Figure 9.
Claudins and JAM-A regulate the conformation of ZO-1. (A) Schematic illustration of the experimental design. MDCK II cells or claudin/JAM-A KO cells stably expressing ZO-1 with an N-terminal HA tag (green) and a C-terminal Flag tag (magenta) were cocultured with non-transfected cells, and the junctions between the transfected and the non-transfected cells were observed. A line scan was performed across paired signals of the HA/Flag tag puncta, and the distance between the HA and Flag tags was measured. (B) STED observation of double staining for the N-terminal HA tag (green) and C-terminal Flag tag (magenta) of HA-ZO-1-Flag expressed in MDCK II cells. The N-terminal HA tag was localized proximal to the membrane, while the C-terminal Flag tag was located more distally on the cytoplasmic side. (C) STED observation of double staining for the N-terminal HA tag (green) and C-terminal Flag tag (magenta) of HA-ZO-1-Flag expressed in claudin/JAM-A KO cells. The N-terminus localized at the membrane-proximal region, while the C-terminus was located on the cytoplasmic side. (D) Measurement of the distance between the N-terminal HA tag (green) and C-terminal Flag tag (magenta) in MDCK II cells and claudin/JAM-A KO cells. ZO-1 tended to adopt an elongated conformation (>60 nm) in MDCK II cells, while it preferred a closed conformation (<60 nm) in claudin/JAM-A KO cells (n = 100 particles from 10 to 11 junctions). (E) Schematic illustration of the method to quantify the proximity between the N-terminal HA tag (green) and C-terminal Flag tag (magenta) in MDCK II cells and claudin/JAM-A KO cells. The fluorescent intensity curves of the line scan were normalized, and the overlapping area between the two curves was quantified. (F) Quantification of the signal overlap between the N-terminal HA tag and C-terminal Flag tag in MDCK II cells and claudin/JAM-A KO cells. The signal overlap between the N-terminal HA tag and C-terminal Flag tag was significantly greater in claudin/JAM-A KO cells, suggesting that the two epitopes were more proximal to each other in claudin/JAM-A KO cells. Data are shown as mean ± SD (n = 100 particles from 10 to 11 junctions). ***P < 0.0005, t test. The plasma membrane outlines are indicated by the dotted lines in B and C. Scale bar: 200 nm.

Claudins and JAM-A regulate the conformation of ZO-1. (A) Schematic illustration of the experimental design. MDCK II cells or claudin/JAM-A KO cells stably expressing ZO-1 with an N-terminal HA tag (green) and a C-terminal Flag tag (magenta) were cocultured with non-transfected cells, and the junctions between the transfected and the non-transfected cells were observed. A line scan was performed across paired signals of the HA/Flag tag puncta, and the distance between the HA and Flag tags was measured. (B) STED observation of double staining for the N-terminal HA tag (green) and C-terminal Flag tag (magenta) of HA-ZO-1-Flag expressed in MDCK II cells. The N-terminal HA tag was localized proximal to the membrane, while the C-terminal Flag tag was located more distally on the cytoplasmic side. (C) STED observation of double staining for the N-terminal HA tag (green) and C-terminal Flag tag (magenta) of HA-ZO-1-Flag expressed in claudin/JAM-A KO cells. The N-terminus localized at the membrane-proximal region, while the C-terminus was located on the cytoplasmic side. (D) Measurement of the distance between the N-terminal HA tag (green) and C-terminal Flag tag (magenta) in MDCK II cells and claudin/JAM-A KO cells. ZO-1 tended to adopt an elongated conformation (>60 nm) in MDCK II cells, while it preferred a closed conformation (<60 nm) in claudin/JAM-A KO cells (n = 100 particles from 10 to 11 junctions). (E) Schematic illustration of the method to quantify the proximity between the N-terminal HA tag (green) and C-terminal Flag tag (magenta) in MDCK II cells and claudin/JAM-A KO cells. The fluorescent intensity curves of the line scan were normalized, and the overlapping area between the two curves was quantified. (F) Quantification of the signal overlap between the N-terminal HA tag and C-terminal Flag tag in MDCK II cells and claudin/JAM-A KO cells. The signal overlap between the N-terminal HA tag and C-terminal Flag tag was significantly greater in claudin/JAM-A KO cells, suggesting that the two epitopes were more proximal to each other in claudin/JAM-A KO cells. Data are shown as mean ± SD (n = 100 particles from 10 to 11 junctions). ***P < 0.0005, t test. The plasma membrane outlines are indicated by the dotted lines in B and C. Scale bar: 200 nm.

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