S S symmetry

Review Coupling Form and Function: How the Oligomerisation Symmetry of the SAS-6 Contributes to the Architecture of Organelles

Jodie E. Ford, Phillip J. Stansfeld and Ioannis Vakonakis *

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK; [email protected] (J.E.F.); [email protected] (P.J.S.) * Correspondence: [email protected]; Tel.: +44-1865-275725

Academic Editor: John H. Graham Received: 9 April 2017; Accepted: 12 May 2017; Published: 16 May 2017

Abstract: make up the and basal bodies in animals and as such play important roles in cell division, signalling and motility. They possess characteristic 9-fold radial symmetry strongly influenced by the protein SAS-6. SAS-6 is essential for canonical centriole assembly as it forms the central core of the organelle, which is then surrounded by microtubules. SAS-6 self-assembles into an oligomer with elongated spokes that emanate towards the outer microtubule wall; in this manner, the symmetry of the SAS-6 oligomer influences centriole architecture and symmetry. Here, we summarise the form and symmetry of SAS-6 oligomers inferred from crystal structures and directly observed in vitro. We discuss how the strict 9-fold symmetry of centrioles may emerge, and how different forms of SAS-6 oligomers may be accommodated in the organelle architecture.

Keywords: centriole; oligomer symmetry; SAS-6; spiral; cartwheel

1. Introduction Centrioles are conserved eukaryotic organelles essential for correct cellular behaviour. In animals, a pair of centrioles together with the comprise the centrosome, which is the main microtubule organising centre of cells and directs the formation of the mitotic spindle during division [1–3], thereby ensuring correct segregation. Centrioles are also essential for organising cilia and flagella at the surface of cells. As such, they play a crucial role in cellular motility, for example of sperm, cell signalling via the primary cilium, and in the interaction of cells with their surroundings, as seen in tracheal lining cells that sweep mucus and dirt out of the respiratory system. Thus, aberrations in centriole structure or numbers are linked to disease conditions such as ciliopathies, male sterility, ectopic pregnancies, primary and possibly cancer [4–10]. Centrioles are cylinders characterised by 9-fold radial symmetry in the vast majority of organisms [11–13] that are approximately 100–250 nm in diameter and 150–400 nm in length, depending on species and cell type. In centrioles, single, double or triple microtubule ‘blades’ form the outer wall of the cylinder and they are arranged around a central scaffold, termed the ‘cartwheel’ [14]. The cartwheel typically comprises a stack of 9-fold symmetric rings consisting of a ‘central hub’ with ‘spokes’ emanating radially. These spokes interact with microtubules at the outer centriole wall via a structure known as the ‘pinhead’, thereby linking the cartwheel radial symmetry to that of the entire organelle. Thus, cartwheels provide an essential building block for centriole assembly that influences organelle symmetry.

Symmetry 2017, 9, 74; doi:10.3390/sym9050074 www.mdpi.com/journal/symmetry Symmetry 2017, 9, 74 2 of 10

