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

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.


Introduction
Tight junctions play a crucial role in epithelial barrier function and epithelial transport by regulating the free diffusion of solutes via the paracellular pathway, contributing to the homeostasis of fluid compartments in the body (Anderson and Van Itallie, 2009;Otani and Furuse, 2020;Zihni et al., 2016).The claudin family of membrane proteins is responsible for the backbone structure and function of TJs (Furuse et al., 1998b).Claudins have four transmembrane domains, two cytoplasmic domains, two extracellular loops and a cytoplasmic turn between the second and third transmembrane domains (Furuse et al., 1998a).
Claudins assemble into cell-cell contacts at both of the adjacent cells and polymerize within the plasma membrane to form a fibril-like structure, designated as TJ strands (Furuse et al., 1998a(Furuse et al., , 1999)).A network of TJ strands continuously circumscribes the cell and seals the intercellular space to form paracellular diffusion barriers (Staehelin, 1973).Claudins comprise a multigene family of 27 members, which have functional diversity (Mineta et al., 2011).They can be briefly categorized into two types: barrier-forming claudins, which act as barriers to small molecules such as electrolytes, and poreforming claudins, which can allow electrolytes and water to permeate through as paracellular channels (Tsukita et al., 2019;Günzel and Yu, 2013;Van Itallie and Anderson, 2014).Poreforming claudins are further divided into cation-selective claudins, including claudin-2, -10b, and -15, and anion-selective claudins, including claudin-10a, -17, and -21 (Günzel and Yu, 2013).
Each epithelium expresses a certain combination and proportion of claudin subtypes to create functional diversity of the paracellular pathway depending on the physiological requirement of each epithelium (Furuse and Tsukita, 2006;Van Itallie and Anderson, 2014;Günzel and Yu, 2013;Tsukita et al., 2019).
Almost without exception, multiple subtypes of claudins are co-expressed in various types of epithelial cells.Even allowing for functional regulation of TJs by barrier-forming and poreforming claudins, it seems that an excessive number of claudin subtypes are often co-expressed in certain epithelial types.For instance, epithelial cells of the thick ascending limb in the kidney express claudin-3, -10b, -14, -16, and -19 (Milatz et al., 2017), while those of the small intestine express claudin-2, -3, -4, -7, -8, -12, and -15 (Tamura et al., 2008).There are several possible explanations for the significance of the co-expression of many claudin subtypes.First, fine-tuning the permeability property of the paracellular pathway may need a complex combination of claudin subtypes, each of which may have a unique function.
Second, excessive expression of multiple claudin subtypes may guarantee TJ formation by securing the necessary amount of the material for TJs.Third, the expression of a set of multiple claudin subtypes may be required for the generation of TJs with specific properties.In the thick ascending limb, claudin- 16 and -19 interact with each other to make the paracellular pathway permeable to divalent cations (Hou et al., 2008).It was shown that the lack of either claudin-16 or -19 affects the localization of the other at TJs, suggesting that these two claudins work as a set (Hou et al., 2008).Such an interdependency was also reported between claudin-4 and claudin-8 to give TJs an anion channel property (Hou et al., 2010).However, it remains elusive why so many claudin subtypes are co-expressed in certain cell types.
Regarding pore-forming claudins, their channel properties were demonstrated by their overexpression in cultured epithelial cells, which also express endogenous claudins (Van Itallie et al., 2006;Krug et al., 2012b;Amasheh et al., 2002;Günzel et al., 2009;Tanaka et al., 2016;Colegio et al., 2002;Van Itallie et al., 2003).In these experiments, the detailed interaction of the introduced pore-forming claudins with endogenous claudins within TJ strands remains elusive.Claudin-2 and claudin-15 form TJ strands when overexpressed in claudin-negative fibroblasts (Furuse et al., 1998b) and insect Sf-9 cells (Suzuki et al., 2015), respectively, indicating that they assemble in a homophilic manner.Consistently, a homophilic assembly model for the cation pores of claudin-15 has been proposed because of the structural biology of the molecule (Suzuki et al., 2015).However, it has not been demonstrated yet whether these pore-forming claudins display their properties by homophilic assembly or by possible heterophilic assembly with other claudin subtypes.At least in in vitro experiments, claudin-2 can assemble in TJ strands in a heterophilic manner with claudin-1 or claudin-3 (Furuse et al., 1999).
In studies of the barrier/channel properties of individual claudin subtypes and their combinations using cultured epithelial cells, it would be ideal to use epithelial cell lines that lack TJs  (Otani et al., 2019).Claudin quinKO cells lack TJ strands as well as the barrier function to small molecules, although they retain an intercellular membrane attachment with barrier function to macromolecules, which is mediated by JAM-A, a TJ-associated membrane protein with two Ig-like domains (Otani et al., 2019).
We have recently established mouse claudin-3-expressing claudin quinKO cells and confirmed that they reconstituted functional TJs (Fujiwara et al., 2022).Here, we further attempted to use claudin quinKO cells as 'TJ-competent epithelial cells' to reconstitute TJs through the introduction of individual claudins.
As the first step to do this, we examined the expression of

