Many cells have tiny hair-like structures called cilia on their surface that are important for communicating with other cells and for detecting changes in the cell’s surroundings. Some cilia also beat to move fluids across the cell surface—for example, to move mucus out of the lungs—or act as flagella that undergo rapid whip-like movements to propel cells along. Cilia are formed when a small cylindrical structure in the cell called a centriole docks against the cell membrane and subsequently grows out. However, many of the details of this process are poorly understood.

One of the earliest events in centriole assembly is the formation of a central structure that looks like a cartwheel. This cartwheel acts as a scaffold onto which the rest of the centriole is then added. It has been proposed that a protein called SAS-6 can build this cartwheel just by interacting with itself. However, this has so far not been shown clearly. Now, using a technique called X-ray crystallography, van Breugel et al. directly confirm this hypothesis. This is significant because it demonstrates that the simple self interaction of a protein could lie at the heart of building a complex structure like a centriole.

The single-celled human parasites that spread diseases such as Leishmaniasis, Chagas disease, and sleeping sickness rely on flagella to move around and interact with their surroundings. If SAS-6 cannot assemble into the cartwheel structure, flagella cannot form correctly, potentially stopping the parasites. By screening a library of small molecules, van Breugel et al. found one that partially disrupted the interactions of SAS-6 with itself in the test tube. This small molecule interacted only very weakly with SAS-6 and was not specific for SAS-6 from the disease-causing organism. These unfavourable properties therefore make this compound of no immediate use. However, this result nevertheless shows that small molecules can impair SAS-6 function at least in the test tube and that the development of a more efficient inhibitor might therefore be possible.

Eukaryotic flagella and cilia have essential roles in cell locomotion, fluid movement, and sensing. Their formation strictly requires the presence of centrioles, cylindrical organelles with a radially ninefold symmetric, peripheral microtubule array (Bettencourt-Dias and Glover, 2007; Azimzadeh and Marshall, 2010; Gonczy, 2012). During ciliogenesis/flagellogenesis, centrioles (then referred to as basal bodies) dock to the cell membrane via their distal ends and nucleate the flagellar/ciliar axoneme from their microtubule array (Sherwin and Gull, 1989; Dawe et al., 2007; Ishikawa and Marshall, 2011; Avasthi and Marshall, 2012; Chemes, 2012). The symmetry and diameter of centrioles (and thereby of flagella/cilia) is, to a large extent, established through scaffolding by the centriolar cartwheel, a structure with a central ring-like hub and nine radially projecting spokes that contact the peripheral centriolar microtubules (Azimzadeh and Marshall, 2010; Brito et al., 2012; Gonczy, 2012).

The cartwheel is organized through the homo-oligomerization of the highly conserved centriolar protein SAS-6 (Nakazawa et al., 2007; Kitagawa et al., 2011; van Breugel et al., 2011). High-resolution crystal structures of zebrafish and C. reinhardtii SAS-6 fragments together with biochemical and biophysical characterizations (Kitagawa et al., 2011; van Breugel et al., 2011) demonstrated that two dimerization interfaces in SAS-6 mediate this oligomerization: a coiled-coil domain that forms a rod-like parallel dimer; and a globular N-terminal domain that forms a curved head-to-head dimer. Modeling these two dimer interactions together in silico resulted in ring assemblies that were compatible with the symmetry and dimension of cartwheels observed in vivo. In these models, the N-terminal head domains constitute the cartwheel hubs from which the coiled-coil rods project to form the cartwheel spokes (Kitagawa et al., 2011; van Breugel et al., 2011). However, so far, no high-resolution structure of the SAS-6 cartwheel is available. Although rotary shadowing EM studies suggested that SAS-6 might be able to form cartwheels, the available resolution was insufficient to determine the symmetry of the observed assemblies directly (Kitagawa et al., 2011). Another, higher resolution, EM study with recombinant SAS-6 revealed cartwheel-like assemblies with an eightfold, not a ninefold symmetry (van Breugel et al., 2011), while a third EM study showed the presence of SAS-6 tetramers (Gopalakrishnan et al., 2010). Furthermore, the available biochemical and biophysical data do not provide evidence for efficient cartwheel formation by SAS-6 in solution (Gopalakrishnan et al., 2010; Kitagawa et al., 2011; van Breugel et al., 2011), raising the question of whether SAS-6 alone is sufficient to organize ninefold symmetric cartwheels and, if so, under what conditions.

