice-cold Neutralizing Buffer, 0.5 M Tris-HCl, pH 8.25.
• ice-cold Triton I Soln, 25 mM Tris-HCl, pH 7.6, 3 mM MgCl₂, 1 mM EGTA, 0.1 mM DTT , 0.2% Triton X-100 (from a 10 w/v stock), 0.005% PMSF added just before use.

A) Differentiation of Amebae.

• 1) Put 20 ml of 2 mM Tris-HCl, pH 7.6 (Tris) into a 125 ml Erlenmeyer flask and put the flask in a reciprocating water bath set at 25.0 ^oC. Allow the buffer to reach the bath temperature before starting to harvest cells. Meanwhile collect an ice bucket with a 50 ml polycarbonate centrifuge tube, a similar tube for use as a balance, and ice-cold Tris with a 10 ml pipette.

IMPORTANT, set up the tubes in Part B, steps (1), (2) and (3) before proceeding with the differentiation.
 
• 2) Suspend each plate of amebae in 9 ml ice-cold Tris using a glass spreader. DO NOT push down on the spreader, it will break. Pour the cell suspension into a 50 ml polycarbonate centrifuge tube in ice. One tube will hold the cells from 5 plates.

• 3) Prepare a balance tube with an equal volume of water then wash the amebae free of bacteria by centrifugation at a setting of 6 in the Clinical centrifuge for 45 sec. Bring the head to a stop rapidly using the brake in the cover. DUMP the supernatant from the tube, place the tube in ice, and rapidly resuspend the cells by pipetting in 10 ml of ice-cold Tris. NOTE: In contrast to most cases, the supernatant is not carefully decanted because this results in a loss of the cells in the loose pellet. DUMP means to rapidly invert the tube and then immediately return it to an upright position. This may not remove all of the supernatant but that is not a concern. Your instructor will demonstrate.

• 4) Vortex the cell suspension to remove bacteria adhering to the amebae. Repeat the centrifugation, DUMP, and resuspension as in (2). Note: The intention is to minimize the time amebae are in a pellet and the time from initial resuspension until the cells are put into warm buffer.

• 5) Repeat the centrifugation and DUMP as in (2) but this time resuspend the cells in 10 ml of 25 ^oC Tris. START a timer, this is zero time for the differentiation. Pour the cell suspension into the 125 ml Erlenmeyer flask with the remaining 10 ml of warm Tris and place the flask on a reciprocating shaker water bath at 25 ^oC and 80-100, 1 inch strokes per min.

• 1) Prepare a set of 16, 10 x 75 mm or 13 x 100 mm test tubes at room temperature with one drop of Lugol's iodine in each. When adding the iodine soln it is IMPORTANT to hold the dropper well away from the lip of the tube and try and get the drop to fall to the bottom of the tube without touching the walls. Any iodine that is present on the lip of the tube may be carried back to the cell suspension and this would kill all the cells. Label the tubes 0, 10 ,20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 100, 110, and 120 min.

• 2) Prepare three 10 x 75 mm disposable test tubes in ice with 250 µl of MgCF in each. Use care to place the fixative in the bottom of the tube so as not to contaminate the lip (see above). Label these tubes 10, 70, and 120 min.

• 3) Prepare a set of 6, 1.5 ml microcentrifuge tubes with boiling resistant labels at room temperature. Label the tubes 5, 20, 40, and 80 min.

• 4) Using a wide bore sampling pipette to prevent shear, take a sample from the flask of cells and add 3 drops to a tube containing Lugol's iodine at the times indicated. Try and add the cells so that they fall directly into the iodine soln rather than down the side of the tube but take special CARE not to touch the sampling pipette to the tubes containing Lugol's iodine. Gently shake each tube after addition of the cells. Note that the "0" time point will be a little late. Record this time in your notebook.

IMPORTANT: DO NOT return unused cells to the flask. Discard any cells remaining in the sampling pipette into the DUMP. After each sample, rinse the sampling pipette three times in DDW and store in DDW.
 
• 5) At 5, 20, 40, and 80 min. after the initiation of the differentiation remove 1 ml of the cell suspension to a microcentrifuge tube with a P1000. Centrifuge for 30 sec. and carefully aspirate the supernatant. Immediately place the tube with the cell pellet at -20 °C. We will run the gels next week.

