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. 2023 Jan 20;48(1):1-17.
doi: 10.1247/csf.22068. Epub 2022 Dec 9.

Reconstitution of functional tight junctions with individual claudin subtypes in epithelial cells

Mikio Furuse  1   2   3 , Daiki Nakatsu  4 , Wendy Hempstock  5   6 , Shiori Sugioka  6 , Noriko Ishizuka  6 , Kyoko Furuse  1 , Taichi Sugawara  7 , Yugo Fukazawa  8 , Hisayoshi Hayashi  6
Affiliations

Reconstitution of functional tight junctions with individual claudin subtypes in epithelial cells

Mikio Furuse et al. Cell Struct Funct. .

Abstract

The claudin family of membrane proteins is responsible for the backbone structure and function of tight junctions (TJs), which regulate the paracellular permeability of epithelia. It is thought that each claudin subtype has its own unique function and the combination of expressed subtypes determines the permeability property of each epithelium. However, many issues remain unsolved in regard to claudin functions, including the detailed functional differences between claudin subtypes and the effect of the combinations of specific claudin subtypes on the structure and function of TJs. To address these issues, it would be useful to have a way of reconstituting TJs containing only the claudin subtype(s) of interest in epithelial cells. In this study, we attempted to reconstitute TJs of individual claudin subtypes in TJ-deficient MDCK cells, designated as claudin quinKO cells, which were previously established from MDCK II cells by deleting the genes of claudin-1, -2, -3, -4, and -7. Exogenous expression of each of claudin-1, -2, -3, -4, and -7 in claudin quinKO cells resulted in the reconstitution of functional TJs. These TJs did not contain claudin-12 and -16, which are endogenously expressed in claudin quinKO cells. Furthermore, overexpression of neither claudin-12 nor claudin-16 resulted in the reconstitution of TJs, demonstrating the existence of claudin subtypes lacking TJ-forming activity in epithelial cells. Exogenous expression of the channel-forming claudin-2, -10a, -10b, and -15 reconstituted TJs with reported paracellular channel properties, demonstrating that these claudin subtypes form paracellular channels by themselves without interaction with other subtypes. Thus, the reconstitution of TJs in claudin quinKO cells is advantageous for further investigation of claudin functions.Key words: tight junction, claudin, paracellular permeability, epithelial barrier.

Keywords: claudin; epithelial barrier; paracellular permeability; tight junction.

