Intraflagellar transport (IFT) of ciliary precursors such as tubulin from the cytoplasm to the ciliary tip is involved in the construction of the cilium, a hairlike organelle found on most eukaryotic cells. However, the molecular mechanisms of IFT are poorly understood. Here, we found that the two core IFT proteins IFT74 and IFT81 form a tubulin-binding module and mapped the interaction to a calponin homology domain of IFT81 and a highly basic domain in IFT74. Knockdown of IFT81 and rescue experiments with point mutants showed that tubulin binding by IFT81 was required for ciliogenesis in human cells.Cilia are microtubule-based organelles that function in motility, sensory reception, and signaling (1). Ciliary dysfunction results in numerous diseases and disorders commonly known as ciliopathies. Intraflagellar transport (IFT) is involved in cilium formation (2, 3) but also functions in other cellular processes, such as the recycling of Tcell receptors at the immune synapse (4). IFT relies on kinesin-2 and IFT-dynein molecular motors moving along the microtubule-based axoneme of cilia (5-7) and on the IFT complex, which contains at least 20 different protein subunits. Although ~600 proteins are known to reside in the cilium (8), we know very little about how they are recognized as ciliary cargo by the IFT machinery (9-11).To identify potential cargo-binding sites on the IFT complex, we carried out bioinformatical and biochemical screening and identified conserved domains that were not required for IFT complex formation. We reasoned that such domains could protrude from the IFT particlecore structure and would thus be in a prime position for cargo recognition. The two IFT core (12). Given that the cilium consists of a MT-based axoneme, IFT of large quantities of tubulin is required for cilium formation (13). We thus tested the tubulin-binding properties of HsIFT81N using affinity pull-downs ( Fig. 1D and fig. S4E) and microscale thermophoresis (MST) with unpolymerized bovine αβ-tubulin (Fig. 1, E S5F). Thus, the tubulin-binding module is formed by the IFT74/81 complex rather than by IFT81N alone.To dissect the binding mode in the IFT74/81:αβ-tubulin complex, samples were prepared from MT and unpolymerized αβ-tubulin lacking the highly acidic C-terminal tails, often referred to as E-hooks (12) ( fig. S5A). αβ-tubulin lacking E-hooks had similar affinity for IFT81N as intact tubulin ( fig. S5, B and C), which suggested that IFT81N recognizes the globular domain of αβ-tubulin with no substantial interaction with the E-hooks. IFT74/81 displayed robust MT binding in sedimentation assays, which was, however, reduced to background levels in the absence of the β-tubulin E-hook ( fig. S5E). Thus, IFT81N appears to bind the globular domain of tubulin to provide specificity, and IFT74N recognizes the β-tubulin tail to increase affinity (Fig. 1H).To examine the role of tubulin binding by IFT74/81 in a cellular system, we transiently expressed Flag-HsIFT81 or Flag-HsIFT81ΔN in human RPE-1 cells and induced formation ...

Primary cilia play critical roles in development and disease. Their assembly is triggered by mature centrioles (basal bodies) and requires centrosomal protein 164kDa (Cep164), a component of distal appendages. Here we show that loss of Cep164 leads to early defects in ciliogenesis, reminiscent of the phenotypic consequences of mutations in TTBK2 (Tau tubulin kinase 2). We identify Cep164 as a likely physiological substrate of TTBK2 and demonstrate that Cep164 and TTBK2 form a complex. We map the interaction domains and demonstrate that complex formation is crucial for the recruitment of TTBK2 to basal bodies. Remarkably, ciliogenesis can be restored in Cep164-depleted cells by expression of chimeric proteins in which TTBK2 is fused to the C-terminal centrioletargeting domain of Cep164. These findings indicate that one of the major functions of Cep164 in ciliogenesis is to recruit active TTBK2 to centrioles. Once positioned, TTBK2 then triggers key events required for ciliogenesis, including removal of CP110 and recruitment of intraflagellar transport proteins. In addition, our data suggest that TTBK2 also acts upstream of Cep164, contributing to the assembly of distal appendages.primary cilium | centrosome

Wnts are a family of secreted proteins that regulate multiple steps of neural development and stem cell differentiation.

Wnt5a is a morphogen that activates the Wnt/planar cell polarity (PCP) pathway and serves multiple functions during development. PCP signaling controls the orientation of cells within an epithelial plane as well as convergent extension (CE) movements. Wnt5a was previously reported to promote differentiation of A9–10 dopaminergic (DA) precursors in vitro. However, the signaling mechanism in DA cells and the function of Wnt5a during midbrain development in vivo remains unclear. We hereby report that Wnt5a activated the GTPase Rac1 in DA cells and that Rac1 inhibitors blocked the Wnt5a-induced DA neuron differentiation of ventral midbrain (VM) precursor cultures, linking Wnt5a-induced differentiation with a known effector of Wnt/PCP signaling. In vivo, Wnt5a was expressed throughout the VM at embryonic day (E)9.5, and was restricted to the VM floor and basal plate by E11.5–E13.5. Analysis of Wnt5a−/− mice revealed a transient increase in progenitor proliferation at E11.5, and a precociously induced NR4A2+ (Nurr1) precursor pool at E12.5. The excess NR4A2+ precursors remained undifferentiated until E14.5, when a transient 25% increase in DA neurons was detected. Wnt5a−/− mice also displayed a defect in (mid)brain morphogenesis, including an impairment in midbrain elongation and a rounded ventricular cavity. Interestingly, these alterations affected mostly cells in the DA lineage. The ventral Sonic hedgehog-expressing domain was broadened and flattened, a typical CE phenotype, and the domains occupied by Ngn2+ DA progenitors, NR4A2+ DA precursors and TH+ DA neurons were rostrocaudally reduced and laterally expanded. In summary, we hereby describe a Wnt5a regulation of Wnt/PCP signaling in the DA lineage and provide evidence for multiple functions of Wnt5a in the VM in vivo, including the regulation of VM morphogenesis, DA progenitor cell division, and differentiation of NR4A2+ DA precursors.

Lrp5/6 are crucial coreceptors for Wnt/beta-catenin signaling, a pathway biochemically distinct from noncanonical Wnt signaling pathways. Here, we examined the possible participation of Lrp5/6 in noncanonical Wnt signaling. We found that Lrp6 physically interacts with Wnt5a, but that this does not lead to phosphorylation of Lrp6 or activation of the Wnt/beta-catenin pathway. Overexpression of Lrp6 blocks activation of the Wnt5a downstream target Rac1, and this effect is dependent on intact Lrp6 extracellular domains. These results suggested that the extracellular domain of Lrp6 inhibits noncanonical Wnt signaling in vitro. In vivo, Lrp6-/- mice exhibited exencephaly and a heart phenotype. Surprisingly, these defects were rescued by deletion of Wnt5a, indicating that the phenotypes resulted from noncanonical Wnt gain-of-function. Similarly, Lrp5 and Lrp6 antisense morpholino-treated Xenopus embryos exhibited convergent extension and heart phenotypes that were rescued by knockdown of noncanonical XWnt5a and XWnt11. Thus, we provide evidence that the extracellular domains of Lrp5/6 behave as physiologically relevant inhibitors of noncanonical Wnt signaling during Xenopus and mouse development in vivo.