Centrioles are critical for the formation of centrosomes, cilia and flagella in eukaryotes. They are thought to assemble around a nine-fold symmetric cartwheel structure established by SAS-6 proteins. Here, we have engineered Chlamydomonas reinhardtii SAS-6-based oligomers with symmetries ranging from five-to ten-fold. Expression of a SAS-6 mutant that forms six-fold symmetric cartwheel structures in vitro resulted in cartwheels and centrioles with eight-or nine-fold symmetries in vivo. In combination with Bld10 mutants that weaken cartwheel-microtubule interactions, this SAS-6 mutant produced six-to eight-fold symmetric cartwheels. Concurrently, the microtubule wall maintained eight-and nine-fold symmetries. Expressing SAS-6 with analogous mutations in human cells resulted in nine-fold symmetric centrioles that exhibited impaired length and organization. Together, our data suggest that the self-assembly properties of SAS-6 instruct cartwheel symmetry, and lead us to propose a model in which the cartwheel and the microtubule wall assemble in an interdependent manner to establish the native architecture of centrioles.The formation of centrosomes in animal cells, as well as that of cilia and flagella in eukaryotic cells, critically depends on the evolutionarily conserved microtubule-based cylindrical centriole. In most species, newly formed centrioles are organized around a nine-fold radially symmetric 'cartwheel' structure. This cartwheel comprises a central hub ∼25 nm in diameter from which nine spokes emanate and connect through a pinhead structure to nine microtubule triplets, which are linked together and constitute a peripheral 'microtubule wall' (reviewed in refs 1,2). A generally acknowledged model posits that the cartwheel acts as a molecular scaffold for the formation of the nine-fold symmetric architecture of centrioles (reviewed in refs 1-7). In its strictest sense, this 'scaffold model' of centriole formation postulates that the nine-fold symmetric cartwheel is formed first and then dictates the assembly of a nine-fold symmetric microtubule wall.A key prediction of the scaffold model is that changes in cartwheel symmetry should dictate likewise changes in the architecture of the centriole. A central component of the cartwheel is SAS-6, a protein that is essential for cartwheel formation across eukaryotes (reviewed in refs 1,2). In Chlamydomonas, however, despite the absence of cartwheels, ∼20% of cells bearing a deletion of the sole SAS-6 gene nevertheless assemble centrioles that comprise a circular microtubule wall with a nine-fold symmetry in ∼70% of cases, but with seven-, eight-, ten-or eleven-fold symmetries in the remaining cases 8 . These observations suggest that the cartwheel is critical for proper assembly of the microtubule wall in Chlamydomonas, but also that in this species the centriolar microtubules have an inherent ability to assemble into near-nine-fold symmetrical structures. In contrast, knockdown of SAS-6 proteins in other systems, including human cells 9,10 , results in a failure...

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases (VEGFRs). The extracellular domain of VEGFRs consists of seven Ig-homology domains, of which D2-3 form the ligand-binding site, while the membrane proximal domains D4-7 are involved in homotypic interactions in ligand-bound receptor dimers. Based on low-resolution structures, we identified allosteric sites in D4-5 and D7 of vascular endothelial growth factor receptor 2 (VEGFR-2) accomplishing regulatory functions. Allosteric inhibition of VEGFR-2 signaling represents an attractive option for the treatment of neovascular diseases. We showed earlier that DARPin binders to domains D4 or D7 are potent VEGFR-2 inhibitors. Here we investigated in detail the allosteric inhibition mechanism of the domain D4 binding inhibitor D4b. The 2.38 Å crystal structure of D4b in complex with VEGFR-2 D4-5, the first high-resolution structure of this VEGFR-2 segment, indicates steric hindrance by D4b as the mechanism of inhibition of receptor activation. At the cellular level, D4b triggered quantitative internalization of VEGFR-2 in the absence of ligand and thus clearance of VEGFR-2 from the surface of endothelial cells. The allosteric VEGFR-2 inhibition was sufficiently strong to efficiently inhibit the growth of human endothelial cells at suboptimal dose in a mouse xenograft model in vivo, underlining the therapeutic potential of the approach.

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