Figure 1.

Formation of a tricellular actin meshwork during epithelial junction formation. (A–C) WT EpH4 cells (EpH4 WT) were cultured overnight in low-calcium medium then in normal culture medium containing calcium for 0, 12, 24, and 48 h. Cells were fixed and stained with phalloidin (green) and either anti-tricellulin mAb (N24-69 was used throughout for immunofluorescence unless noted otherwise; A) or anti-VASP mAb (magenta; B). The boxed region is magnified in C. Magenta arrowheads indicate tricellulin or VASP localized at tricellular junctions. Scale bar: 20 μm. (D) WT EpH4 cells expressing Clover-myosin IIA were stained with anti-myosin IIB mAb (magenta). Arrowheads indicate that myosin II localized at tricellular junctions. Scale bar: 20 μm. (E) Schematic showing the process of junction formation focusing on the changes in VASP localization (magenta circles) and distribution of actin filaments (green lines). Appearance of sparse, spot-like AJs between nearby cells signals the initiation of epithelial adhesion formation (stage 1). AJs increase in density, which triggers the formation of mature, belt-like AJs (stage 2). VASPs accumulate at these primordial AJs, which anchor actin filaments that are perpendicularly associated. During these initial stages, tricellulins do not localize to spot-like AJs (stage 1) but gradually accumulate at maturing bicellular junctions (Fig. 1 A, 12 h, stage 2). The maturation of belt-like AJs and the formation of bicellular TJs alter tricellulin localization to tricellular junctions only (Fig. 1 A, 24 h, stage 3). At the same time, VASPs strongly accumulate to the ends of bicellular junctions in the vicinity of tricellular junctions. Two actin cables running along converging bicellular junctions crisscross at VASP-positive junctions, resulting in the formation of a finely interlaced mesh-like actin network at tricellular corners, which we call “tricellular actin meshwork.” Finally, when tricellular TJs are sealed, VASPs are no longer concentrated around tricellular junctions, and the actin meshwork is bundled to linear actomyosin fibers (stage 4). The region circled in red is enlarged to show a schematic of the orientation of the actin filaments that crisscross at the tricellular junction. The actin filaments (green lines) that run along bicellular junctions intersect at the tricellular corner to form partially overlapping, antiparallel strands. Myosin (purple circles) concentrate where actin filaments overlap. Based on the observation of actin distribution at tricellular junctions (Fig. 1 C).