SymmetryGenetic2017 and, 9, 74 functional studies in Caenorhabditis elegans aiming to isolate 2 ofspindle 10 assembly-defective mutants originally identified five , SAS-6, SAS-4, SAS-5, SPD-2 and ZYG-1, essential for centriole formation [15–22]; relatives of these proteins were quickly located Genetic and functional studies in Caenorhabditis elegans aiming to isolate spindle across eukaryotic evolution [23,24]. SAS-6 was perceived as a key protein for centriole symmetry, as assembly-defective mutants originally identified five proteins, SAS-6, SAS-4, SAS-5, SPD-2 and immunolocalisationZYG-1, essential forelec centrioletron microscopy formation [15and–22 ];su relativesb-diffraction of these fluorescence proteins were microscopy quickly located studies placedacross this eukaryotic protein evolutionin cartwheels [23,24 ].[25–27]. SAS-6 was X-ray perceived crystallography as a key protein studies for centriole showed symmetry, that SAS-6 comprisesas immunolocalisation a globular N-terminal electron domain microscopy followed and sub-diffractionby a long coiled fluorescence coil (Figure microscopy 1) and a studiesC-terminal disorderedplaced thisregion. protein SAS-6 in cartwheels strongly [25 dimerises–27]. X-ray in crystallography vitro and in studiesvivo via showed its coiled-coil that SAS-6 comprisesdomain, and proteina globular dimers N-terminal further interact domain vi followeda their N-terminal by a long coiled domains coil (Figure to form1) and oligomers a C-terminal with disordered 9-fold radial symmetryregion. on SAS-6 average strongly [28–31]. dimerises The SAS-6in vitro N-terminaland in vivo domainvia its coiled-coil bears strong domain, structural and protein resemblance dimers to similarfurther domains interact of via XRCC4 their N-terminal and XLF domains (Figure to form 2), oligomersproteins withinvolved 9-fold radialin DNA symmetry repair on and non-homologousaverage [28–31 ].end The joining. SAS-6 N-terminal Further, domainsimilar bears to SAS-6, strong structural XRCC4 resemblanceforms dimers to similar via a domains coiled-coil of XRCC4 and XLF (Figure2), proteins involved in DNA repair and non-homologous end joining. domain, and it hetero-oligomerises with XLF via the globular N-terminal domains to form a spiral Further, similar to SAS-6, XRCC4 forms dimers via a coiled-coil domain, and it hetero-oligomerises [32–36]. with XLF via the globular N-terminal domains to form a spiral [32–36].

Figure 1. SAS-6 oligomerisation. A SAS-6 ring containing nine protein dimers is shown. SAS-6 Figureself-assembles 1. SAS-6 oligomerisation. into rings via two A dimerisation SAS-6 ring interfaces containing mediated nine protein by (A) andime N-terminalrs is shown. globular SAS-6 self-assemblesdomain (NN) into and rings (B) a via coiled two coil dimerisation (CC) 35 to 50 nminterfaces long, depending mediated on by species. (A) an The N-terminal full extent of globular the domainSAS-6 (NN) coiled and coil (B and) a coiled a disordered coil (CC) C-terminal 35 to 50 protein nm long, region depending are not shown. on species. Hinge regionsThe full connecting extent of the the SAS-6 N-terminal domains with the coiled coil are indicated by red circles in (B). SAS-6 coiled coil and a disordered C-terminal protein region are not shown. Hinge regions connecting the SAS-6 N-terminal domains with the coiled coil are indicated by red circles in (B).

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Figure 2.2. ComparisonComparison of SAS-6SAS-6 toto structurallystructurally similarsimilar proteins.proteins. (A) SAS-6;SAS-6; ((B) XRCC4;XRCC4; andand ((C)) XLFXLF proteinsproteins displaydisplay structurallystructurally related N-terminal globularglobular domains and helical dimerisation regions, whichwhich comprisecomprise coiledcoiled coilscoils inin SAS-6SAS-6 andand XRCC4.XRCC4. SuperpositionSuperposition of the N terminalterminal domaindomain ofof SAS-6SAS-6 withwith XLFXLF ((DD)) andand XRCC4XRCC4 ((EE)) (a(a singlesingle chainchain isis shownshown forfor clarity).clarity).

