Experiment 6: Parts 1 and 2: Under Agarose Aggregation of Dictyostelium wild-type and myosin II mutant cells


We have been working with vegetative Dictyostelium amoebae. This organism nourishes by eating solid particles such as bacteria or through the media and divides indefinitely as long as conditions are conducive. In nature, this organism exhibits chemotaxis that enables it to move toward bacteria where it is further engulfed via phagocytosis. When food is scarce, Dictyostelium has a survival strategy to prevent extermination.  They create spores that can survive desiccation, lack of food, high temperature and other environmental insults.  Eventually, when conditions are favorable again, an amoeba will hatch out of the spore case and resume growth.


In some species, individual amoebae can encapsulate and become spores.  Dictyostelium discoideum uses a more complex pathway.  When food becomes limited, the cells enter a developmental pathway, such that new genes are expressed and others cease to be expressed and the cells aggregate together to form a multicellular organism.  This pseudoplasmodium or slug is sensitive to light (phototactic), and temperature (thermotactic) allowing it to migrate from the subsurface soil to a near surface.  There the slug transforms itself into a fruiting body with a spore case atop a long column of stalk cells.  You will watch and record this process over the 24 hours following initiation of starvation and compare mutant with wild-type. 


There are links to review articles of Dictyostelium development and pattern formation on the main web site (Dictyostelium discoideum: a model system for differentiation and patterning, by Escalante and Vicente and Developmental Decisions in Dictyostelium discoideum, by Strmecki).  You should read these so you understand what is happening in your experiment.


Suggested guide to sequence of events:  Plan experiment. Pour agarose dishes first, so they are hardening while you prep the cells.  Prep the cells and set up the agarose plates.  Then do Part II (the bacterial growth plates) with the leftover cells.


PART 1: Wash GFP labelled and unlabeled Wild-type and Mutant Cells 2X in Starvation Bufferto remove the nutrients and starve the cells to initiate development 


1.Use a pipet to triturate your cells and transfer them to a 15ml centrifuge tube.  Take a small volume out and place in your hemocytometer so that you can be counting while the cells are spinning.  Pellet the cells to the bottom of the tube by spinning in the centrifuge for 5 minutes at 1000rpm at 4°C.


2.Aspirate the HL-5 being careful not to remove your cell pellet.  Add 5mL MCPB (a buffer containing Mg, Ca++, and phosphate buffer adjusted to pH6.5).  Vortex tube to resuspend the cells and centrifuge the cells for 5min.


3.Remove the medium and resuspend the cells to 107 cells/ml or higher.  Keep them on ice while setting things up.



PART I: Development Under Agarose:  The under agarose assay will focus on the cAMP pulsatile aggregation phase. The cells will not develop beyond aggregates unless the agarose is removed (which you can try to do).  Because the cells are under agarose, the cells are flattened, and the cAMP gradients that drive aggregation are well formed and cells can be seen responding to the periodic waves.


Preparing Agarose


1.In a flask, combine make up a 1.5% agarose solution (1.5g agar per 100ml MCPB). Figure out how much you need for the number of plates you are setting up (below), but make up a bit more than you need since you lose some volume in boiling (minimum  is 20 ml in the flasks we use).  Make sure the screw cap is loose!  Heat by using short bursts in microwave.  Stop as soon as the solution starts to boil, swirl it and repeat until agarose is melted and liquid appears clear (no floating particles).


2.Let the flask cool a little, then pipet 5 ml into each 60mm Petri dishes and allow to harden on a level surface. Once it has hardened, cut a small wedge near the outside edge with a pointed spatula and remove the wedge.


Setting up under agarose development plates


1.For each Petri dish, combine 1x107 unlabeled cells with about 5% GFP-labeled cells  (the percentage to use depends on the proportion of your GFP-labeled cells that are actually fluorescent) in a total of 5ml of MCPB. Vortex and then add to the dish and allow cells to attach. Check them on your benchtop microscope to know when they are well attached. You are aiming for a monolayer of cells, so if you have too few, add more cells.  Too many is OK unless absurdly high.


2.Use a spatula to loosen an agarose sheet from one of your dishes.


3.Once cells are attached, pick up the agarose sheet from the dish you poured it in  (I use a combination of fingers and spatula) and gently lay the agarose sheet on top of the buffer over the cells.  The agarose will slowly sink to the bottom of the dish.  The wedge you cut will allow the buffer to flow from below the agarose to the top as it settles. You may need to gently push down on the agarose to get it to settle. Then gently remove the excess buffer with your P1000. Do not move the dish while doing this, but eventually you will want to tilt the dish up and allow the residual buffer to flow into the wedge area. Keep removing buffer until there is nothing left in the wedge area.


4.Check your plate under your benchtop light microscope to make sure your cells looks OK.


5.Incubate at 22oC.