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Figures

Fig. 1
Fig. 1
RNA-seq analyses of the expression of claudin subtypes in claudin quinKO cells Analyses of the density plots of log2-transformed TPM values (A–C) and FPKM values (D–F) of each gene in claudin quinKO cells. In all graphs, the x-axis shows the log2-transformed TPM or FPKM values of each gene, while the y-axis shows the number of genes. Blue lines (A, B, D, E) and black bars (C, F) represent the density plots. The TPM and FPKM values were rounded off to the first decimal place. Orange lines (A, D) represent the results of the double gaussian model fit to the density plot. Light green lines (B, E) represent a gaussian model of active genes fit to the main peak of the right side of the density plot. Light yellow lines (B, E) represent the results of the gaussian model of inactive genes fit to the main peak of the left side of the density plot. The y-axis values of the orange line (A, D) are the sum of the y-axis values of the light green and light yellow lines (B, E). Dashed black lines (B, E) and white arrows (C, F) represent the level of gene expression where the fraction of active genes is estimated to be five times higher than that of inactive genes, and is evaluated in FPKM or TPM. In (C) and (F), numbers in the graphs correspond to subtype numbers of claudin family genes and arrows indicate their log2-transformed TPM or FPKM values. Red and blue labels indicate active genes and inactive genes, respectively.
Fig. 2
Fig. 2
Subtle expression of claudin-12 and -16 proteins in MDCK II cells and claudin quinKO cells (A) Western blots of MDCK II cells, claudin quinKO cells, and two clones of claudin quinKO cells expressing exogenous dog claudin-12 with anti-claudin-12 antibody or anti-α-tubulin antibody. (B) Western blot of MDCK II cells, claudin quinKO cells, and two clones of claudin quinKO cells expressing exogenous dog claudin-16 with anti-claudin-16 antibody or anti-α-tubulin antibody. (C) Double immunofluorescence staining of MDCK II cells, claudin quinKO cells, and claudin quinKO cells expressing exogenous dog claudin-12 with anti-claudin-12 and anti-occludin antibodies. (D) Double immunofluorescence staining of MDCK II cells, claudin quinKO cells, and claudin quinKO cells expressing exogenous dog claudin-16 with anti-claudin-16 and anti-occludin antibodies. Claudin is abbreviated as Cldn. Scale bars: 10 μm.
Fig. 3
Fig. 3
Exogenous expression of dog claudins-1, -2, -3, -4 and -7 in claudin quinKO cells (A) Western blots of MDCK II cells, claudin quinKO cells, and claudin quinKO cell clones expressing exogenous claudin-1, -2, -3, -4, or -7 with antibodies to respective claudins and α-tubulin. (B) Double immunofluorescence staining of claudin quinKO cell clones expressing exogenous dog claudin-1, -2, -3, -4, or -7 with antibodies to each respective claudin and occludin. Scale bar: 10 μm.
Fig. 4
Fig. 4
Absence of endogenous claudin-12 and -16 proteins at cell–cell contacts in claudin quinKO cells expressing exogenous dog claudin-1, -2, -3, -4, or -7 (A) Double immunofluorescence staining of claudin quinKO cell clones expressing exogenous dog claudin-1, -2, -3, -4, -7, or -12 with anti-claudin-12 and anti-occludin antibodies. (B) Double immunofluorescence staining of claudin quinKO cell clones expressing exogenous dog claudin-1, -2, -3, -4, -7, or -16 with anti-claudin-16 and anti-occludin antibodies. In claudin quinKO cell clones expressing exogenous dog claudin-1, -2, -3, -4, and -7, neither claudin-12 nor claudin-16 was detected at cell–cell contacts colocalizing with occludin. Claudin quinKO cells expressing exogenous dog claudin-12 or -16 were immunostained as positive controls (A, B). Scale bars; 10 μm.
Fig. 5
Fig. 5
Freeze-fracture replica images of claudin quinKO cells and claudin quinKO cell clones expressing exogenous dog claudin-1, -2, -3, -4, -7, -12, or -16 (A, B) Continuous belts of TJ strands were observed at the most apical region of the lateral membrane in claudin quinKO cell clones expressing exogenous dog claudin-1, -2, -3, -4, and -7, but not in claudin quinKO cells (A) and those expressing exogenous dog claudin-12 and -16. (B) Stepping stone-like structures were often observed along the most apical part of the lateral membrane in the claudin-16-expressing cells, (B, arrowheads). All images are placed with microvilli on top. Scale bars: 500 nm.
