Proc. Natl. Acad. Sci. USA Vol. 80, pp. 5646-5649, September 1983 Developmental Biology Thermotaxis of Dictyostelium discoideum amoebae and its possible role in pseudoplasmodial thermotaxis (temperature/sensory transduction/slime mold) CHOO B. HONG*, DONNA R. FONTANAt, AND KENNETH L. POFFt Michigan State University-Department of Energy, Plant Research Laboratory, -Michigan State University, East Lansing, Michigan 48824 Communicated byJ. T. Bonner, June 6, 1983 ABSTRACT Thermotaxis by individual amoebae of Dictyo- bae. For example, the amoebae respond to cyclic AMP and fo- stelium discoideum on a temperature gradient is described. These late, whereas the multicellular pseudoplasmodia do not, and amoebae showpositive thermotaxis attemperatures between 14°C the action spectra for phototaxis by the amoebae substantially and 28WC shortly (3 hr) after food depletion. Increasing time on the differ from those for phototaxis by the pseudoplasmodia, sug- gradient reduces the positive thermotactic response at the lower gesting different photoreceptor pigments regulating the re- temperature gradients (midpoint temperatures of 14, 16, and 18°C), sponses to light. and amoebae show an apparent negative thermotactic response Thermotaxis by the pseudoplasmodia has been knownfor many after 12 hr on the gradient. The thermotaxis response curve for years (8). The characteristics of this response include (i) a very "wild-type" amoebae after 16 hr on the gradient is similar to that high sensitivity (response to less than 0.0005'C across an in- shown for the pseudoplasmodia. Growth of the amoebae at a dif- and a relatively narrow temper- ferent temperature causes a shift in the thermotaxis response curve dividual pseudoplasmodium) for the amoebae. This adaptation is similar to that shown for the ature range across which the response is given, (ii) negative pseudoplasmodia. Two mutants in thermotaxis, H0428 and H0813, thermotaxis at temperatures several degrees below the tem- show changes in amoebal thermotaxis similar to the observed perature at which the amoebae were grown and permitted to changes in pseudoplasmodial thermotaxis. On the basis of the sim- develop into pseudoplasmodia and positive thermotaxis at higher ilarities between these responses, thermotaxis by the amoebae is temperatures, and (iii) adaptation-dependence of the direc- proposed to be the basis for thermotaxis by the multicellular pseu- tion of thermotaxis on the recent thermal history of the or- doplasmodium. ganism. This work was undertaken to determine whether or not in- Amoebae of the cellular slime mold Dictyostelium discoideum dividual amoebae of D. discoideum also exhibit thermotaxis, live and grow in the mulch on the forest floor, where they feed and it was expanded after the affirmative answer to examine on bacteria. Upon depletion of the food supply, the amoebae 'the relationship between thermotaxis by the individual amoe- aggregate, forming a multicellular pseudoplasmodium that bae and thermotaxis by the multicellular pseudoplasmodia. This eventually develops into a sorocarp consisting of a stalk bearing paper reports the evidence supporting the conclusion that ther- a sorus containing spores. Under the proper conditions, the motaxis by the amoebae is the basis for the thermotaxis by the spores may germinate, releasing amoebae, thus repeating the pseudoplasmodia. developmental cycle (1). Much of the work with this organism has been centered upon its development and the fact that one MATERIALS AND METHODS can easily separate growth and cell division from development. Amoebae of D. discoideum, strains HL50, H0428, and H0813 Dictyostelium is also being increasingly recognized as an ideal (14), were grown in association with Klebsiella aerogenes (15) system for the study of sensory transduction in a eukaryotic or- for about 22 hr at 23.5 ± 0.50C. At this time, the bacteria had ganism. Sensory responses thus far described are chemotaxis by been cleared from about 80% of the agar plate. In some ex- the amoebae to cyclic AMP (2) and folate (3), both positive and periments designed to test for thermal adaptation, amoebae of negative phototaxis by the amoebae (4-6), chemotaxis by the strain HL50 were grown for about 22 hr at 27.50C, at which pseudoplasmodia to an endogenous "slug turning factor" (7), time the plates were usually about 50% cleared of bacteria. phototaxis by the pseudoplasmodia (8-11), and positive and The amoebae were freed of bacteria by washing three or four negative thermotaxis by the pseudoplasmodia (8, 12, 13). In ad- times with 15 mM potassium phosphate buffer, pH 6.1, with dition to the phenomenology, work is also progressing toward intervening centrifugations of 1 or 2 min at about 500 X g, and an understanding both of the primary steps and of the trans- resuspended in phosphate buffer at 1 x 107 amoebae per ml. duction sequence for each of these responses. Moreover, the Aliquots (1 ml) of the suspension were mixed with 0.3 ml of a use of mutants is permitting