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Chemotaxis, aggregation behavior, and foot formation in Dictyoselium discoideum amoeba controlled by microbeam uncaging of cyclic-AMP.

At a certain stage of development, amoebae of the cellular slime mold Dictyostelium discoideum signal to each other by secreting c-AMP [cyclic-3'5' adenosine monophosphate (1, 2, 3)] -- and then aggregate. We analyzed the responses of aggregation-competent amoebae to brief applied pulses of c-AMP under high-resolution video DIC (differential interference or Nomarski contrast) microscopy.

Miniature sources of c-AMP pulses were generated by illuminating caged c-AMP (4) with a 366-nm-wavelength UV (ultraviolet) microbeam delivered as 3-ms flashes repeated every 0.65 s; we had added the caged c-AMP to the buffer and agar layer overlying the amoebae (5). A Zeiss Ultrafluar (UV- and visible light-transmitting, 100X/1.25 NA, glycerol immersion objective) lens equipped with a DIC prism replaced a conventional condenser to focus a highly reduced image of a first-surface micromirror, placed in front of the field diaphragm, superimposed with the DIC image of the specimen. The UV-reflecting micromirror was located at the focus of the UV source, an auxiliary 100-Watt Hg-arc lamp with quartz collector, 366-nm bandpass filter, and electrically activated shutter. They 2.2 x 3.0 [mu] [m.sup.2] / UV image can be seen as a bright rectangle at the tip of the dark shadow of the mirror support in Figure 1A and B, slightly off center from the visible (546 nm) light image of the specimen in DIC. Moving the micromirror or specimen carrier placed the source of c-AMP in different locations relative to one or more amoebae.

Migrating aggregation-stage amoebae responded to the c-AMP pulses by turning towards the source (Fig. 1A) and migrating it. The first amoeba to reach the source engulfed it, and the others spiraled and aggregated around this first amoeba, which remained at the source (Fig. 1B). When the artificial source of c-AMP was removed by shutting off the UV flashes, all the amoebae dispersed and headed towards their natural source, an aggregate formed by many amoebae located outside the induced territory.

Early during aggregation, most amoebae remained in loose contact with each other and slowly spiralled around the center of the aggregate. Whether in a naturally formed aggregate made up of many scores of cells or in an artificially induced aggregate made up of only a few cells, the amoeba located in the center became stationary relative to the substratum. Through-focus observations indicated that such an amoeba anchored itself to the soft substrate by protruding knobby "feet" into the agar surface. In the artificially induced aggregate, the central amoeba generated and maintained its feet (small arrows in Fig. 1C; the UV micromirror is retracted and focus is now on the agar surface) so long as the c-AMP pulses (UV flashes) were continued. Once the UV flashes were turned off, the feet were gradually retracted (Fig. 1D). If the UV flashes were restarted within a minute or so, the feet reformed, and the departing outer amoebae rejoined the aggregate. The process was completely reversible and could be repeated many times. Thus we have established a tool for investigating localized and reversible c-AMP-mediated modulation of chemotaxis and cell response.

Supported by NIH grants R01 GM 39548 to YF and R37 GM 31617 to SI.

Literature Cited

1. Bonner, J. T. 1971. Ann. Rev. Microbiol. 25: 75-92.

2. Gerisch, G. 1987. Annu. Rev. Biochem. 56: 853-879.

3. Devreotes, P. 1989. Science 245: 1054-1058.

4. Nerbonne, J. M., S. Richard, J. Nargeot and H. A. Lester. 1984. Nature 310: 74-76.

5. Fukui Y. and S. Inoue. 1991. Cell Motil. Cytoskel. 18: 41-54.
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Author:Fukui, Yoshio; Inoue, Shinya
Publication:The Biological Bulletin
Date:Oct 1, 1995
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