Figure 2.
Figure 2. In vivo imaging of EGFP-occludin endocytosis in jejunal enterocytes. (A) The opened jejunum, with intact neurovascular supply, is placed in a dish with the mucosa resting on a coverslip. (B) EGFP-occludin (green) is targeted to tight junctions and, to a lesser degree, lateral membranes of jejunal enterocytes. Villous capillaries within the lamina propria can be identified by confocal reflectance (red) based on the presence of blood flow. Portions of four villi are shown in cross section. Nuclei (blue) are shown in the merged image. (bottom) High speed imaging shows erythrocytes (arrows) flowing through villus capillaries (outlined by dashed lines). Images are taken from Video 1. Bars: (top) 20 µm; (bottom) 5 µm. (C) XY plane images at the indicated relative z positions show the targeting of mRFP1–ZO-1 (red) and EGFP-occludin (green) in jejunal enterocytes. Both proteins are concentrated at the tight junction. EGFP-occludin is also present in lateral membranes. The full z stack is shown in Video 2. Bar, 20 µm. (D) 125 confocal sections collected at 0.1-µm intervals were used to create this reconstruction of three villi from a transgenic mouse expressing mRFP1–ZO-1 (red) and EGFP-occludin (green). Nuclei are blue. A rotating view of the reconstruction is available as Video 3. Bar, 20 µm. (E) High magnification images of villous epithelium show that mRFP1–ZO-1 (top; red in merge) and EGFP-occludin (middle; green in merge) were collected at the plane of the tight junction as determined by the location of mRFP1–ZO-1 (pink arrows). EGFP-occludin endocytosis occurs during the interval from 85 to 185 min after TNF injection (blue arrows). The complete time-lapse series is shown in Video 4. Bar, 10 µm. (F) The relatively low magnification image is shown for orientation. Images were collected from a small area of the tight junction (pink arrows). The higher magnification images of the boxed area show focal EGFP-occludin enrichment before endocytosis, vesicle budding, separation, and movement out of the focal plane (blue arrows). The entire process takes ∼15 min. Time after TNF injection is indicated. Bars: (left) 5 µm; (middle) 1 µm. (G) The lower magnification image is shown for orientation. The tight junction (pink arrows) appears as a bright spot of EGFP-occludin. Higher magnification images of the boxed area show EGFP-occludin–containing endocytic vesicles (blue arrows) leaving the basal aspect of the tight junction. Time after TNF injection is indicated. Bars: (left) 10 µm; (middle) 2 µm.

In vivo imaging of EGFP-occludin endocytosis in jejunal enterocytes. (A) The opened jejunum, with intact neurovascular supply, is placed in a dish with the mucosa resting on a coverslip. (B) EGFP-occludin (green) is targeted to tight junctions and, to a lesser degree, lateral membranes of jejunal enterocytes. Villous capillaries within the lamina propria can be identified by confocal reflectance (red) based on the presence of blood flow. Portions of four villi are shown in cross section. Nuclei (blue) are shown in the merged image. (bottom) High speed imaging shows erythrocytes (arrows) flowing through villus capillaries (outlined by dashed lines). Images are taken from Video 1. Bars: (top) 20 µm; (bottom) 5 µm. (C) XY plane images at the indicated relative z positions show the targeting of mRFP1–ZO-1 (red) and EGFP-occludin (green) in jejunal enterocytes. Both proteins are concentrated at the tight junction. EGFP-occludin is also present in lateral membranes. The full z stack is shown in Video 2. Bar, 20 µm. (D) 125 confocal sections collected at 0.1-µm intervals were used to create this reconstruction of three villi from a transgenic mouse expressing mRFP1–ZO-1 (red) and EGFP-occludin (green). Nuclei are blue. A rotating view of the reconstruction is available as Video 3. Bar, 20 µm. (E) High magnification images of villous epithelium show that mRFP1–ZO-1 (top; red in merge) and EGFP-occludin (middle; green in merge) were collected at the plane of the tight junction as determined by the location of mRFP1–ZO-1 (pink arrows). EGFP-occludin endocytosis occurs during the interval from 85 to 185 min after TNF injection (blue arrows). The complete time-lapse series is shown in Video 4. Bar, 10 µm. (F) The relatively low magnification image is shown for orientation. Images were collected from a small area of the tight junction (pink arrows). The higher magnification images of the boxed area show focal EGFP-occludin enrichment before endocytosis, vesicle budding, separation, and movement out of the focal plane (blue arrows). The entire process takes ∼15 min. Time after TNF injection is indicated. Bars: (left) 5 µm; (middle) 1 µm. (G) The lower magnification image is shown for orientation. The tight junction (pink arrows) appears as a bright spot of EGFP-occludin. Higher magnification images of the boxed area show EGFP-occludin–containing endocytic vesicles (blue arrows) leaving the basal aspect of the tight junction. Time after TNF injection is indicated. Bars: (left) 10 µm; (middle) 2 µm.

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