Endogenously expressed claudins in claudin quinKO cells
Claudin quinKO cells were generated from MDCK II cells by introducing mutations into the genes of five major claudin subtypes (Otani et al., 2019).However, transcripts of some other claudin subtypes were detected in an RNA sequencing (RNAseq) analysis of MDCK II cells in a previous study (Shukla et al., 2015).To examine the expression of endogenous claudins in claudin quinKO cells, we performed RNA-seq analysis.

Reconstitution of functional TJs in claudin quinKO cells by individual claudin subtypes expressed in MDCK II cells
Next, we examined whether each of the five claudin subtypes deleted from MDCK II cells during the establishment of claudin quinKO cells can reconstitute TJs in claudin quinKO cells.We constructed expression vectors for each dog claudin-1, -2, -3, -4, and -7 under a strong CAG promoter (Niwa et al., 1991), and finally obtained claudin quinKO cell clones stably expressing each of these claudins.Two independent clones for claudin-1, -2, -3, and -4 were picked based on homogeneous expression of exogenous claudins in immunostaining and analyzed further, but only one cell clone that evenly expressed a sufficient amount of claudin-7 was obtained (Fig. 3A).Immunofluorescence staining showed that each claudin subtype was localized at cell -cell contacts circumscribing the cell and overlapped well with occludin, while additional signals were detected at intracellular vesicles (Fig. 3B).These observations suggest that each of the dog claudin-1, -2, -3, -4, and -7 reconstitutes TJs.In these transfectants, neither endogenous claudin-12 nor claudin-16 was detected at cell-cell contacts (Fig. 4A, B).Furthermore, freezefracture replica electron microscopy revealed that TJ strands are reconstituted at the most apical part in the lateral membrane in claudin quinKO cells expressing either claudin-1, -2, -3, -4, or -7 (Fig. 5A).To examine the epithelial barrier function of these cells, We also examined whether the claudin quinKO cells overexpressing dog claudin-12 or claudin-16 have TJs.In freezefracture replica electron microscopy, neither TJ strand-like fibrils on the P-face nor grooves on the E-face were found in these cells, while stepping stone-like structures were often observed along the most apical part of the lateral membrane in claudin-16-expressing cells (Fig. 5B).Consistently, neither claudin-12-  (cation selective) or -10a (anion selective) (Fig. 7A).Immunofluorescence staining revealed that all of these claudins were localized at cell-cell contacts and well overlapped with occludin in addition to localization at intracellular vesicles (Fig. 7B).TER measurements revealed that the claudin quinKO cells expressing each of claudin-2, -10b, -15 or -10a showed low TER similar to that of claudin quinKO cells (Fig. 8A).On the other hand, paracellular fluorescein flux of the claudin quinKO cells expressing each of these pore-forming claudin subtypes showed highly reduced paracellular flux compared with claudin quinKO cells (Fig. 8B).These results suggest that the claudin quinKO cells expressing each of mouse claudin-2, -10b, -15 or -10a reconstituted continuous TJs with paracellular channel properties.
To investigate their channel properties in more detail, we measured electrophysiological parameters, including transepithelial conductance and dilution potential, of the claudin quinKO cells expressing claudin-2, -10b, -15, or -10a in Ussing chambers (Fig. 9).Cells containing TJs with an ion channel property were expected to show not only moderately reduced transepithelial conductance compared to claudin quinKO cells, but also remarkably increased dilution potential.In our dilution potential experiment, the potential difference was measured upon diluting the apical side of the cell layer with a solution containing half the concentration of NaCl (75 mM) compared to the basolateral side (150 mM).Thus, TJs containing cationselective channels or anion-selective channels should have shown a negative or positive dilution potential, respectively.As shown in Fig. 9A, transepithelial conductance decreased in all the claudin quinKO cells expressing claudin-2, -10b, -15, or -10a compared to claudin quinKO cells.Furthermore, a negative dilution potential was observed in the cells expressing claudin-2, -10b or -15, while a positive dilution potential was observed in the cells expressing claudin-10a (Fig. 9B).These results suggest that TJs formed with claudin-2, claudin-10b or claudin-15 have a paracellular cation channel property, while those formed with claudin-10a have a paracellular anion channel property.To analyze the channel properties in more detail, ion selectivity (P Na /P Cl ) was calculated from the dilution potential using the Goldman-Hodgkin-Katz equation.Comparing the diffusion coefficient (molar ionic conductivity) of Na + and Cl -in solution, Na + mobility is 50.11Scm 2 eq -1 and Cl -mobility is 75.23 Scm 2 eq -1 (Marlon and Lando, 1974).Thus, the ratio of P Na /P Cl through a simple pore without charge selectivity is 0.66, and TJs containing cation-selective pores or anion-selective pores show P Na / P Cl >0.66 or P Na /P Cl <0.66, respectively.Based on this criterion, claudin-2, -10b and -15-expressing cells showed cation selectivity (P Na /P Cl >0.66) (Fig. 9C), while claudin-10a-expressing cells showed anion selectivity (P Na /P Cl <0.66) (Fig. 9C, D), consistent with previous reports (Tamura et al., 2011;Yu et al., 2009;Van Itallie et al., 2006).Claudin quinKO cells have a P Na /P Cl of about 1 (Fig. 9C, D), meaning these cells basically show no permselectivity for cations or anions, which could be due to the large pore size of JAM-A-mediated cell -cell contacts lacking claudin-based TJs (Otani et al., 2019).Claudin-2-expressing cells showed larger P Na /P Cl than claudin-15-expressing cells revealed by the significant difference in the dilution potential between them (Fig. 9C).These results demonstrate that the expression of only a single claudin subtype is sufficient to reconstitute a charge-selective paracellular ion channel at TJs.

