•Loss of Tctn3 gene results in defects in ciliogenesis and Hedgehog signaling.•Overexpression of Tctn3, but not Tctn1 or Tctn2, can rescue ciliogenesis.•Tctn1 or Tctn2 cannot replace Tctn3 in ciliogenesis and Hedgehog signaling in vivo.•Tctn1 or Tctn2 can replace Tctn3 in neural tube patterning and Gli3 processing.Tctn1, Tctn2, and Tctn3 are membrane proteins that localize at the transition zone of primary cilia. Tctn1 and Tctn2 mutations have been reported in both humans and mice, but Tctn3 mutations have been reported only in humans. It is also not clear whether the three Tctn proteins are functionally conserved with respect to ciliogenesis and Hedgehog (Hh) signaling. In the present study, we report that loss of Tctn3 gene function in mice results in a decrease in ciliogenesis and Hh signaling. Consistent with this, Tctn3 mutant mice exhibit holoprosencephaly and randomized heart looping and lack the floor plate in the neural tube, the phenotypes similar to those of Tctn1 and Tctn2 mutants. We also show that overexpression of Tctn3, but not Tctn1 or Tctn2, can rescue ciliogenesis in Tctn3 mutant cells. Similarly, replacement of Tctn3 with Tctn1 or Tctn2 in the Tctn3 gene locus results in reduced ciliogenesis and Hh signaling, holoprosencephaly, and randomized heart looping. Surprisingly, however, the neural tube patterning and the proteolytic processing of Gli3 (a transcription regulator for Hh signaling) into a repressor, both of which are usually impaired in ciliary gene mutants, are normal. These results suggest that Tctn1, Tctn2, and Tctn3 are functionally divergent with respect to their role in ciliogenesis and Hh signaling but conserved in neural tube patterning and Gli3 processing.

•Hedgehog signaling inhibits PKA-mediated Gli2 and Gli3 phosphorylation in cilia.•Gli2 and Gli3 processing is reduced in Talpid3 (Ta3) mutant mouse.•PKA-mediated Gli2 and Gli3 phosphorylation is reduced in Ta3 mutant mouse.•Ta3 protein interacts and colocalizes with PKA at centrosome.•PKARIIβ is mislocalized in some of Ta3 mutant cells.Hedgehog (Hh) signaling is thought to occur in primary cilia, but the molecular basis of Gli2 and Gli3 activation by Hh signaling in cilia is unknown. Similarly, how ciliary gene mutations result in reduced Gli3 processing that generates a repressor is also not clear. Here we show that Hh signaling inhibits Gli2 and Gli3 phosphorylation by protein kinase A (PKA) in cilia. The cilia related gene Talpid3 (Ta3) mutation results in the reduced processing and phosphorylation of Gli2 and Gli3. Interestingly, Ta3 interacts and colocalizes with PKA regulatory subunit PKARIIβ at centrioles in the cell. The centriolar localization and PKA binding regions are located in the N- and C-terminal regions of Ta3, respectively. PKARIIβ fails to localize at centrioles in some Ta3 mutant cells. Therefore, our study provides the direct evidence that Gli2 and Gli3 are dephosphorylated and activated in cilia and that impaired Gli2 and Gli3 processing in Ta3 mutant is at least in part due to a decrease in Gli2 and Gli3 phosphorylation.

•A 199 residue recombinant spider wrapping silk subunit (W1) is characterized.•Structural integrity is maintained with temperature and pH variation and storage.•Reversible denaturation demonstrates a midpoint of ∼71 °C with minimal aggregation.•W1 self-assembles into ∼20–300 nm sized particles in near-physiological buffer.•Nanoparticle self-assembly occurs above a critical concentration of ∼20 μM.Artificial spider silk proteins may form fibers with exceptional strength and elasticity. Wrapping silk, or aciniform silk, is the toughest of the spider silks, and has a very different protein composition than other spider silks. Here, we present the characterization of an aciniform protein (AcSp1) subunit named W1, consisting of one AcSp1 199 residue repeat unit from Argiope trifasciata. The structural integrity of recombinant W1 is demonstrated in a variety of buffer conditions and time points. Furthermore, we show that W1 has a high thermal stability with reversible denaturation at ∼71 °C and forms self-assembled nanoparticle in near-physiological conditions. W1 therefore represents a highly stable and structurally robust module for protein-based nanoparticle formation.