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Immotile cilia of crown cells at the node of mouse embryos are required for sensing of a leftward fluid flow1 that gives rise to the breaking of left-right (L-R) symmetry2. The flow-sensing mechanism has long remained elusive, however, with both mechanosensing and chemosensing models having been proposed1, 3–5. Here we show that immotile cilia at the mouse node respond to mechanical force. In the presence of a leftward flow, immotile cilia on the left side of the node bend toward the ventral side whereas those on the right side bend toward the dorsal side. Application of mechanical stimuli to immotile cilia along the dorsoventral axis by optical tweezers induced Ca2+ transients and degradation of Dand5 mRNA—the first known L-R asymmetric molecular events—in the targeted cells. The Pkd2 channel protein was found to be preferentially localized to the dorsal side of immotile cilia on both left and right sides of the node, and the observed induction of Ca2+ transients preferentially by mechanical stimuli directed toward the ventral side could explain the differential response of immotile cilia to the directional flow. Our results thus suggest that immotile cilia at the node sense the direction of fluid flow in a manner dependent on a flow-generated mechanical force.

Collective migration of epithelial cells plays crucial roles in various biological processes such as cancer invasion. In migrating epithelial sheets, leader cells form lamellipodia to advance, and follower cells also form similar motile apparatus at cell–cell boundaries, which are called cryptic lamellipodia (c-lamellipodia). Using adenocarcinoma-derived epithelial cells, we investigated how c-lamellipodia form and found that they sporadically grew from around E-cadherin–based adherens junctions (AJs). WAVE and Arp2/3 complexes were localized along the AJs, and silencing them not only interfered with c-lamellipodia formation but also prevented follower cells from trailing the leaders. Disruption of AJs by removing αE-catenin resulted in uncontrolled c-lamellipodia growth, and this was brought about by myosin II activation and the resultant contraction of AJ-associated actomyosin cables. Additional observations indicated that c-lamellipodia tended to grow at mechanically weak sites of the junction. We conclude that AJs not only tie cells together but also support c-lamellipodia formation by recruiting actin regulators, enabling epithelial cells to undergo ordered collective migration.

Collective migration of epithelial cells plays crucial roles in various biological processes such as cancer invasion. In migrating epithelial sheets, leader cells form lamellipodia to advance, and follower cells also form similar motile apparatus at cell-cell boundaries, which are called cryptic lamellipodia (c-lamellipodia). Using adenocarcinoma-derived epithelial cells, we investigated how c-lamellipodia are generated, and found that they sporadically grew from Ecadherin-based adherens junctions (AJs). WAVE and Arp2/3 complexes were localized along the AJs, and silencing them not only interfered with c-lamellipodia formation but also prevented follower cells from trailing the leaders. Disruption of AJs by removing αE-catenin resulted in uncontrolled c-lamellipodia growth, and this was brought about by myosin II activation and the resultant contraction of AJ-associated actomyosin cables. Additional observations indicated that c-lamellipodia tended to grow at mechanically weak sites of the junction. We conclude that AJs not only tie cells together but also generate c-lamellipodia by recruiting actin regulators, enabling epithelial cells to undergo ordered collective migration.

Nishimura et al. show that DAAM1, a formin family actin polymerization regulator, stabilizes epithelial cell junctions by counteracting the WAVE complex, another actin regulator. Loss of DAAM1 promotes the motility of junctional membranes and thereby enhances their invasion of neighboring environments., Epithelial junctions comprise two subdomains, the apical junctional complex (AJC) and the adjacent lateral membrane contacts (LCs), that span the majority of the junction. The AJC is lined with circumferential actin cables, whereas the LCs are associated with less-organized actin filaments whose roles are elusive. We found that DAAM1, a formin family actin regulator, accumulated at the LCs, and its depletion caused dispersion of actin filaments at these sites while hardly affecting circumferential actin cables. DAAM1 loss enhanced the motility of LC-forming membranes, leading to their invasion of neighboring cell layers, as well as disruption of polarized epithelial layers. We found that components of the WAVE complex and its downstream targets were required for the elevation of LC motility caused by DAAM1 loss. These findings suggest that the LC membranes are motile by nature because of the WAVE complex, but DAAM1-mediated actin regulation normally restrains this motility, thereby stabilizing epithelial architecture, and that DAAM1 loss evokes invasive abilities of epithelial cells.

Significance Each neuron forms a single axon and multiple dendrites, and this configuration is important for wiring the brain. How only a single axon extends from a neuron, however, remains unknown. This study demonstrates that CAMSAP3, a protein that binds the minus-end of microtubules, preferentially localizes along axons in hippocampal neurons. Remarkably, mutations of CAMSAP3 lead to production of multiple axons in these neurons. In attempts to uncover mechanisms underlying this abnormal axon extension, the authors found that CAMSAP3-anchored microtubules escape from acetylation, a process mediated by α-tubulin acetyltransferase-1, and depletion of this enzyme abolishes abnormal axon formation in CAMSAP3 mutants. These findings reveal that CAMSAP3 controls microtubule dynamics, preventing tubulin acetylation; this mechanism is required for single-axon formation., The molecular mechanisms that guide each neuron to become polarized, forming a single axon and multiple dendrites, remain unknown. Here we show that CAMSAP3 (calmodulin-regulated spectrin-associated protein 3), a protein that regulates the minus-end dynamics of microtubules, plays a key role in maintaining neuronal polarity. In mouse hippocampal neurons, CAMSAP3 was enriched in axons. Although axonal microtubules were generally acetylated, CAMSAP3 was preferentially localized along a less-acetylated fraction of the microtubules. CAMSAP3-mutated neurons often exhibited supernumerary axons, along with an increased number of neurites having nocodazole-resistant/acetylated microtubules compared with wild-type neurons. Analysis using cell lines showed that CAMSAP3 depletion promoted tubulin acetylation, and conversely, mild overexpression of CAMSAP3 inhibited it, suggesting that CAMSAP3 works to retain nonacetylated microtubules. In contrast, CAMSAP2, a protein related to CAMSAP3, was detected along all neurites, and its loss did not affect neuronal polarity, nor did it cause increased tubulin acetylation. Depletion of α-tubulin acetyltransferase-1 (αTAT1), the key enzyme for tubulin acetylation, abolished CAMSAP3 loss-dependent multiple-axon formation. These observations suggest that CAMSAP3 sustains a nonacetylated pool of microtubules in axons, interfering with the action of αTAT1, and this process is important to maintain neuronal polarity.

Summary Cell migration plays a pivotal role in morphogenetic and pathogenetic processes. To achieve directional migration, cells must establish a front-to-rear axis of polarity. Here we show that components of the cadherin-catenin complex function to stabilize this front-rear polarity. Neural crest and glioblastoma cells undergo directional migration in vivo or in vitro . During this process, αE-catenin accumulated at lamellipodial membranes and then moved toward the rear with the support of a tyrosine-phosphorylated β-catenin. This relocating αE-catenin bound to p115RhoGEF, leading to gathering of active RhoA in front of the nucleus where myosin-IIB arcs assemble. When catenins or p115RhoGEF were removed, cells lost the polarized myosin-IIB assembly, as well as the capability for directional movement. These results suggest that, apart from its well-known function in cell adhesion, the β-catenin/αE-catenin complex regulates directional cell migration by restricting active RhoA to perinuclear regions and controlling myosin-IIB dynamics at these sites.