Micron-scale supramolecular myosin arrays help mediate cytoskeletal assembly at mature adherens junctions

Abstract

Epithelial cells assemble specialized actomyosin structures at E-Cadherin–based cell–cell junctions, and the force exerted drives cell shape change during morphogenesis. The mechanisms that build this supramolecular actomyosin structure remain unclear. We used ZO-knockdown MDCK cells, which assemble a robust, polarized, and highly organized actomyosin cytoskeleton at the zonula adherens, combining genetic and pharmacologic approaches with superresolution microscopy to define molecular machines required. To our surprise, inhibiting individual actin assembly pathways (Arp2/3, formins, or Ena/VASP) did not prevent or delay assembly of this polarized actomyosin structure. Instead, as junctions matured, micron-scale supramolecular myosin arrays assembled, with aligned stacks of myosin filaments adjacent to the apical membrane, overlying disorganized actin filaments. This suggested that myosin arrays might bundle actin at mature junctions. Consistent with this idea, inhibiting ROCK or myosin ATPase disrupted myosin localization/organization and prevented actin bundling and polarization. We obtained similar results in Caco-2 cells. These results suggest a novel role for myosin self-assembly, helping drive actin organization to facilitate cell shape change.

Actin-dependent recruitment of AGO2 to the zonula adherens

“…4 A,D). Furthermore, E-cadherin re-populates areas of cell-cell contact to re-form adherens junctions during recovery in the LatA cells, although these cells do not contain filamentous actin, again in agreement with previous observations (Ivanov et al, 2004; Tang and Brieher, 2012; Yu-Kemp et al, 2022) (Fig. 4 A).…”

“…4 A). However, as previously reported (Ivanov et al, 2004; Yu-Kemp et al, 2022), LatA-treated cells fail to round following calcium depletion, due to the lack of contraction, and pools of E-cadherin, as well as punctate actin pools, are retained at areas of cell-cell contact (Abe and Takeichi, 2008; Cavey et al, 2008; Tang and Brieher, 2012; Yamada et al, 2004) (Fig. 4 A,D).…”

“…These cells also displayed extended, widened apical myosin arrays with an appearance previously described as “sarcomeric” (Choi et al, 2016; Yu-Kemp et al, 2022). This actomyosin organization is indicative of incomplete maturation of apical actin bundles (Choi et al, 2016; Yu-Kemp et al, 2022). LIMCH1 knockdown cells showed a different pattern, where the apical filamentous actin ring is severely fragmented or completely absent (Fig.…”

“…3 B). These cells also displayed extended, widened apical myosin arrays with an appearance previously described as “sarcomeric” (Choi et al, 2016; Yu-Kemp et al, 2022). This actomyosin organization is indicative of incomplete maturation of apical actin bundles (Choi et al, 2016; Yu-Kemp et al, 2022).…”

Multi-level force-dependent allosteric enhancement of αE-catenin binding to F-actin by vinculin

“…At each step, polarization of F-actin is anticipated to increase the efficiency of force generation by nonmuscle myosin II, leading to a positive feedback loop between myosin contractility, catch bond formation, and F-actin polarization. This feedback loop would result in the ordered sarcomeric assemblies observed in epithelia [13, 15, 58] and cardiomyocytes [57] (Fig. 6C).…”