Fig. 6
Fig. 6
Epithelial barrier function of claudin quinKO cells expressing exogenous dog claudin subtypes (A) TER measurements of claudin quinKO cells, claudin quinKO cell clones with an empty expression vector (Cont), and claudin quinKO cell clones expressing exogenous dog claudin-1, -2, -3, -4 or -7. (B) The paracellular flux of fluorescein in claudin quinKO cells, claudin quinKO cell clones with an empty expression vector (Cont), and claudin quinKO cell clones expressing exogenous dog claudin-1, -2, -3, -4 or -7. (C) TER measurements of claudin quinKO cells, and claudin quinKO cell clones expressing exogenous dog claudin-3, -12, or -16. (D) The paracellular flux of fluorescein in claudin quinKO cells, and claudin quinKO cell clones expressing exogenous dog claudin-3, -12, or -16. (A–D) Data are shown as mean ± standard deviation (SD) (n = 3). Data from claudin quinKO cells were compared with data from other clones by Dunnett’s test. **: P<0.01, ns: not significant.
Fig. 7
Fig. 7
Exogenous expression of pore-forming mouse claudin subtypes in claudin quinKO cells (A) Western blots of claudin quinKO cells, and claudin quinKO cell clones expressing mouse claudin-2, -10b, -15, and -10a with antibodies to respective claudin and α-tubulin. (B) Double immunofluorescence staining of claudin quinKO cell clones expressing mouse claudin-2, -10b, -15, and -10a with antibodies to respective claudin and occludin. Double staining of claudin quinKO cells with antibodies to occludin and claudin-10 or -15 are also shown as negative controls. Scale bar: 10 μm.
Fig. 8
Fig. 8
Epithelial barrier function of claudin quinKO cells expressing pore-forming mouse claudin subtypes (A) TER measurements of claudin quinKO cell clones with an empty expression vector (Cont) and those expressing mouse claudin-2, -10b, -15, -10a, or -3. (B) The paracellular flux of fluorescein in claudin quinKO cell clones with an empty expression vector (Cont) and those expressing mouse claudin-2, -10b, -15, -10a, or -3. (A,B) Data are shown as mean ± SD (n = 3). Data from a claudin quinKO cell clone with an empty expression vector (Cont_1) were compared with data from other clones by Dunnett’s test. **: P<0.01, ns: not significant.
Fig. 9
Fig. 9
Electrophysiological measurements of claudin quinKO cells expressing pore-forming mouse claudin subtypes in Ussing chambers Cells cultured to confluence on Transwell filters were mounted in Ussing chambers. (A) The electrical conductance of claudin quin KO cells and those expressing mouse claudin-2, -10a, -10b, or -15. (B) NaCl dilution potential measurements of claudin quinKO cells and those expressing mouse claudin-2, -10a, -10b, or -15. (C) The permeability ratio of Na+ to Cl–, (PNa/PCl) of claudin quinKO cells and those expressing mouse claudin-2, -10a, -10b, or -15. (D) The permeability ratio of Cl– to Na+, (PCl/PNa) of claudin quinKO cells and those expressing mouse claudin-10a. (A–D) Each dot represents an individual data point, the horizontal bar indicates the mean value, and the error bars represent SD. quinKO (n = 8), +mCldn-2_1 (n = 4), +mCldn-2_2 (n = 4), +mCldn-10a_1 (n = 4), +mCldn-10b_4 (n = 2), +mCldn-10b_5 (n = 2), +mCldn-15_1 (n = 4), +mCldn-15_2 (n = 4). Data were analyzed by one-way ANOVA, and Tukey’s multiple comparison test was used for the post hoc test. **: P<0.01; ***: P<0.001; ****: P<0.0001.
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References

    1. Alexandre, M.D., Lu, Q., and Chen, Y.H.. 2005. Overexpression of claudin-7 decreases the paracellular Cl– conductance and increases the paracellular Na+ conductance in LLC-PK1 cells. J. Cell Sci., 118: 2683–2693. - PubMed
    1. Amasheh, S., Meiri, N., Gitter, A.H., Schöneberg, T., Mankertz, J., Schulzke, J.D., and Fromm, M.. 2002. Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells. J. Cell Sci., 115: 4969–4976. - PubMed
    1. Anderson, J.M. and Van Itallie, C.M.. 2009. Physiology and function of the tight junction. Cold Spring Harb. Perspect. Biol., 1: a002584. - PMC - PubMed
    1. Chomczynski, P. and Sacchi, N.. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162: 156–159. - PubMed
    1. Colegio, O.R., Van Itallie, C.M., McCrea, H.J., Rahner, C., and Anderson, J.M.. 2002. Claudins create charge-selective channels in the paracellular pathway between epithelial cells. Am. J. Physiol. Cell Physiol., 283: 142–147. - PubMed
-