Summary As a cellular organelle, the cilium contains a unique protein composition [1, 2]. Entry of both membrane [3–5] and cytosolic components [6–8] is tightly regulated by gating mechanisms at the cilium base, however, the mechanistic details of ciliary gating are largely unknown. We previously proposed that entry of cytosolic components is regulated by mechanisms similar to those of nuclear transport and is dependent on nucleoporins (NUPs) which comprise a ciliary pore complex (CPC) [6, 9]. To investigate ciliary gating mechanisms, we developed a system to clog the pore by inhibiting NUP function via forced dimerization. We targeted NUP62, a component of the central channel of the nuclear pore complex (NPC) [10], for forced dimerization by tagging it with the homodimerizing Fv domain. As proof of principle, we show that forced dimerization of NUP62-Fv attenuated active transport of bovine serum albumin into the nuclear compartment and of the kinesin-2 motor KIF17 into the ciliary compartment. Using the pore clogging technique, we find that forced dimerization of NUP62 attenuated the gated entry of cytosolic proteins but did not affect entry of membrane proteins or diffusional entry of small cytosolic proteins. We propose a model in which active transport of cytosolic proteins into both nuclear and ciliary compartments requires functional NUPs of the central pore whereas lateral entry of membrane proteins utilizes a different mechanism that is likely specific to each organelle’s limiting membrane.

Centriole duplication occurs once per cell cycle to ensure robust formation of bipolar spindles and chromosome segregation. Each newly-formed daughter centriole remains connected to its mother centriole until late mitosis. The disengagement of the centriole pair is required for centriole duplication. However, the mechanisms underlying centriole engagement remain poorly understood. Here, we show that Cep57 is required for pericentriolar material (PCM) organization that regulates centriole engagement. Depletion of Cep57 causes PCM disorganization and precocious centriole disengagement during mitosis. The disengaged daughter centrioles acquire ectopic microtubule-organizing-center activity, which results in chromosome mis-segregation. Similar defects are observed in mosaic variegated aneuploidy syndrome patient cells with cep57 mutations. We also find that Cep57 binds to the well-conserved PACT domain of pericentrin. Microcephaly osteodysplastic primordial dwarfism disease pericentrin mutations impair the Cep57-pericentrin interaction and lead to PCM disorganization. Together, our work demonstrates that Cep57 provides a critical interface between the centriole core and PCM.

In the node of mouse embryo, rotational movements of cilia generate an external liquid flow known as nodal flow, which determines left-right asymmetric gene expression. How nodal flow is converted into asymmetric gene expression is still controversial, but the increase of Ca(2+) levels in endodermal cells to the left of the node has been proposed to play a role. However, Ca(2+) signals inside the node itself have not yet been described. By our optimized Ca(2+) imaging method, we were able to observe dynamic Ca(2+) signals in the node in live mouse embryos. Pharmacological disruption of Ca(2+) signals did not affect ciliary movements or nodal flow, but did alter the expression patterns of the Nodal and Cerl-2 genes. Quantitative analyses of Ca(2+) signal frequencies and distributions showed that during left-right axis establishment, formerly symmetric Ca(2+) signals became biased to the left side. In iv/iv mutant embryos that showed randomized laterality due to ciliary immotility, Ca(2+) signals were found to be variously left-sided, right-sided, or bilateral, and thus symmetric on average. In Pkd2 mutant embryos, which lacked polycystin-2, a Ca(2+)-permeable cation channel necessary for left-right axis formation, the Ca(2+) signal frequency was lower than in wild-type embryos. Our data support a model in which dynamic Ca(2+) signals in the node are involved in left-right patterning.

SUMMARY The motility and signaling functions of the primary cilium require a unique protein and lipid composition that is determined by gating mechanisms localized at the base of the cilium. Several protein complexes localize to the gating zone and may regulate ciliary protein composition, however, the mechanisms of ciliary gating and the dynamics of the gating components are largely unknown. Here we used the BiFC (bimolecular fluorescence complementation) assay and report for the first time on the protein-protein interactions that occur between ciliary gating components and transiting cargoes during ciliary entry. We find that the nucleoporin Nup62 and the C-termini of the nephronophthisis (NPHP) proteins NPHP4 and NPHP5 interact with the axoneme-associated kinesin-2 motor KIF17 and thus spatially map to the inner region of the ciliary gating zone. Nup62 and NPHP4 exhibit rapid turnover at the transition zone and thus define dynamic components of the gate. We find that B9D1, AHI1 and the N-termini of NPHP4 and NPHP5 interact with the transmembrane protein SSTR3 and thus spatially map to the outer region of the ciliary gating zone. B9D1, AHI1 and NPHP5 exhibit little to no turnover at the transition zone and thus define components of a stable gating structure. These data provide the first comprehensive map of the molecular orientations of gating zone components along the inner-to-outer axis of the ciliary gating zone. These results advance our understanding of the functional roles of gating zone components in regulating ciliary protein composition.