Chemotaxis Comments

Chemotaxis Practical - Comments

It has been our experience that folate chemotaxis is much more fool proof than cAMP chemotaxis due to the ability of Dicty to respond to a wide range of folate concentrations. We simply spot droplets (1-2 ml) of washed cells on phosphate buffered agar plates and then spot (1-2 ml) droplets of 10 µM to 10 mM folate next to (~2 mm gap) the cell droplets. With enough cells (we use > 10e7 cells/ml) you can easily observe cells chemotaxing beyond the original cell droplet perimeter in about 2-3 hours. There is no need to keep the plates fully hydrated - we just let the droplets absorb into the agar. You can use a regular compound or disecting microscope to observe the cells. Also, there are easy-to-grow (no selection in HL5 medium) mutants (e.g., Ga4 mutants) available that absolutely do not respond to folate (for controls). We observe a much more robust chemotactic response to folate than to cAMP. Let me know if you would like more information.

Best regards,

Jeff Hadwiger
Associate Professor, Microbiology and Molecular Genetics, Oklahoma State University.

Dr Yoshida told me that you are seeking advice about experiments suitable for laboratory course, and asked me if I have any suggestion. I don't think I know more than you about Dicty experiments, but you might find some of what I can recall from my limited experience useful. For a class over 40 students, real-time observation of chemotaxis requiring high magnification is not easy, so I use more macroscopic sorts of experiment. The easiest is something everyone knows; to just include a chemoattractant in agar and put small drops of starved cells on it. Use of washed agar (which is water repellent) is a key to success. For t6 cells, micromolar cAMP would be fine (1). An interesting variation of this method is to use Dictyostelium cells spreading on bacterial lawn. For this, spot vegetative cells or spores on bacterial lawn, incubate until it makes a sizable plaque (say 5 cm diameter), cut out two or more rectangular agar blocks of similar size in the radial direction so that both vegetative cells and starved cells are on the same agar block, and place them upside-down on agar plates containing either folate or cAMP. If done carefully, cells in the growth zone spread outwards on the folate agar while cells on the clear zone do so only on the cAMP plate. Great care must be taken when placing teh agar block, otherwise you would get cells spreading outside the block from the beginning. Also the amount of liquid on the plates (both growth plate and chemoattractant plate) is important; too much water would mix up the results. In my experience usually about half of the students succeed. This method by the way was developed in Kyoto and used for the screening of the KI mutants (2). There are some other methods (3), like the classical Konijn test, but more skill is required. A setup with inverted microscope with a manipulater and time-laps video may be used only in a smaller scale class, but if you have a projector in the classroom, it may be possible to show experiments done by the teacher or volunteers. I haven't done this myself.

I have not done phagocytosis experiments for lab courses. A fluorescent microscope and labelled bacteria or particles would be necessary, but this would be difficult for a large class. With phase-contrast, you can see a slime mould amoeba crawling over a bacterium and move away with it, but that's about all. With Nomarski, you may be able to see a bit more. Perhaps this is all what you have already known well, in which case I am sorry for not being of help. By the way, in lab courses with many students, I used Physarum and Phycomyces very much, since they are large, move or respond fast in chemotaxis, phototaxis and phototropism experiments, and as a consequence give strong impressions to the students.

References

(1) Finney et al. (1979), Dev. Biol. 73, 290-303.
(2) Kuwayama et al. (1993), JCB 123, 1453-1462.
(3) Konijn and van Haastert (1987), Methods in Cell Biology 28, Chapter 16.

If you have more questions, please feel free to ask me. Yours sincerely,

Kei Inouye
Department of Botany, Graduate School of Science, Kyoto University.

We do the phago assay with quenched fluorescent yeasts during the course, but NOT for 80 students. You could use trypan blue quenching in the microscope. We also use neutral red to identify acidic and neutral yeasts (which are not fluorescently labelled of course). If you had transmission microscopes only, there must be a protocol for congo red staining somewhere (which we actually have never done). This in brief(s). But I don't know whether it is feasible.

