Effects of TAMP family on the tight junction strand network and barrier function in epithelial cells

Abstract

Occludin, tricellulin, and marvelD3 belong to the tight junction (TJ)-associated MARVEL protein family. Occludin and tricellulin jointly contribute to TJ strand branching point formation and epithelial barrier maintenance. However, whether marvelD3 has the same function remains unclear. Furthermore, the roles of the carboxy-terminal cytoplasmic tail, which is conserved in occludin and tricellulin, on the regulation of TJ strand morphology have not yet been explored in epithelial cells. We established tricellulin/occludin/marveld3 triple-gene knockout (tKO) MDCK II cells and evaluated the roles of marvelD3 in the TJ strand structure and barrier function using MDCK II cells and a mathematical model. The complexity of TJ strand networks and paracellular barrier did not change in tKO cells compared to that in tricellulin/occludin double-gene knockout (dKO) cells. Exogenous marvelD3 expression in dKO cells did not increase the complexity of TJ strand networks and epithelial barrier tightness. The expression of the carboxy-terminal truncation mutant of tricellulin restored the barrier function in the dKO cells, whereas occludin lacking the carboxy-terminal cytoplasmic tail was not expressed on the plasma membrane. These data suggest that marvelD3 does not affect the morphology of TJ strands and barrier function in MDCK II cells and that the carboxy-terminal cytoplasmic tail of tricellulin is dispensable for barrier improvement.

EpCAM proteolysis and release of complexed claudin-7 repair and maintain the tight junction barrier

“…The restriction sites in the cDNA sequences were disrupted by introducing a silent mutation using specific primer sets (5′-GTG​GAC​GGC​A AGA​T T T GCA​CGG​TGA​CCG​GC-3′ and 5′-GCC​GGT​CAC​CGT​GC A A ATC​T TGC​CGT​CCA​C-3′ [ TP1 ]; 5′-CAG​CCC​AGA​CCA AGA C CT GGA​TGT​TGT​TGG-3′ and 5′-CCA​ACA​ACA​TCC AG G TCT TGG​TCT​GGG​CTG-3′ [ TP4 ]; 5′-CGG​GTT​CAG​A AGA​T T T TCA​ATG​GCT​ACC​TG-3′ and 5′-CAG​GTA​GCC​ATT​GA A A ATC​T TCT​GAA​CCC​G-3′ [ TP14 ]; 5′-GGA​ACC​TTC​ACG G C ATC​C TGC​GGG​ACT​TC-3′ and 5′-GAA​GTC​CCG​CA GGA​T G C CGT​GAA​GGT​TCC-3′ [ CLDN2 ]). The fragments were digested with restriction enzymes, purified using NucleoSpin Gel and PCR Clean-up kit (U0609C; Takara-Bio), and cloned into the pCAG, pCAG-IRES-mCherry-nls, pCAG-cGFP, or pCAG-cmCherry vectors ( Saito et al, 2021 ; Saito et al, 2022 ) at Bgl II and EcoR I restriction sites using T4 DNA ligase (#2011B, Takara-Bio). The DNA sequences were verified (Macrogen).…”

Protocol for establishing knockout cell clones by deletion of a large gene fragment using CRISPR-Cas9 with multiple guide RNAs

“…This protocol can delete gene regions over 100 kbp, including GC-rich domains, and is applicable to various cell lines. For complete details on the use and execution of this protocol, please refer to Saito et al., 1 Saito and Endo et al., 2 and Higashi et al. 3 …”

“…For complete details on the use and execution of this protocol, please refer to Saito et al., 1 Saito and Endo et al., 2 and Higashi et al. 3 …”