Dynamics of GFP-coronin and Eupodia in live Dictyostelium observed with real-time confocal optics.
In the current study, we have analyzed the dynamic integration of GFP-coronin into the actin cytoskeleton, in particular the eupodia, to probe the architectural dynamics of actin in live Dictyostelium amoebae. Amoebae prepared by the agar-overlay method form actin-rich anchorage structures, or "eupodia" (true feet), that contain actin-binding proteins, such as myosin-IB and [Alpha]-actinin (6), as well as coronin and ABP-120 (Fukui, unpub.). As we document here, coronin, presumably bound to actin, appears in exceptionally high concentration each time a eupodium is transiently formed at the base of an extending pseudopodium. Because eupodia are small ([approximately]1 [[micro]meter] in diameter), dynamic (average life is less than 3 min), and are located at the amoeba-agarose interface, we observed the agar-overlaid amoebae with a real-time confocal system (7) to achieve high temporal and spatial resolution. The amoebae express either an S65T-GFP-coronin fusion protein in wild type Ax2, or a wild type GFP-coronin in the null coronin mutant. S65T-GFP-coronin was integrated into a vector, whereas GFP-coronin was integrated into chromosomes by gene replacement.
We found that Dictyostelium amoebae expressing GFP-coronin are very sensitive to the 488-nm blue laser beam used for confocal fluorescence imaging; they rounded up within a few minutes of continuous or shorter than 5 seconds' observation. To minimize this effect, we first located the amoeba by nonconfocal epifluorescence, using reduced intensity blue excitation, for 10 to 20 seconds, on a Nikon E800 microscope equipped with a 60x/1.4 N. A., or 100x/1.4 N. A., plan apo oil immersion objective lens. As quickly as possible, we focused on a cell, closed the epifluorescence shutter, and switched out the epifluorescence dichromatic mirror. We then opened the electric shutter in the real-time, spinning-disk confocal scanning unit (Yokogawa CSU-10) attached to the microscope. The video-rate confocal image was integrated on-chip for 6 to 15 video frames (0.2-0.5 s) in a Hamamatsu Chilled CCD camera (C-5985). Synchronous opening of the electric shutter, and timelapse acquisition (at an interval of 10 s) of the image, were controlled with an image processing computer (MetaMorph: Universal Imaging Corp.). All images were saved as tiff files into the hard drive and processed for the production of video movies and image panels using MetaMorph and Photoshop (Adobe).
High-resolution confocal video images of motile Dictyostelium amoeba showing the dynamic accumulation of GFP-coronin, as well as its absence in several organelles (nucleus, mitochondria, contractile vacuoles) were shown at the 1997 MBL General Scientific Meetings. Our observations demonstrate that GFP-coronin is dynamically integrated into the actin cytoskeleton in the pseudopodia, especially the eupodia, and apparently in endocytotic cortical structures. The brightly fluorescent eupodia do not move relative to the substratum. Instead, as shown in Figure 1, when a new row of eupodia appears, the previous row disappears in a well-coordinated manner. Both the assembly and disassembly of GFP-coronin into a single eupodium takes place in less than 30 s. It is unlikely that the observed dynamics of GFP-coronin in eupodia was caused by exposure of the amoeba to 488 nm light because identical pattern of eupodia dynamics can be recognized without fluorescence excitation by their high refraction index (6).
Since coronin binds to F-actin (1); the eupodia contain a high concentration of F-actin (6); and injected rhodamine-actin pattern is similar to that of GFP-coronin (Yumura and Fukui, in prep.), the intense fluorescence of GFP-coronin in the eupodia is likely to reflect the high concentration of F-actin filaments that appear in these transient organelles. However, detailed interpretation must await clarification of the physiological role of coronin and the physico-chemical conditions that regulate the binding of coronin to actin.
We thank Nikon USA, Yokogawa Electronic, and Hamamatsu Photonics for loan of equipment to S. I. and Universal Imaging for technical support. Supported in part by NIH grant GM39548 to Y. F.
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|Author:||Fukui, Yoshio; de Hostos, Eugenio L.; Inoue, Shinya|
|Publication:||The Biological Bulletin|
|Date:||Oct 1, 1997|
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