Markus Maniak

I recently put together a chemotaxis lab practical using axenically grown Dicty that might be of use to you. In my case, it was used as part of a multi-week module, in which the students did a basic assay in the first week and then did some variations on the theme in the next couple of weeks, to give them some practice in experimental design. The results can be visualized with a teaching grade dissecting scope. The only catch is the time factor. What I do is to starve the cells ahead of time, have the students put the cells on the chemotaxis plates and then have them come back 3-5 hrs later to score the assay. That works OK at a small residential university, where all the students live on or near the campus. You can get some idea of the basic assay if you visit the web site that I put together for the students:

http://merlin.alleg.edu/employee/m/mnelson/courses/Bio290/Dicty.html

The "experimental protocol" link shows the handout that I distribute to students for their use in the first week. The "additional info on methods" link is fairly self-descriptive -- basically some more info about how cells were prepared that wasn't included in initial handouts (but that students needed to know). The "potential jumping off points" link was to help students brainstorm about independent projects. In the first term that I ran this, we had success with time course ( varying hrs of starvation), folic acid vs. cAMP (often introducing different hrs of starvation), and expts in which we incorporated caffiene into the agar. It one had access to some chemotaxis mutants, those might work too. We found that this assay, although admittedly low tech and qualitative in nature worked OK for folic acid too. In either case the students developed some sort of arbitrary but internally consistent scale in which they scored the extent of the chemotactic response (+, ++, +++, ++++ or 1,2,3,4,5 or something of that nature). In most cases, this worked fine for detecting trends. I actually put together a poster about this lab for the educational poster session at this summer's Society for Devt. Bio meeting. Attaching the whole thing will probably make this message too large, but I will attach a few items that may give you some idea of what the data look like (allowing for the fact that I did take these photos on a research grade dissecting scope). They show the basic assay (Fig 3A & 3B) and a variation (Fig. 4). If this looks of use to you and you need more information, please let me know.

Margaret Nelson
Assistant Professor of Biology, Allegheny College, Meadville, PA 16335 USA.

Attached is a paper (Gauthier & O'Day, 2001) in which a radial bioassay for chemotaxis is shown. I developed this simple agar based assay in 1978 for assessing sexual chemotaxis. It is based on the methods developed originally by Bonner and Konijn (detailed in O'Day, D.H., 1979. Aggregation during sexual development in Dictyostelium discoideum. Can. J. Microbiol. 25: 1416-1426). Agents that affect signal transduction can be incorporated into the agar and the amount of chemotaxis to folate or cAMP can be quantified (i.e., measure the halo diameter vs time) to give an accurate measurement of rate of chemotaxis. Recently others have used similar approaches to assess chemotaxis. I hope this helps.

Danton H. O'Day, Ph.D.
Professor of Zoology, Cell & Developmental Molecular Biology,UTM

I wrote a chapter in Current Protocols in Cell Biology titled "Dictyostelium cell dynamics" (see complete reference at the end of the text). It describes how one goes about culturing and imaging Dictyostelium for cell motility and aggregation. It is meant for first users and has step by step procedures. It describes the micropipette assay and the microscopy settings used. I do not think this is exactly what you need, but it might be useful.
Complete reference: Parent C.A. Dictyostelium cell dynamics, Current Protocols in Cell Biology (J.S. Bonifacino, M. Dasso, J. Lippincott-Schwartz, J.B. Harford, K.M. Yamada; Eds. John Wiley & Sons Press), Vol. 1, Unit 12.5 (2000).

Carole A. Parent, Ph.D.
Principal Investigator, Laboratory of Cellular and Molecular Biology, NCI, NIH.

Please see ee my article in 'cell motility and cytoskel.' from earlier this year (51:39-48, 2002)- has easy, cheap, quantitative/semiquantitative/qualitative assay that can be adjusted to give results in as little as 2 hrs.

Dr. Jared L. Rifkin
Biology Department, Queens College, Flushing, NY.

It depends what level of equipment you have. The micropipet assay is the best, and works best with cAMP on starved cells. The droplet assay of Van Haastert and De Wit is rapid but requires some practice. The agar well assay with folate is the least demanding technically but is relatively slow (6 hours to get the ring of responding cells). You are welcome to take any of the movies on my web site that you would like, of course.

Jeffrey E. Segall
Professor, Histology Course Director, Department of Anatomy and Structural Biology, Albert Einstein College of Medicine.

Including some mathematics/computational work in your course is a good idea...since Dicty is one of the best studied and best modeled system for understanding the dynamics of chemotaxis and pattern formation. As well, Britain has many experts in this field: Phillip Many at Oxford and some fellows from Dundee come to mind. Don't leave the mathematics out of chemotaxis and signalling. There's many books...one by Leah Edelstein Keshet called Mathematical Models in Biology has an extensive section on Dicty.

Chad Shaw, BCM

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