Characterization of claudin quinKO cells
In this study, we used claudin quinKO cells as TJ-competent epithelial cells to reconstitute TJs of individual claudin subtypes.
Here, we further showed that each claudin subtype deleted from MDCK II cells during the establishment of claudin quinKO cells, including dog claudin-1, -2, -3, -4, and -7, reconstitutes functional TJs when exogenously expressed under a strong promotor in claudin quinKO cells.However, RNA-seq analysis revealed that a significant amount of claudin-12 and claudin-16 were still expressed in claudin quinKO cells.After careful examination of claudin-12 and claudin-16 proteins, we showed the following.(Gonschior et al., 2022).Because it was reported that claudin-16 and claudin-19 cooperatively generate TJs through their interaction with each other (Hou et al., 2008), endogenous claudin-16 would be likely to be incorporated into TJs reconstituted by claudin-19 in claudin quinKO cells.It may also possible for claudin-12 to be recruited to TJs by a partner claudin subtype, the identity of which, however, is still unknown.In this sense, further deletion of claudin-12 and -16 genes in claudin quinKO cells would provide more appropriate TJ-competent epithelial cells.

Reconstitution of TJs with individual claudin subtypes in claudin quinKO cells
Among the claudin quinKO cells expressing dog claudin-1, -2, -3, -4, or -7, only claudin-2-expressing cells showed low TER.This is consistent with claudin-2 being a pore-forming claudin with cation selectivity (Amasheh et al., 2002).In contrast, the cells expressing claudin-1, -3, -4, or -7 showed high TER, demonstrating that they are barrier-forming claudins.These results are consistent with our previous study in which MDCK II cells with a deletion of the claudin-2 gene showed high TER with clearer localizations of claudin-1, -3, -4, and -7 at cell -cell contacts (Tokuda and Furuse, 2015).Among the cells expressing claudin-1, -3, -4 or -7 used in this study, the TER values of the claudin-1-expressing cells were relatively low compared to the others.To date, variations in the barrier properties between barrier-forming claudin subtypes have not been demonstrated.However, RNAi-mediated knockdown of claudin-4 in a cortical collecting duct cell line M1 and an inner medullar collecting duct cell line mIMCD3 increased TER, i.e. decreased ion permeability, and these studies suggested an anion-preferring channel property of claudin-4 (Hou et al., 2010).Knockouts of claudin-4 in MDCK I cells (Shashikanth et al., 2022), MDCK II cells, and claudin-2-deleted MDCK II cells (Tokuda et al., 2017) did not influence the barrier properties.Similarly, it was reported that overexpression of claudin-7 in LLC-PK1 cells increased TER with a decrease in the paracellular conductance of Cl -and an increase in the paracellular conductance of Na + (Alexandre et al., 2005), while knockdown studies of claudin-7 in MDCK II cells and LLC-PK1 cells indicated a role for claudin-7 as a barrier to Na + and a channel to Cl -, respectively (Hou et al., 2006).These contradictory results appear to be attributed to the variations in different cell systems used, which express unique sets of endogenous claudins (Van Itallie et al., 2003;Krug et al., 2012a).
It was reported that the interaction of claudin-4 with claudin-8 is necessary for the localization of claudin-4 at TJs (Hou et al., 2010).Furthermore, two recent studies described that claudin-4 alone could not generate TJ strand-like structures when expressed in non-epithelial cells (Gonschior et al., 2022;Shashikanth et al., 2022).In contrast, claudin-4 reconstituted functional TJs in claudin quinKO cells in this study.However, whether the amount of claudin-4 or another unknown mechanism underlies the discrepancies between these studies, remains unknown.In regard to the specific function of claudin-4, it has been reported that claudin-4 negatively regulates claudin-2 function in MDCK I cells by destabilizing claudin-2based TJs via a previously unrecognized mechanism, designated as " interclaudin interference " (Shashikanth et al., 2022).
Reconstitution of TJs co-expressing claudin-2 and claudin-4 or their mutants in claudin quinKO cells may provide a useful experimental system to further examine the detailed mechanism of interclaudin interference.