• 6) At 10, 70, and 120 min. also add a sample of four drops of cell suspension to a tube containing MgCF using the same CARE as above to avoid contaminating the flask of cells.

Note: Before starting this section make sure you understand the steps and that you have all the reagents and pipettes on hand. It is IMPORTANT that steps (1) and (2) be carried out quickly.

• 1) After you have taken samples to Lugol's iodine and MgCF at 120 min., pour the cells remaining in the flask into a 50 ml screw cap centrifuge tube (you do not need to put the cap on at this step) and centrifuge at a setting of 6 for 2 min. in the Clinical centrifuge. Decant the supernatant and place the tube in ice.

• 2) Remove the flagella by adding 10 ml of ice-cold Detailing medium, cap the tube, and shake vigorously for 15 sec. IMMEDIATELY add 0.5 ml of Neutralizing buffer and mix.

• 3) Remove the cell bodies by centrifuging at a setting of 6 for 2 min. in the Clinical centrifuge.

• 4) Pour the supernatant into an ice-cold 15 ml centrifuge tube and centrifuge at 600 x g for 1 min. in the cold to remove residual cell bodies and food vacuoles.

• 5) Pour the supernatant into a second ice-cold 15 ml centrifuge tube and centrifuge at 600 x g for 4 min. in the cold.

• 6) Pour the supernatant into a third ice-cold 15 ml centrifuge tube. Remove a small drop of the flagellar suspension to a slide and examine it as described under (D). Record your observations. Centrifuge the suspension at 20,000 x g for 10 min. in the cold to pellet the flagella.

• 7) Discard the final supernatant and resuspend the flagella, using a Pasteur pipette, in 5 ml of ice-cold Triton I solution to remove the flagellar membrane from the flagellar axoneme. Transfer this suspension to an ice-cold 12 ml Corex centrifuge tube. Incubate on ice for 15 min.

• 8) Pellet the axonemes by centrifugation at 40,000 x g for 30 min. in the cold.

• 9) Discard the supernatant and resuspend the pellet in 250 µl of ice-cold Triton I solution using a Pasteur pipette.

• 1) Using a diamond pencil, label the end of two slides for each of the three time points with the time and your group number.

• 2) Lay the slides flat under the hood with the labeled side up. Gently shake the cells fixed in MgCF to suspend the cells and then place 60 µl of the cell suspension as a puddle in the middle of the slide. Allow to dry thoroughly under the hood.

• 3) Rinse the slides of each time point briefly in PBS, drain for a min. and then transfer to MeOH in ice for 10 min. After the MeOH fixation drain the slides briefly and then fix in acetone in ice for 10 min. At the end of the acetone fixation, allow the slides to dry under the hood at room temperature.

• 4) Store the dry slides at -20 ^oC over desiccant.

• To stain the slides, warm them to room temperature, place a drop of anti-tubulin on the slide and spread gently  with the tip of a pipette. Incubate in a damp box at 37 ^oC for 60 min, wash by passing through 4 changes of TBS or PBS for 2-3 min. each. Add second antibody (Alexa488 from Molecular probes is very good at 1/200) in the same way and incubate . After washing, mount slides in 90% glycerol, 10% 0.1 M NaCO₃, pH 9.0 containing 100 mg/ml of 1,4 - diazabicyclo-[2.2.2] octane (DABCO) (3).

1) Pick up an EM grid, coated with Parlodian and carbon, in the tips of a fine locking forceps. Place a small drop of the axoneme suspension on the grid and allow to stand for 30 sec. Remove the drop by touching the edge of a piece of filter paper to the edge of the grid and allowing the drop to be soaked into the paper. Immediately place a drop of 1% uranyl acetate on the grid allow to stand for about 15 sec. Remove the uranyl acetate drop with the edge of a piece of filter paper. Allow the grid to air dry.

G) Counting Flagellates. (Probably save for later. Cover the tubes tightly with Parafilm).

• 1) Prepare a counting slide by placing three 18x18 mm cover slips supported by small clay feet on the slide. Resuspend the cells fixed in Lugol's iodine by gently shaking the tube and then place a drop of fixed cells under a cover slip. Repeat for two other samples. You may find it easier to start counting with the 120 min. sample and working backward.

