Abstract
Cyclic adenosine monophosphate (cAMP), which is derived from adenosine triphosphate through adenylyl cyclase A (acaA), acts as an intracellular secondary messenger and an extracellular chemotactic substance in important biological processes. In the social amoebae Dictyostelium discoideum, cAMP mediates cell aggregation, development, and differentiation to spore and stalk cells during fruiting body formation. The acaA gene is transcribed under the control of three different alternative promoters. This study aimed to develop a promoter assay for acaA in D. discoideum using bioluminescence microscopy. Here, we inserted green- and red-emitting luciferase genes into downstream of promoter regions 1 and 3, respectively. Promoter activities were visualized by bioluminescence microscopy. We confirmed the differential expression of acaA under the control of promoters 1 and 3 at the different stages of D. discoideum development. We also demonstrated the application of dual-color bioluminescence imaging in the development of an imaging promoter assay.
Keywords
- Dictyostelium discoideum
- adenylyl cyclase A promoter
- dual-color luciferase
- bioluminescence microscopy
- imaging promoter assay
- fruiting body formation
1. Introduction
Gene expression and regulation are essential processes in cellular proliferation and differentiation and are involved in morphogenesis and embryogenesis. The social amoebae
Adenylyl cyclase A is considered a development-specific enzyme [8, 9]. Galardi-Castilla et al. [10] characterized the promoter region of the
Promoter assays using histochemical techniques are quite cumbersome, as the samples have to be fixed, stained, and observed over time sequentially for each promoter. For this purpose, many samples must be prepared. On the other hand, a promoter assay using bioluminescence microscopy can be used to obtain time-lapse image data from a single experiment using one sample. This method is often used in the study of clock genes [11, 12, 13] and developmental biology [14, 15, 16]. In this chapter, we applied bioluminescence microscopy and used two luciferases in the development of an
2. Materials and methods
2.1 Dictyostelium discoideum
Under the National BioResource Project (NBRP), the National Institute of Advanced Industrial Science and Technology (AIST) in Japan provided the
2.2 Firefly luciferase gene
Green- and red-emitting luciferases were used for the dual-color bioluminescence promoter assay. The green-emitting luciferase gene
2.3 Construction of adenylyl cyclase A reporter vector
The
2.4 Transformation and fruiting body formation
The Ax2 cells were co-transfected with the two constructed vectors by electroporation using a MicroPulser (Bio-Rad, California, USA) according to the protocol for
Millicell Cell Culture Insert PICM 0RG50 (Merck, Darmstadt, Germany) was placed onto a 35-mm glass bottom dish, and 1.2 mL of D buffer (saline solution for
2.5 Bioluminescence microscopy
The bioluminescence images of the cells were captured using an LV200 bioluminescence microscope (Olympus, Tokyo, Japan) [19, 20] equipped with a UCPLFLN 20XPH objective lens (Olympus) and an ImagEM C9100-13 EM-CCD camera (Hamamatsu Photonics, Shizuoka, Japan). The activities of
2.6 Fluorescence microscopy
The autofluorescence images of the cells were captured using an IX83 inverted microscope (Olympus) equipped with a U-HGLGPS excitation light source (output level 100 with ND25 filter), a U-FGFP mirror unit, and a DP74 color CCD camera. A UCPFLN 10XPH objective lens was used for mound and slug observations, and a UCPFLN 4xPH objective lens was used for fruiting body observation. Exposure time was 500 ms for all experiments.
The irradiation power of the excitation light was measured at 480 nm using a PM100D optical power meter (Thorlabs, New Jersey, USA) with an S170C sensor probe for microscopy.
3. Results and discussion
Bright-field and bioluminescence images of the activities of promoters 1 and 3 are shown in Figure 1. According to the bright-field image, the amoeba cells began to aggregate after 10 h of seeding (Figure 1A) and formed the mound (Figure 1D), slug, and fruiting body (Figure 1F) after 16, 18, and 20 h, respectively. The image of the slug is not shown in Figure 1, since the moving slug disappeared from the field of view. In the case of Figure 1, the slug stage was extremely short and the fruiting body formation occurred immediately from the mound.
According to the bioluminescence image, promoter 1 activity was observed in single amoeba cells. It increased gradually (Figure 1A–C) and peaked at the mound stage after 16 h (Figure 1D). Then, it decreased during fruiting body formation (Figure 1E–G) and eventually disappeared (Figure 1H). On the other hand, promoter 3 activity increased during the cell aggregation stage (Figure 1C), peaked during the fruiting body stage (Figure 1F), and then decreased (Figure 1G and H). Thereafter, the activity of the stalk cells disappeared. Histochemical detection of promoter activity using a
To show the time course of the promoter activities, two regions of interest (ROI) (ROI-1 and ROI-2) were assigned to cover the process from cell aggregation to fruiting body formation, as shown in Figure 1. Figure 2 shows the time course of luminescence intensity reflecting the activities of promoters 1 and 3 in ROI-1 (Figure 2A) and ROI-2 (Figure 2B) using 961 time-lapse images captured at 90s intervals for 24 h. The intensity of promoter 1 activity increased after 13 h, peaked after 16 h, decreased, and disappeared after 24 h in ROI-1 and ROI-2. On the other hand, the intensity of promoter 3 activity increased to the same timing as that of promoter 1 after 13 h, but peaked after around 20–22 h. Then, the intensity decreased gradually in ROI-2, but rapidly decreased and recovered in ROI-1. Since the measurement of the intensities involves live imaging, the discrepancy may be caused by the movement of the spores in the ROI during fruiting body formation. The measurement of the time courses of the activities of promoters 1 and 3 by bioluminescence imaging and by a β-galactosidase reporter system [10] showed similar results.
The results of the promoter assay using bioluminescence microscopy were the same as those of the promoter assays using histochemistry and β-galactosidase, confirming the convenience of this imaging promoter assay for
One of the advantages of bioluminescence microscopy is that it is not affected by autofluorescence background. Figure 4 shows the bright-field and autofluorescence images of the mound, slug, and fruiting body stages of
4. Conclusion
The imaging promoter assay of the
Acknowledgments
We thank Prof. H. Kuwayama (Tsukuba University, Japan) for providing the materials needed in the
This work was conducted in association with Nimura Genetic Solutions and Perak State Development Corporation for luciferase gene cloning under a contract with the Convention on Biological Diversity in Malaysia. This research did not receive grants from funding agencies in the public, commercial, or non-profit sectors.
Conflict of interest
The authors T. Hayashi, K. Ogoh, and H. Suzuki are employees of Olympus Corporation (Tokyo, Japan).
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