We also demonstrated that individual pore-forming claudin subtypes, including mouse claudin-2, -10b, -15, and -10a create paracellular channels by themselves, i.e. via their homophilic assembly.The ion selectivity of the claudin quinKO cells expressing each of these claudins was consistent with previous reports on the characterization of their channel properties (Yu et al., 2009;Tamura et al., 2011;Van Itallie et al., 2006).In addition, we showed that these pore-forming claudins create barriers against fluorescein.These results are consistent with a recent study on claudin quinKO cells expressing human claudin-2, -15, and -10a conducted in parallel with this study (Gonschior et al., 2022).Furthermore, super-resolution microscopy demonstrated that claudin-2 and claudin-10a are segregated as patches but constitute continuous TJ strands overall in proximal tubules, suggesting that these pore-forming claudins assemble in a homophilic manner in vivo (Gonschior et al., 2022).Notably, the claudin-2-expressing cells showed larger P Na /P Cl than the claudin-15-expressing cells.This difference may be caused by a different degree of the cation selectivity between claudin-2 and -15, but the detailed mechanism should be investigated in future studies.

Cell and antibodies
MDCK II cells were provided by Dr. Masayuki Murata (Tokyo Institute of Technology).Claudin quinKO cells and claudin quinKO cell clones expressing mouse claudin-3 were established and characterized previously (Fujiwara et al., 2022;Otani et al., 2019) performed using the characteristic that the density plot of log2transformed TPM and FPKM values derived from RNA-seq analysis approximate a mixed gaussian distribution consisting of two gaussian distributions of active and inactive gene (Hart et al., 2013;McGowan et al., 2021).Also, the calculations for this analysis were performed using the R software and Perl version 5.18.4.Genes with a higher value than the calculated value were determined to be active genes.
caused by the loss of claudin expression but are able to reconstitute TJs if claudin subtypes of interest are exogenously expressed.Using such cell lines, the function of TJs can be investigated with individual claudin subtypes or any combinations of multiple claudin subtypes without needing to consider the interventions of the other endogenously expressed claudins.Recently, we have succeeded in the establishment of a TJ-negative epithelial cell line from MDCK II cells by deleting the genes for claudin-1, -2, -3, -4, and -7, and they have been designated as claudin quintuple knockout (quinKO) cells To examine the role of endogenous claudin-12 and claudin-16 in TJ formation, we analyzed their expression and localization in MDCK II cells, claudin quinKO cells, and claudin quinKO cell clones stably expressing exogenous dog claudin-12 or claudin-16.Western blots showed strong signals for claudin-12 and claudin-16 in claudin quinKO cells expressing exogenous claudin-12 and claudin-16, respectively, while faint signals (possibly of the endogenous proteins) were detected in the same band locations (sizes) in MDCK II cells and claudin quinKO cells (Fig. 2A, B).In immunofluorescence staining, we could not detect claudin-12 or claudin-16 at cell-cell contacts in MDCK II cells and claudin quinKO cells except faint dot-like signals (Fig. 2C, D).In claudin quinKO cells expressing exogenous claudin-12, claudin-12 was distributed throughout the cell including the plasma membrane and intracellular vesicles, but not concentrated at the apical cell -cell contacts where occludin accumulates (Fig. 2C).In claudin quinKO cells expressing exogenous claudin-16, the distribution of claudin-16 on the plasma membrane appeared more evident than claudin-12, but the colocalization of claudin-16 with occludin was not clear (Fig. 2D).These results suggest that neither endogenous claudin-12 nor claudin-16 contributes to the structure of TJs in MDCK II cells.It is likely that the faint dot-like signals are due to the background staining.
we measured transepithelial electrical resistance (TER) and paracellular flux, which reflect the resistance to electrolyte permeation and passage of a water-soluble tracer, respectively, across the cellular sheet.All the transfectants except one expressing pore-forming claudin-2 showed higher TER values Reconstituting tight junctions in epithelial cells Cell Structure and Function 48: 1-17 (2023) https://doi.org/10.1247/csf.22068compared to claudin quinKO cells although the TER varies depending on claudin subtypes and clones (Fig. 6A).The paracellular flux of fluorescein (332Da) in all the transfectants was much lower than in claudin quinKO cells (Fig. 6B).Taken together, we conclude that each of the claudin subtypes deleted from MDCK II cells during the generation of claudin quinKO cells can reconstitute functional TJs and that the contribution of endogenous claudin-12 or claudin-16 to these reconstituted TJs is negligible.

