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

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 expressions 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 investigations of claudin functions.

Claudin-4 polymerizes after the incorporation of just two extracellular claudin-3 residues

“…Other factors that could influence Cldn4 polymerization are the different N-or C-terminal tags used in the studies and the species origin of the protein. The importance of all of these factors is especially evident when comparing our data to a recent study by Furuse et al (41). In the same qKO cell line, they find that Cldn4, which did not create a barrier in our experiments, makes TJ strands and introduces a strong barrier against solutes.…”

“…Due to the absence of TJ strands, small molecules and electrolytes can easily permeate between qKO cells (38). However, it has been shown that reconstitution of barrier Cldns like Cldn3 can rescue these barrier deficiencies (36, 41). As done in these past studies, we measured the permeability of a small fluorescent tracer molecule (fluorescein, 332 D) [Fig.…”

Integrating Continuous Transepithelial Flux Measurements into an Ussing Chamber Set-Up

“…Others studies concluded that Cldn4 might act as a paracellular channel, either on its own [28,37] or in combination with Cldn8 [29]. In contrast, barrier-forming properties of dog Cldn4 were demonstrated by Furuse et al [38], who reported TER values of up to 4000 Ω•cm 2 when overexpressing dog Cldn4 in claudin quin KO cells. Average TER values of about 2500 Ω•cm 2 were obtained in our experiments with claudin quin KO cells expressing mouse claudin-4, whereas the TER was very low in claudin quin KO cells expressing mouse claudin-8 (averaging about 20 Ω•cm 2 ; for baseline values, see Supplemental Table S1; for an example, see Supplemental Figure S1).…”

Claudins in Cancer: A Current and Future Therapeutic Target

“…The barrier characteristics of a given tissue and a given CLDN seem to be reliant on the combination of the CLDNs that are expressed and on the manner in which they copolymerize [17]. Some CLDNs exert their function via interaction with other CLDNs, e.g., CLDN23, which functions via heteromeric cis and heterotypic trans interactions with CLDN3 and CLDN4 [18], while others function independently, as in the case of channelforming CLDNs −2, 10a, 10b, and −15 [19].…”