Figure 1.
Bioluminescence imaging of GLase as a reporter protein to visualize proteins on the surface of mammalian cells.
(A) Schematic representation of imaging principles by GLase bioluminescence. (B) Detectable area of exocytosis in a single mammalian cell using GLase bioluminescence imaging method. (C) Schematic representation of the secretion of GLase and the fusion protein of GLase from mammalian cells and its binding on the cell surface. (D) Schematic representation of expression vectors for GLase and MMP2-GLase. GLuc (pcDNA3-GLuc); GLase with the signal peptide sequence, MMP2-GLuc (pcDNA3-hMMP2-GLuc); human MMP2 preproprotein fused to GLase. (E) GLase activity in the conditioned medium of HeLa cells. HeLa cells were transfected with pcDNA3-GLuc or pcDNA3-hMMP2-GLuc and cultured for 24 hr followed by incubation with HBSS for 60 min at 37°C. The luminescence activity in the conditioned medium was determined using a luminometer.
Figure 2.
Video-rate bioluminescence imaging of GLase secretion from a single HeLa cell using an EM-CCD camera.
HeLa cells were transfected with pcDNA3-GLuc and cultured for 24 h. (A) Bright-field image of a single cell. Objective lens; 40×. (B) Bioluminescence image of GLase secretion obtained from all area of bright-field image (A). Luminescence signals were recorded with an exposure time of 500 ms per image for 75 s (Video image is in Video S1). Yellow arrows in the images at 0.5 s indicate the luminescence spots in three spots 1–3. (C) Time-dependent changes of the average (red line) and maximum (green line) of luminescence intensities from GLase secretion. The arrows with numbers of 1–3 indicate the luminescence positions, shown as yellow arrows at 0.5 s in (B). (D) Magnified luminescence image of GLase secretion obtained from the area of yellow square in (A). Luminescence signals were acquired with an exposure time of 30.5 ms per image for 4.7 s (Video image is in Video S3).
Figure 3.
Time-dependent decrease of luminescence intensity of MMP2-GLase with coelenterazine.
Luminescence intensity of MMP2-GLase with coelenterazine (3 µg/ml) is recorded for 60 s. HeLa cells were transfected with pcDNA3-hMMP2-GLuc and cultured for 24 h. After removing the medium, the cells were incubated with HBSS for 60 min at 37°C and the luminescence activity in HBSS was determined using a luminometer, as described in Materials and Methods.
Figure 4.
Characterization of MMP2-GLase expressed in HeLa cells with immunochemical analyses.
MMP2-GLase expressed in HeLa cells was identified by Western blot (A and B) and immunofluorescence (C) analyses. (A) Western blot analysis of MMP2-GLase in cell lysate using anti-MMP-2 and anti-GLase antibodies (MMP-2 and GLase for lane 1 and lane 2, respectively). HeLa cells were transfected with pcDNA3-hMMP2-GLuc for the expression of MMP2-GLase (MMP2-GLase) and cultured for 24 h, and proteins in the cell lysate were separated in a 5–20% gel. The numbers on the left margin represent the molecular weight of size maker proteins (XL ladder, Promega). The molecular sizes of two bands by arrow 1 and 2 correspond to the pro-form (89.4 kDa) and processed form (80.3 kDa) of MMP2-GLase, respectively. (B) Western blot analyses of the cell lysate and the conditioned medium expressing MMP2-GLase, MMP2-FLAG, and wild type MMP-2 using anti-MMP-2 antibody (MMP-2). HeLa cells transfected with pcDNA3 (as a control vector: lane 1 and 2), pcDNA3-hMMP2 (lane 3 and 4), pcDNA3-hMMP2-Flag (lane 5 and 6), or pcDNA3-hMMP2-GLuc (lane 7 and 8) (Cont, MMP-2, MMP2-FLAG, and MMP2-GLase, respectively) were cultured for 24 h, and further cultured for 24 h in HBSS. Proteins in the cell lysate (lane 1, 3, 5, 7) and the concentrated proteins from the conditioned medium of HBSS (lane 2, 4, 6, 8) were separated in a 10% gel. The numbers on the left margin represent the molecular weight of size maker proteins (Precision Plus Protein All Blue standards, Bio-Rad). Two bands indicated by arrow 1 and 2 at the right margin correspond to pro- and processed forms of MMP2-GLase, respectively. Two bands indicated by arrow 3 and 4 at the right margin correspond to pro- and processed forms of MMP-2, respectively. Endogenous pro- and processed MMP-2 (71.0 kDa and 62.0 kDa) slightly detected in lane 1 and 2, exogenous pro- and processed MMP-2 in lane 3 and 4, and pro- and processed MMP2-FLAG (72.2 kDa and 63.2 kDa) in lane 5 and 6. (C) Immunofluorescence images of MMP2-FLAG (Green) and MMP2-GLase (Magenta) co-expressed in HeLa cells. Overlay; Merged image of the both fluorescence images. IB, immunoblot; TF, transfection; DF, dye front.