a study of the interconnections suspension of washed charcoal in phosphate buffer (2%, wt/vol). between these various sensory responses. The charcoal was added to mark the point of cell spotting. The Because of the ease with which the single-celled and mul- cell/charcoal suspension was agitated and deposited in a line ticelled stages may be separated in Dictyostelium, one would on 2% (wt/vol) water agar which was about 1 mm thick covering also expect this to be an excellent system for studying the a 1 X 25 X 75 mm microscope slide.'The line of amoebae (Fig. expression of a particular sensory transduction system in the 1A) was formed with the aid of two parallel strands of fine plat- two motile forms, the amoebae and the pseudoplasmodia. inum wire, 0.1 mm in diameter, which were attached at their However, neither chemotaxis nor phototaxis appears to use the same system in the pseudoplasmodia that is used in the amoe- * Present address: Chemistry Dept., Texas Tech Univ., Lubbock, TX 79409. The publication costs of this article were defrayed in part by page charge t Present address: Dept. of Biochemistry, Johns Hopkins Univ., Bal- payment. This article must therefore be hereby marked "advertise- timore, MD. ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. t To whom reprint requests should be addressed. 5646 Downloaded by guest on October 2, 2021 Developmental Biology: Hong et al. Proc. Natl. Acad. Sci. USA 80 (1983) 5647 ends to two metal bars. The bars were attached to a rack and pinion gear so that the wires could be raised and lowered. After B spotting, the agar slides were placed on a temperature gradient A A (0.220C/cm), formed as described by Poff and Skokut (12), and left there for 3, 6, 9, 12, 16, or 20 hr. After the exposure to the thermal gradient, the number of amoebae that moved in each direction away from the original P. : .. .:. .: .:.. : : ae . .. 0 line was determined. This was done by examining five ran- P domly chosen microscope fields along the original line of about 3 x 5 mm on each of ten slides. The response (A percentage) for each replicate was calculated as: A percentage = 100(no. of cells moving up gradient - no. of cells moving down gradient)/ :,:*...,s 0@ t total no. of cells that moved. Thus, A percentage is a measure of the directed movement on a thermal gradient. For thermal response curves, relative A percentage was determined for thermal gradients with various midpoint temperatures. c D The experiments on the thermotaxis of pseudoplasmodia were carried out as described by Schneider et al. (14) with a slight modification. Some of the HL5O amoebae, used for the ad- aptation experiments, were grown at 27.50C for 22 hr. How- ever, the inoculum was reduced so that no clearing was visible. This change appeared to delay the formation of fruiting bodies from the pseudoplasmodia. Development in this case also oc- curred at 27.50C. RESULTS Amoebae of D. discoideum strains HL50, H0428, and H0813 regularly showed amoeboid movement for up to 20 hr without any sign of aggregation. In the absence of a thermal gradient, the amoebae moved randomly on the agar surface. Depending FIG. 1. Thermotactic responses of D. discoideum amoebae. Tem- on the midpoint temperature of the thermal gradient used, there peratures are higher at the top. Larger dots represent amoebae and were conditions that resulted in more cells on the warmer side, smaller dots are charcoal. Tracings from photomicrographs showing cooler side, or no directed movement (Fig. 1 C, D, and B, re- original spotting (A), random response (B), positive thermotaxis (C), spectively). Thus individual amoebae are capable of positive and negative thermotaxis (D). and negative thermotaxis. Amoebae of strain at were then ex- HL50, grown 23.50C, grown and allowed to develop into pseudoplasmodia at 27.5'C, posed to thermal gradients for six different time intervals. After also demonstrates both and thermotaxis 3 was seen entire tem- positive negative (Fig. hr, positive thermotaxis throughout the 3B), with the transition between the two responses occurring were perature range (Fig. 2A). When the amoebae removed around 20.40C. this transition is consistent with that seen a Again, from the gradient after 6 hr, the amoebae did not show sig- with amoebae. Strains H0428 and H0813 are mutants in pseu- nificant positive thermotactic response on gradients with mid- doplasmodial thermotaxis derived from strain HL50 (14). Amoe- point temperatures of 14, 16, and 18'C (Fig. 2B). This trend continued, as can be seen from the results for amoebae exposed to the gradients for 9 and 12 hr (Fig. 2 C and D). After 12 hr A 8BXC on thermal gradients with midpoint temperatures of 14 and 160C, significant negative thermotaxis was evident. After 16 hr, the amoebae demonstrated negative thermotaxis on gradients with 0~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~0 midpoint temperatures of 14, 16, and 18'C and positive ther- -8 motaxis when the midpoint temperature was above 20'C (Fig.