The bicellular tensile force sorts the localization of LSRs in bicellular and tricellular junctions

Abstract

Lipolysis-stimulated lipoprotein receptors (LSRs) localize to tricellular tight junctions. Recent studies have shown that changes in the localization and expression profiles of LSRs are associated with malignancy of endometrial carcinomas, although the precise mechanisms by which malignant progression induces changes in the localization of LSRs are still unknown. In this study, we found that changes in cell tension correlated with alterations in the junctional localization of LSRs in endometrial cancer Sawano cells. At high cell densities, myosin phosphatase target subunit 1 (MYPT1) localized to bicellular junctions, whereas activated myosin regulatory light chain 2 (MRLC2) was dislocated from these regions, suggesting that circumferential tensile forces decreased at high cell densities. Under these conditions, LSRs localized to tricellular junctions. In contrast, a phosphorylated form of MRLC2 localized to bicellular regions, while MYPT1 was excluded from these regions, suggesting that tensile forces formed along the circumferential edge at low cell densities. It is noteworthy that, when cells were cultured under these conditions, LSRs localized to bicellular regions. Upon treatment with a myosin inhibitor, LSR localization in bicellular junctions decreased at low cell densities. Overall, our results indicate that the modulation of cellular tension was involved in the translocation of LSRs from bicellular to tricellular tight junctions.

Role of Tricellular Tight Junction Protein Lipolysis-Stimulated Lipoprotein Receptor (LSR) in Cancer Cells

“…Conversely, at low cell densities, cells spread thinly and cell motility increases. In Sawano cells, LSR and tricellulin localized in tricellular contacts under high cell density conditions, while these proteins migrated to the bicellular region under low cell density conditions despite the presence of tricellular contacts [83]. Under these conditions, TER measurements indicated that an epithelial barrier was present even at low cell densities.…”

“…An increase in cell density affects the intracellular tension mediated by actomyosin [84]. MRLC2 is localized at bicellular regions in the low density culture of Sawano cells [83]. Phosphorylated form of MRLC2, which represents activated myosin, is also localized in these regions.…”

“…Myosin, a cross-linked protein of actin fibers, generates cellular tension by contracting actin fibers [88]. Sawano cells cultured at low density conditions were spread thin and wide, and significant stress fiber formation was induced [83]. This suggested that high cellular tension is present under these conditions.…”

“…In Sawano cells, LSR is reversibly translocated in a cellular tension- and/or cell size-dependent manner, whereas the localizations of OCLN and CLDN-4 were not affected by these conditions. Interestingly, accumulation of F-actin was observed not only along the lateral region of tricellular contacts but also the circumferential subapical region, leading to increase in cell thickness with the high cell density culture of Sawano cells [83]. Moreover, the actin polymerization inhibitor, Cytochalasin D, excluded LSR from tricellular contacts, resulting in partial stratification of monolayered Sawano cell sheets.…”

“…MYPT1 is known to activate Merlin [68,79]. The localization of MYPT1 was sensitive to changes in cell density [83]. Therefore, in endometrial cancer cells, the activity of Merlin might be altered by changes in cell density.…”