SAS-6 is a highly conserved protein that is found in all eukaryotes that have cilia/flagella during some of their life-cycle stages (Carvalho-Santos et al., 2010; Hodges et al., 2010). Inhibiting SAS-6 self-assembly by point mutations in vivo abolishes the formation of centrioles (Kitagawa et al., 2011; van Breugel et al., 2011; Lettman et al., 2013) and thereby flagella (van Breugel et al., 2011). Thus, targeting SAS-6 oligomerization by inhibitors could be a strategy to disable flagellogenesis in organisms that cause human disease and rely on flagella for their pathogenicity. The Trypanosomatids are of special interest in this respect. They consist of parasitic, eukaryotic protozoa with a single flagellum and comprise members that cause major human diseases, such as sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and Leishmaniasis (Leishmania spec.) (Simpson et al., 2006). These organisms display complex life cycles in which they shuttle between an insect vector and human or animal hosts. Their flagellum provides key roles in this life cycle through its multiple functions in essential cellular processes such as motility, signalling, sensing, and attachment (Vaughan and Gull, 2003; Ralston et al., 2009; Gluenz et al., 2010). Furthermore, the flagellar pocket, a membrane invagination from which the flagellum emerges, is the exclusive site of vesicular membrane traffic in these organisms (Overath et al., 1997; Landfear and Ignatushchenko, 2001; Field and Carrington, 2004, 2009; Overath and Engstler, 2004). It is therefore not surprising that flagella have been demonstrated or are proposed to be key factors in the pathogenicity of these organisms (Vaughan and Gull, 2003; Ralston et al., 2009; Gluenz et al., 2010).

As in other eukaryotes, the flagellum of the Trypanosomatids is templated from centrioles (basal bodies). Tomographic EM studies demonstrated the presence of canonical, ninefold symmetric cartwheel structures in the centre of their centrioles (Lacomble et al., 2009). Currently, no high-resolution structures of SAS-6 homologues from Trypanosomatids are available, and it is unclear whether oligomerization of their SAS-6 homologues could be inhibited.

SAS-6 organizes the rotationally ninefold symmetric cartwheel (Nakazawa et al., 2007; Kitagawa et al., 2011; van Breugel et al., 2011), an early assembly intermediate of centrioles that participates in establishing their symmetry and diameter (Nakazawa et al., 2007; Brito et al., 2012; Gonczy, 2012). Based on high-resolution structures of SAS-6 fragments and in silico modeling, detailed models of how SAS-6 self-associates to organize these cartwheels have been proposed (Nakazawa et al., 2007; Kitagawa et al., 2011; van Breugel et al., 2011). However, these models have so far not been confirmed; EM studies with recombinant SAS-6 constructs were either of too low resolution (Kitagawa et al., 2011) or showed assemblies that were not ninefold symmetric (Gopalakrishnan et al., 2010; van Breugel et al., 2011). Furthermore, several studies found no evidence for complete ring-formation by recombinant SAS-6 in solution (Gopalakrishnan et al., 2010; Kitagawa et al., 2011; van Breugel et al., 2011). Thus, it has been unclear if and under what conditions SAS-6 is sufficient to assemble ninefold symmetric cartwheels and thereby assist in dictating centriole symmetry.

We were unable to detect cartwheel formation by recombinant L. major SAS-6 in solution, confirming these previous findings. However, we demonstrate that under high protein and precipitant concentrations, a L. major SAS-6 construct crystallized as ninefold symmetric rings with a diameter very similar to cartwheel hubs in vivo; this provides the first unambiguous experimental evidence that SAS-6 is able to form ninefold symmetric cartwheels on its own. Our results suggest that SAS-6 needs to be highly concentrated locally in order to be able to form cartwheels. Intriguingly, in vivo, SAS-6 is indeed highly enriched at the site of centriole formation (Kleylein-Sohn et al., 2007; Strnad et al., 2007; Dammermann et al., 2008; Blachon et al., 2009; Sonnen et al., 2012; Lettman et al., 2013). However, in vivo, SAS-6 assembly occurs in the context of other essential centriole duplication factors, some of which can be found in a complex with SAS-6 (Stevens et al., 2010; Tang et al., 2011; Lin et al., 2013). It is therefore likely that additional centriole proteins assist SAS-6 assembly, especially in light of the fact that several studies (Gopalakrishnan et al., 2010; Kitagawa et al., 2011; van Breugel et al., 2011), and our study presented here, have been unable to demonstrate efficient cartwheel assembly by SAS-6 in solution.

Why does SAS-6 not form cartwheels efficiently? The interaction interfaces that are critical for ring formation (i.e., the head-to-head dimer interface and the head-domain–coiled-coil interface) are both relatively small (Figures 1, 2; Kitagawa et al., 2011; van Breugel et al., 2011). In solution, SAS-6 oligomers are therefore likely to show some ‘wobble’ that would make ring closure inefficient and assembled rings unstable. However, in our ring structure, both of these critical interfaces are similar in their orientations to those observed in the crystals of the Lm SAS-6^97–320 F257E and the Lm SAS-6^97–274 constructs that did not crystallize as rings (Figure 2—figure supplement 2). Thus, although clearly not occurring efficiently in solution, our data suggest that ninefold symmetric ring formation might correspond to a weakly favored SAS-6 conformation that needs to be stabilized. In our crystal structure, rings are stabilized by crystal packing interactions and, in vivo, probably by SAS-6 interacting proteins. This model could explain why SAS-6 is required for the faithful establishment of centriole symmetry, while not being the sole determinant of their ninefold symmetry in vivo (Nakazawa et al., 2007).

