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Molecular left-right (L-R) asymmetry is established at the node of the mouse embryo as a result of the sensing of a leftward fluid flow by immotile cilia of perinodal crown cells and the consequent degradation of Dand5 mRNA on the left side. We here examined how the fluid flow induces Dand5 mRNA decay. We found that the first 200 nucleotides in the 3′ untranslated region (3′-UTR) of Dand5 mRNA are necessary and sufficient for the left-sided decay and to mediate the response of a 3′-UTR reporter transgene to Ca2+, the cation channel Pkd2, the RNA-binding protein Bicc1 and their regulation by the flow direction. We show that Bicc1 preferentially recognizes GACR and YGAC sequences, which can explain the specific binding to a conserved GACGUGAC motif located in the proximal Dand5 3′-UTR. The Cnot3 component of the Ccr4-Not deadenylase complex interacts with Bicc1 and is also required for Dand5 mRNA decay at the node. These results suggest that Ca2+ currents induced by leftward fluid flow stimulate Bicc1 and Ccr4-Not to mediate Dand5 mRNA degradation specifically on the left side of the node., Questioning what regulates left-right asymmetry breaking in the mouse node: the authors identify a 200 bp stretch of the Dand5 3’UTR where Bicc1 binds, and Cnot proteins downstream of calcium flow regulate the post-transcriptional regulation of Dand5 by Bicc1.

Immotile cilia sense extracellular signals such as fluid flow, but whether Ca2+ plays a role in flow sensing has been unclear. Here, we examined the role of ciliary Ca2+ in the flow sensing that initiates the breaking of left-right (L-R) symmetry in the mouse embryo. Intraciliary and cytoplasmic Ca2+ transients were detected in the crown cells at the node. These Ca2+ transients showed L-R asymmetry, which was lost in the absence of fluid flow or the PKD2 channel. Further characterization allowed classification of the Ca2+ transients into two types: cilium-derived, L-R-asymmetric transients (type 1) and cilium-independent transients without an L-R bias (type 2). Type 1 intraciliary transients occurred preferentially at the left posterior region of the node, where L-R symmetry breaking takes place. Suppression of intraciliary Ca2+ transients delayed L-R symmetry breaking. Our results implicate cilium-derived Ca2+ transients in crown cells in initiation of L-R symmetry breaking in the mouse embryo.

To study the properties of tracheal cilia beating under various conditions, we developed a method to monitor the movement of the ciliary tip. One end of a demembranated cilium was immobilized on the glass surface, while the other end was capped with a polystyrene bead and tracked in three dimensions. The cilium, when activated by ATP, stably repeated asymmetric beating as in vivo. The tip of a cilium in effective and recovery strokes moved in discrete trajectories that differed in height. The trajectory remained asymmetric in highly viscous solutions. Model calculation showed that cilia maintained a constant net flux during one beat cycle irrespective of the medium viscosity. When the bead attached to the end was trapped with optical tweezers, it came to display linear oscillation only in the longitudinal direction. Such a beating-mode transition may be an inherent nature of movement-restricted cilia.

Cluap1/IFT38 is a ciliary protein that belongs to the IFT-B complex and is required for ciliogenesis. In this study, we have examined the behaviors of Cluap1 protein in nonciliated and ciliated cells. In proliferating cells, Cluap1 is located at the distal appendage of the mother centriole. When cells are induced to form cilia, Cluap1 is found in a novel noncentriolar compartment, the cytoplasmic IFT spot, which mainly exists once in a cell. Other IFT-B proteins such as IFT46 and IFT88 are colocalized in this spot. The cytoplasmic IFT spot is present in mouse embryonic fibroblasts (MEFs) but is absent in ciliogenesis-defective MEFs lacking Cluap1, Kif3a or Odf2. The cytoplasmic IFT spot is also found in mouse embryos but is absent in the Cluap1 mutant embryo. When MEFs are induced to form cilia, the cytoplasmic IFT spot appears at an early step of ciliogenesis but starts to disappear when ciliogenesis is mostly completed. These results suggest that IFT-B proteins such as Cluap1 accumulate in a previously undescribed cytoplasmic compartment during ciliogenesis.

Highly coordinated dyneins generate asymmetric ciliary beating composed of clearly distinguished effective and recovery strokes. We herein aim to understand the mechanism of asymmetric beating of a single cilium by combining multiple optical-microscope techniques: 3-D tracking; optical tweezers; DIC adjusted to the different focal planes. A fluorescent bead was attached to the tip of the surface-immobilized mouse-tracheal cilium that was reactivated with a 100 µM ATP-containing solution, and trapped at various points of the beating stroke to measure the 3-D force (F). Next, after the solution was replaced with 100 µM ATP and 50 µM Vanadate, we measured 2-D structural bending energy in a relaxed state at the same cilium. With the estimation of the 3-D force driven by dyneins (FD) by subtraction of the effect of bending energy from F, the following were obtained: 1) FD periodically changed in the range from −75 to 35 pN, which is about 10 times larger than the force of viscous drag. 2) As the trapping point was displaced from the position of post-effective stroke, maximum values of FD (FDM) of the effective stroke increased from 3 to 35 pN and FDM of the recovery stroke increased from −1 to −75 pN, suggesting that the change in the force depends on the bending deformation of the cilium. 3) Under the trapping condition, in spite of the small curvature change, the direction of FD was spontaneously switched from the recovery direction to the effective direction, and vice versa. This result supports that FD in the effective and the recovery stroke direction, were generated by the different dynein-bridge sets on each side of the axoneme.