• 2) Examine the cells using a 40X phase contrast objective. Count the fraction of cells with one or more flagella in fields chosen at random. Count only cells that are completely within the field and that are not touching another cell. It is IMPORTANT that you focus up and down on each cell as you examine it to see if it has flagella. The flagella are so thin that they can be out of the plane of focus when you can see the cell body. If you are unsure whether a short projection is a flagellum, do not count it. Count cells from fields chosen at random until you have scored 100 cells at each time point. Record the time of the sample and the percent flagellates. When you have all the data, plot the percent flagellates vs. the time of sampling.

H) SDS Gels and Western Blots. (Week 2).
 

• 1) Run the samples on on a 7% SDS gel.
• 2) Blot the gel to nitrocellulose or PVDF
• 3) Develop the blot with an anti-tubulin first antibody and a HRP or alkaline phosphatse second antibody and appropriate substrate.

• 1) Pour NM plates:
• to 800 ml of DIW add:
• 1.2 gm K₂HPO₄
• 0.8 gm KH₂PO₄
• 1.6 gm dextrose
• 1.6 gm Bacto-peptone
• after these are completely dissolved add
• 16 gm Bacto-agar without mixing
• autoclave for 15 min.
• mix well by swirling soon after removal from the autoclave but after bubbling has stopped.
• cool to about 50 ^oC and pour about 40 ml per plate
• dry plates at room temperature for 3 days before use and then store at 3 ^oC
• 2) Prepare Penassay broth:
• to 80 ml of DIW add:
• 1.4 gm of Difco Antibiotic medium #3 and dissolve
• aliquot 8 ml into 16 x 150 mm culture tubes, cap and autoclave for 15 min.
• store at room temperature.
• 3) Inoculate Klebsiella pneumoniae from a slant into Pennassay broth and incubate at 34 ^oC for 24 to 48 hr. This stock is good for 1 week stored at room temperature. Inoculate from a slant to broth every week do not inoculate from broth to broth as the cultures become slimy (5).

• 4) Prepare a Naegleria edge plate.
• Place 0.1 ml of a K. pneumoniae broth culture on an NM plate and spread with a glass spreader.
• Using a sterile inoculating loop place a small dot of Naegleria cysts near one edge of the plate.
• Incubate the plate inverted at 34 ^oC for 2-3 days during which an "edge" of growing amebae will move across the plate as they consume the bacteria.
• When the edge has reached the far side, the plate will be covered with cysts. Wrap the plate tightly in Saranwrap ( You must use the thick plastic.)  and store at room temperature for 1-3 weeks.

• 5) Prepare spread plates:
• A few hours before inoculating, place the NM plates at room temperature to warm up.
• 22 hr. before use:
• Use a sterile inoculating loop to scrape up cysts from an edge plate.
• Suspend the cysts in sterile DIW and vortex vigorously to disperse.
• Count the cysts with a haemocytometer and adjust the concentration to 2.5x10⁶ cysts/ml.
• Place 0.1 ml of K. pneumoniae broth culture on each NM plate.
• Place 0.1 ml of the cyst suspension (vortex just before use as the cysts settle rapidly) on each plate.
• Using a sterile glass spreader, mix and distribute the bacteria and cysts evenly over the plates.
• Incubate the plates, right side up, at 34 ^oC for 1-2 hr and then invert the plates.

•   Using a glass bottle with a ground glass stopper:
• add 2 gm of iodine to the bottle (CAUTION - TOXIC)
• add 3 gm of potassium iodide to the bottle
• add  0.5 - 1.0 ml of DIW ( may need a little more)
• mix until dissolved
• add DIW to a total of 50 ml
• store at room temperature

• 1) Fulton, C. and A.D. Dingle. 1967. The appearance of the flagellate phenotype in populations of Naegleria. Develop. Biol. 15:165-191.
• 2) Dingle, A.D. and C. Fulton. 1966. Development of the flagellar apparatus of Naegleria. J. Cell Biol. 31:43-54.
• 3) Walsh, C. 1984. Synthesis and assembly of the cytoskeleton of Naegleria gruberi flagellates. J. Cell Biol. 98:449-456.
• 4) Fulton, C. 1993. Naegleria: A research partner for cell and developmental biology. J. Euk. Microbiol. 40:520-532.
• 5) Fulton, C. 1970. Amebo-flagellates as research partners: The laboratory biology of Naegleria and Tetramitus. Meth. Cell Physiol. 4: 341-476.