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.

First
, endogenous claudin-12 and claudin-16 could neither be detected at cell-cell contacts in MDCK II cells nor in claudin quinKO cells by immunofluorescence staining.Second, in the reconstitution of TJs in claudin quinKO cells due to the introduction of claudin-1, -2, -3, -4, or -7, neither endogenous claudin-12 nor claudin-16 could be detected at the TJ region by immunofluorescence staining.Third, exogenous overexpression of claudin-12 or claudin-16 did not result in the reconstitution of TJs in claudin quinKO cells with their concentrated localization at cell-cell contacts.These results suggest that we can neglect possible incorporation of endogenous claudin-12 and -16 into reconstituted TJs formed by exogenous claudins in claudin quinKO cells.Thus, we conclude that claudin quinKO cells are useful as TJ-competent epithelial cells.Furthermore, our observations suggest that there are claudin subtypes that cannot generate TJ strands in epithelial cells by themselves, such as claudin-12 and claudin-16.Consistently, neither claudin-12 nor claudin-16 was able to generate TJ strand-like structures when overexpressed in non-epithelial cells

Further
quantitative analyses of the complexity of TJ strands and epithelial barrier function between these cells would clarify functional differences between barrier-forming claudins.The TER values varied between two clones of the dog claudin-4expressing cells although the expression levels of claudin-4
By using claudin quinKO cells as TJ-competent epithelial cells, the barrier/channel properties of claudin-based TJs can be further investigated.Morphology of TJs as well as the barrier function of TJs evaluated by measurements of TER and paracellular flux may vary depending on individual claudins.A certain combination of multiple claudin subtypes may synergistically create a structure and barrier of the TJs that cannot be explained by a simple sum of the properties of the individual claudins.These possibilities should be examined in the future by reconstituting TJs containing certain combinations and proportions of claudin subtypes in claudin quinKO cells.Furthermore, the paracellular permeability of epithelia to which electrophysiological measurements cannot be applied due to their sizes or structures may be reconstituted in claudin quinKO cells and investigated by mimicking their expression pattern of claudin subtypes.These types of studies will contribute to the understanding of the mechanism of epithelial transport in various epithelia.Claudin quinKO cells are also useful for functional analyses of claudin molecules.Claudins undergo various types of postmodification is expected to modify the behavior of claudins and control their functions(Shigetomi and Ikenouchi, 2018).However, the physiological relevance of each modification has not been fully understood yet.To clarify the role of these post-translational modifications, it is common to examine the behaviors of mutant claudins in which the amino acids susceptible to each modification are replaced with other amino acids.When these mutant claudins are expressed in cultured epithelial cells with TJs, they may be incorporated into TJs due to their interaction with endogenous claudins regardless of the influence of the mutations, so that the function or role of the post-translational modifications may be masked.Introduction of these mutant claudins into claudin quinKO cells will enable us to examine the effects of these post-translational modifications of claudins in TJ formation and function directly.