6.In about 6 hours, the cells will begin to aggregate and you will see spiral streams of cells moving toward aggregation centers.  Depending on the magnification you use, you can see the cells more clearly or the larger behavior of the population. 


7.Start a two channel time lapse movie so you can measure the movement of the cells.  It is good to start at low magnification (4-10x) but you may want to change to lower (4x) or higher (20-40x) so you can see a wider field of view or see more detail about the cells.  They will continue to aggregate for 4-6 hours so you have a wide window of time to try different modes of imaging.  You should measure the parameters of movement (speed, persistence, directionality) and compare that to the vegetative and folate chemotaxing cells you have previously measured.  One of the interesting analysis techniques you can use is to measure the periodicity of the cells response to cAMP.  As the cAMP wave moves through your field of view, the cells change shape.  If you phase contrast is set up well, you will see the cells become light and dark as they round up and flatten out again.  You can measure this by calculating the average pixel intensity of the image over time.

a.In ImageJ, import your image stack, then Edit/Select All

b.Go to Analysis/Set Measurements and make sure that mean gray value is selected

c.Go to Plugins/Macros/Measure Stack

d.This will read out the average intensity of each image in the stack

e.Plot these values over time, and you should see a periodic pattern corresponding to the cAMP wave coming through the field of view.



PART II. Growth and Development of Dictyostelium in bacterial growth plates


Dictyostelium normally eats bacteria as its food source. The cell lines we are using can either utilize broth media or bacteria as their food source. Today, you will also set up cells to grow on bacteria (Klebsiella aerogenes) and observe their growth and development. The bacteria grow on the nutrients in the SM agarose plates and the Dicty cells feed on the bacteria.  You will plate a small number of cells on a plate and each cell will divide many times and eventually form a small clear plaque where all the bacteria have been eaten (about 4 days).  The plaque will continue to enlarge as cells spread outward. In the center  of the plaque, the cells will be starving and begin development.  You can see all the stages of development in one plaque.


Procedure:


1.Titer your cells using the hemocytometer.  You can use the left over cells from the above experiment.


2.Set up 2 plates: Plate 1 with 10 Dicty cells on it (to give 10 plaques) while Plate 2 will have 100 cells. Calculate the volume of suspension you will need for each plate. You will probably need a dilution of your cells to make this a reasonable volume.


3.Add about 300 ul of Klebsiella aerogenes into a plastic tube. Add your Dicty cells and mix (vortex).


4.After mixing the cells thoroughly, pipet out all the contents of the tube onto an SM agar plate.  Spread the mixture over the surface well with a sterile spreader.


5.Place the plates in your incubator box and observe growth and development.


6.Over the first two days you will see a film of bacteria form as the ones you spread grow and form a lawn on the plate.


7.Starting about day 3-4, the individual amoebae have propagated to the point where they have eaten all the bacteria around them. This will form tiny plaques: clear circular areas free of bacteria. From this point on, you should make measurements of the plaque diameter. Measure the diameter over time by taking images with the macroscope.  You will have to calibrate the macroscope to convert the images to microns, or you can use pixels as your measurement. This will give you one type of measure of the rate of growth of cells. Think about what you are actually measuring here (growth, motility or both?) and how it differs from the direct hemocytometer titering you did previously to measure growth.


8.After another day, the amoeba will begin to form multicellular aggregates and eventually fruiting bodies. Use the article you were given and the link to the DictyBase movies (http://dictybase.org/tutorial/) to guide you in determining which forms are which. Observe and record the location, diameter size or any other interesting observations you care to make about this process.  Capture images of the different developmental stages for the web site. 


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Reagents/Media:


HL5 media

10 g BBL Thoitone E peptone

5 g yeast extract

10 g glucose

0.35 Na2HPO4

0.35 KH2PO4

Fill up to 1L with H2O

Adjust pH to 6.5-6.7

Autoclave


SM liquid media and SM Plates [prepare about 60 plates]

10 g Difco Bacto-Peptone

10 g glucose

1 g yeast extract

1.9 g KH2PO4

0.6 g K2HPO4

0.43 g MgSO4

Fill up to 1L with H20

Adjust pH to 6.5-6.7

[Add 20 g of agar for SM plates, just adjust the volume of H2O]

Autoclave



MCPB

1.42 g Na2HPO4

1.36 g KH2PO4

0.19 g MgCl2

0.03 g CaCl2

0.5 Streptomycin dihydrosulfate

Fill up to 1L with H20

Adjust pH to 6.5-6.7

Filter-sterilize


Cell Strain(s):

HK321

NC4A2


Other things needed for this experiment:

1.p60 dishes

2.Black and white filters

3.spatula

4.clean flasks (for boiling agarose)

5.microwave

6.bunsen burner

7.spreader

8.ruler

9.Macroscope




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