Article Figures & Data
Figures
- FIG. 1.
Schematic representation of different expression vectors for transient transfection of AhSub-EGFP and AhCP-EGFP fusion proteins in A. healyi. (A) pUbAhSub(f)g, full-length AhSub; pUbAhSub(pre)g, pre region of AhSub; pUbAhSub(pre-pro)g, prepro region of AhSub; pUbAhSub(pro-m)g, promature region of AhSub; pUbAhSub(m)g, mature region of AhSub; pUbAhSub(pro)g, pro region of AhSub. (B) pUbAhCP(f)g, full-length AhCP; pUbAhCP(pre-pro)g, prepro region of AhCP; pUbAhCP(pro)g, pro region of AhCP; pUbAhCP(m)g, for mature region of AhCP; pUbAhCP(pre)g, pre region of AhCP.
- FIG. 2.
(A) Fluorescence (left) and light (right) micrographs of a live Acanthamoeba expressing the full-length AhSub-EGFP fusion protein. Small fluorescent vesicle-like structures were distributed at the periphery of the cytoplasm. (B) Two amoebae with the fluorescence of EGFP alone showed dispersed distribution in the cytoplasm. Bar, 10 μm.
- FIG. 3.
(A) Fluorescence micrographs of Acanthamoeba secreting AhSub-EGFP fusion protein photographed at 20-s time interval. The numbers in the upper left corners denote time in seconds. The arrows indicate the excretion of a secretory vesicle containing the AhSub-EGFP fusion protein. (B) Magnification of the amoeba expelling the fluorescent material taken at 40 s (white box in 40-s panel). Bar, 10 μm.
- FIG. 4.
Fluorescence (left) and light (right) micrographs of live amoeba expressing each deletion mutant construct of AhSub-EGFP fusion protein. (A) Pre region of AhSub-EGFP; (B) prepro region of AhSub-EGFP; (C) promature region of AhSub-EGFP; (D) mature region of AhSub-EGFP; (E) pro region of AhSub-EGFP. Small fluorescent vesicle-like structures were observed in cells expressing the prepro region and pre region, whereas amoeba cells expressing the other 3 constructs demonstrated dispersed localization of fluorescence in the cytoplasm. Bar, 10 μm.
- FIG. 5.
Fluorescence (left) and light (right) micrographs of a live Acanthamoeba expressing the full-length AhCP-EGFP fusion protein. Small fluorescent vesicle-like structures were randomly distributed in the cytoplasm of the amoeba. Bar, 10 μm.
- FIG. 6.
Localization of AhCP-EGFP in A. healyi. (A) Light micrograph; (B) expressed AhCP-EGFP and AhSub-EGFP fusion proteins (green signal); (C) lysosomes of Acanthamoeba stained with Lyso Tracker Red DND-99 (red signal); (D) overlapped micrograph images of panels B and C (yellow signal). The presence of the yellow signals was demonstrated only in the cells expressing AhCP but not in the cells transfected with AhSub. Bar, 10 μm.
- FIG. 7.
Fluorescence (left) and light (right) micrographs of live amoeba expressing each deletion mutant construct of AhCP-EGFP. (A) Prepro region of AhCP-EGFP; (B) pro region of AhCP-EGFP; (C) mature region of AhCP-EGFP; (D) pre region of AhCP-EGFP. Amoeba cells expressing prepro region and pro region displayed the fluorescent vesicle-like structures. In contrast, dispersed distribution of fluorescence in the cytoplasm was observed in cells transfected with mature region and pre region. Bar, 10 μm.
Tables
- TABLE 1.
Primer sequences for amplication of AhSub and AhCP inserted into expression vector
Construct Primer sequencea pUbAhSub(f)g F: CCGCCATGGCCATGCGCGCCGTCACCCCTC R: CCGACTAGTGGCGGTGGGGTACGAAGCAGC pUbAhSub(pre)g F: CCGCCATGGCCATGCGCGCCGTCACCCCTC R: ATAACTAGTAGCGAGAGCGCTAGCGCAGAG pUbAhSub(pre-pro)g F: CCGCCATGGCCATGCGCGCCGTCACCCCTC R: CCGACTAGTAGCAAGACCCTTGAAGAGGCG pUbAhSub(pro-m)g F: AATCCATGGCCACGCACGACCCGCTCACGG R: CCGACTAGTGGCGGTGGGGTACGAAGCAGC pUbAhSub(m)g F: AATCCATGGACTTCGACTACTCCAAGCACG R: CCGACTAGTGGCGGTGGGGTACGAAGCAGC pUbAhSub(pro)g F: AATCCATGGCCACGCACGACCCGCTCACGG R: CCGACTAGTAGCAAGACCCTTGAAGAGGCG pUbAhCP(f)g F: ATTGAATTCATGCGTGCCTACTTCGTGGGT R: TATACTAGTGCACCGGGCGGAGTAAAGCAG pUbAhCP(pre-pro)g F: ATTGAATTCATGCGTGCCTACTTCGTGGGT R: ATAACTAGTCTGCGCGCTGGAGATGGAGAC pUbAhCP(pro)g F: ATTGAATTCATGTCTCTTGCCCCTCTTCACAGG R: ATAACTAGTCTGCGCGCTGGAGATGGAGAC pUbAhCP(m)g F: ATTGAATTCATGAACTGCCTGTCCCAGAGCGGC R: TATACTAGTGCACCGGGCGGAGTAAAGCAG pUbAhCP(pre)g F: ATTGAATTCATGCGTGCCTACTTCGTGGGT R: ATAACTAGTAGCGAGAGCGCTAGCGCAGAG ↵ a F, forward; R, reverse. Restriction enzyme sites are underlined.