In basal bodies, cartwheels are stacked onto each other with a vertical distance between cartwheel hubs of ∼8 nm (Guichard et al., 2012). Although we observed a similar stacking in our cartwheel hub structure (Figure 2—figure supplement 1), the corresponding distance was only ∼4 nm. Furthermore, although a comparable packing was previously observed in a crystal of the N-terminal head domain of zebrafish SAS-6 (van Breugel et al., 2011), the underlying packing interactions are based on a non-conserved interface and are therefore unlikely to be of relevance in vivo. Recent EM tomograms of basal bodies from Trichonympha (Guichard et al., 2012, 2013) rather suggest that ring stacking in vivo is based on parallel interactions between the SAS-6 coiled-coil stalks and vertical interactions of SAS-6 associated components at the periphery of centriolar cartwheels.

Interestingly, we discovered a dimerization-impaired state of Leishmania major SAS-6 that could provide a potential regulatory mechanism for its assembly. In this state, residue Y215 blocks the hydrophobic pocket into which F257 from its homo-dimer partner binds. Mutating Y215 to lysine resulted in an apparent threefold increase in dimerization affinity as measured by analytical ultracentrifugation. If open and closed Y215 conformations are in equilibrium with each other, the dimerization affinity of the closed Y215 state in solution would be even lower than the measured value, since, in this case, ultracentrifugation analyses the association of a mixture of open and closed states. We speculate that a release of this closed SAS-6 state through either a regulatory protein or a direct phosphorylation of Y215 could provide a simple mechanism to trigger SAS-6 assembly locally by increasing the concentration of assembly efficient SAS-6. Should this mechanism indeed be used in vivo (which is currently unknown), it would probably be confined to the Trypanosomatids as other SAS-6 homologues appear not to have tyrosine/aromatic residues in the position equivalent to Y215 that could play an equivalent role.

Finally, as a proof-of-principle we show that oligomerization of SAS-6 can be inhibited by the small molecule PK9119 in vitro. The evolutionary conservation of the hydrophobic pocket involved in dimerization together with PK9119’s relatively broad activity in inhibiting both L. major and (to a lesser extent) D. rerio SAS-6 could suggest that PK9119 targets this pocket. However, our NMR binding studies are most consistent with PK9119 binding adjacent to the head-to-head dimerization interface and thereby altering its structure subtly (Figure 3B). Confirming this notion, when we modeled the SAS-6–PK9119 complex with the HADDOCK software package (available at http://haddock.science.uu.nl/services/HADDOCK/haddock.php) (de Vries et al., 2010; Wassenaar et al., 2012), using high ambiguity restraints derived from the observed chemical shift perturbations in HSQC spectra (6 restraints >0.2 ppm), we found that of the 200 docked structures 98% occupied a single cluster in which PK9119 was bound to helix α1, but not on the side lining the hydrophobic pocket, but on its opposite side, facing away from this pocket (data not shown). Clearly, an elucidation of a high-resolution structure of PK9119 bound to its target would be important to understand how exactly PK9119 functions in inhibiting L. major SAS-6. The binding affinity of our compound is currently low (in the mM range) and it does not show strong species specificity. To establish whether PK9119 has any potential to be improved in these critical aspects, it will be essential to systematically explore chemical modifications of its central indole scaffold. Regardless of these limitations, our demonstration that SAS-6 can be inhibited in vitro is a first, small step towards the goal of developing SAS-6 inhibitors that also could be used in vivo, for example as a cell-biological tool.

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Author details

1. Mark van Breugel

   Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   MB, Crystallized the constructs and solved their X-ray structures, Contributed to writing the manuscript

   For correspondence

   vanbreug@mrc-lmb.cam.ac.uk

   Competing interests

   The authors declare that no competing interests exist.

2. Rainer Wilcken

   Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

   Present address

   Novartis Institutes for BioMedical Research, Basel, Switzerland

   Contribution

   RW, Performed the NMR experiments, Contributed to writing the manuscript

   Competing interests

   The authors declare that no competing interests exist.

3. Stephen H McLaughlin

   Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   SHM, Carried out the analytical ultracentrifugation, Contributed to writing the manuscript

   Competing interests

   The authors declare that no competing interests exist.

4. Trevor J Rutherford

   Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   TJR, Performed the NMR experiments, Contributed to writing the manuscript

   Competing interests

   The authors declare that no competing interests exist.

5. Christopher M Johnson

   Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   CMJ, Performed the SEC-MALS experiments, Contributed to writing the manuscript

   Competing interests

   The authors declare that no competing interests exist.

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