Figure 5.
Identification of MMP2-GLase being secreted and bound on the cell surface of a HeLa cell using bioluminescence imaging.
Bioluminescence imaging of HeLa cells transiently expressing MMP2-GLase. Objective lens; 40×. Luminescence signals recording was started after 20 s from the addition of HBSS buffer containing coelenterazine with an exposure time of 500 ms for 75 s (Video image is in Video S4). (A) The bright-field image (the left panel), and luminescence images acquire at 0, 6, and 25 s. Two continuous luminescence spots (spot 1 and 2) are indicated by yellow arrowheads labeled with 1 and 2, respectively, and two transient diffusive luminescence spots (spot 3 and 4) are indicated by yellow arrowheads labeled with 3 and 4, respectively. (B) Magnified luminescence images of spot 1–4 indicated by arrowheads labeled with 1–4 in (A). (C) Time-dependent changes of the average luminescence intensity of spot 1–4 indicated by arrowheads labeled with 1–4 in (A).
Figure 6.
Bioluminescence images of MMP2-GLase secretion in a migrating HeLa cell.
Luminescence images of MMP2-GLase secretion after the disappearing of luminescence signals of the membrane-associated MMP2-GLase. (A–D) Data of luminescence images after 25 s from the start of recording with 40× objective lens shown in Figure 3 (Video image is in Video S4). (E–G) Data of luminescence images of the leading edge obtained with 100× objective lens, after the recording with 40× objective lens (Video image is in Video S5). (A) Time-dependent luminescence signals of MMP2-GLase secretion. The first image (T = 0) is one frame in Video S4 at 35 s 54 ms after the start of recording. Luminescence spots newly appeared in a frame are indicated by yellow arrowheads. (B) Magnified luminescence images of the leading edge corresponding to the area indicated by a yellow square in (A). The first image (T = 0) is one frame in Video S4 at 51 s 79 ms after the start of recording. Newly appeared luminescence spots are indicated by yellow arrowheads. (C) An image of the maximum luminescence intensity obtained with an exposure time of 500 ms for 50 s after the disappearance of luminescence signals of MMP2-GLase. The image was made from the frame at 26 s 541 ms to the end frame at 1 min 16 s 118 ms (100 frames) after the start of recording. The luminescence signals of maximum intensity are green-colored. (D) Time-dependent changes of the maximum luminescence intensity in the area of red circles (1–4) in (C). (E) A luminescence image of MMP2-GLase on the leading edge obtained with 100× objective lens in the area of a yellow square in (C). The image is one frame in Video S5 with an exposure time of 500 ms at 43 s 567 ms after start of recording. The yellow arrows indicate luminescence signals of MMP2-GLase secretion, and luminescence spots newly appeared in this frame of the video image are numbered (spot 1–3). (F) Time-dependent change of magnified luminescence spots in (E). The magnified images of spot 1–3 at 0.5 s correspond to the spots indicated by arrow 1–3, respectively, in (E). (G) An image of the maximum luminescence intensity on the leading edge obtained with an exposure time of 500 ms for 100 s with 100× objective lens. The luminescence signals of maximum intensity are green-colored and the areas with the strong luminescence spots were red-circled.
Figure 7.
Identification of MMP2-GLase secretion from the bottom side of cell body.
Bioluminescence imaging of HeLa cells transiently expressing MMP2-GLase was performed using an IX81-ZDC Zero Drift microscope system. Objective lens; 60×. Luminescence video images were obtained with an exposure time of 500 ms for 50 s after 4 min from the addition of HBSS buffer containing coelenterazine. On recording the luminescence images of MMP2-GLase secretion from cells attached on the cover slip, the in-focus z-axis positions were adjusted to 0, 0.5, 1.0, or 1.5 µm from the upside of the cover slip on a microscope stage using the Zero Drift system. Images of maximum luminescence intensity were made from all frames (100 images for 50 s) at the z-axis positions (Z) of 0, 0.5, 1.0, and 1.5 µm.
Figure 8.
A schematic representation of MMP-2 secretion and binding on the cell surface in migration cells.
(A) Hot spots of both secretion and binding of MMP-2 on the cell surface in a migrating cell. (B) Inactive pro-form of MMP-2 is frequently secreted on the leading edge and then bind to micro areas on the cell surface.