Most SAS-6 oligomers resolved to date are highly reminiscent of cartwheel rings, with a central Most SAS-6 oligomers resolved to date are highly reminiscent of cartwheel rings, with a central hub derived from the N-terminal globular domains and the SAS-6 coiled coils comprising the hub derived from the N-terminal globular domains and the SAS-6 coiled coils comprising the cartwheel cartwheel spokes [28,29,31]. Furthermore, disruption of SAS-6 oligomerisation abrogates formation spokes [28,29,31]. Furthermore, disruption of SAS-6 oligomerisation abrogates formation of the central of the central centriole scaffold and strongly hinders organelle assembly [29,31]. Thus, SAS-6 centriole scaffold and strongly hinders organelle assembly [29,31]. Thus, SAS-6 oligomers are thought oligomers are thought to comprise the centriole cartwheels, thereby influencing the radial symmetry to comprise the centriole cartwheels, thereby influencing the radial symmetry of this organelle [26,37]. of this organelle [26,37]. Although the tertiary structure of SAS-6 is highly conserved amongst homologues, there exists Although the tertiary structure of SAS-6 is highly conserved amongst homologues, there exists both subtle and large variations in oligomer morphologies. For example, Caenorhabditis elegans SAS-6 both subtle and large variations in oligomer morphologies. For example, Caenorhabditis elegans SAS-6 (CeSAS-6) self-assembles into a spiral [30] rather than the planar rings observed in the Chlamydomonas (CeSAS-6) self-assembles into a spiral [30] rather than the planar rings observed in the reinhardtii (CrSAS-6; [31]), Danio rerio (DrSAS-6; [29]) and Leishmania major (LmSAS-6; [28]) variants of Chlamydomonas reinhardtii (CrSAS-6; [31]), Danio rerio (DrSAS-6; [29]) and Leishmania major (LmSAS-6; the same protein. Furthermore, there is substantial heterogeneity in the intrinsic symmetry of SAS-6 [28]) variants of the same protein. Furthermore, there is substantial heterogeneity in the intrinsic oligomers, both inferred by X-ray crystallography and directly visualised by microscopy. Here, we symmetry of SAS-6 oligomers, both inferred by X-ray crystallography and directly visualised by summarise the structural differences seen across SAS-6 variants, with emphasis on the symmetry of microscopy. Here, we summarise the structural differences seen across SAS-6 variants, with protein oligomers, and discuss how SAS-6 variants may support the formation of a highly conserved emphasis on the symmetry of protein oligomers, and discuss how SAS-6 variants may support the organelle architecture with strict symmetry requirements. formation of a highly conserved organelle architecture with strict symmetry requirements. 2. SAS-6 Rings 2. SAS-6 Rings Initial insight into the symmetry of SAS-6 oligomers was obtained through in silico models, whereInitial oligomer insight symmetry into the was symmetry inferred of by SAS-6 combining oligomers separate was crystal obtained structures through of SAS-6in silico constructs models, comprisingwhere oligomer dimers symmetry of the was N-terminal inferred by globular combining domain separate (NN crystal interface) structures or of coiled-coil SAS-6 constructs dimers (CCcomprising interface). dimers Such of studiesthe N-terminal suggested globular that SAS-6 domain oligomers (NN interface) feature radial or coiled-coil symmetries dimers close (CC to theinterface). canonical Such centriolar studies 9-fold suggested arrangement, that SAS-6 although oligomers significant feature symmetry radial variationssymmetries were close observed to the thatcanonical could centriolar be attributed 9-fold to artefactsarrangement, of crystallisation although significant or protein sy conformationalmmetry variations heterogeneity. were observed For example,that could there be attributed exist three to crystallographic artefacts of crystallis modelsation of the or CrSAS-6 protein NN conformati interfaceonal and oneheterogeneity. of the CrSAS-6 For CCexample, interface there [31 ];exist thus, three three crystallographic different oligomer model variantss of the can beCrSAS-6 built depending NN interface on the and NN one interface of the modelCrSAS-6 employed CC interface resulting [31]; in thus, 9.6- three to 10.1-fold different radial olig symmetry.omer variants Similarly, can be when built DrSAS-6depending oligomers on the NN are interface model employed resulting in 9.6- to 10.1-fold radial symmetry. Similarly, when DrSAS-6 oligomers are built from available crystal structures a variation in oligomer architecture is observed, with DrSAS-6 forming either an 8-fold symmetric shallow spiral or an 8-fold symmetric ring [29],

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Symmetrybuilt from 2017 available, 9, 74 crystal structures a variation in oligomer architecture is observed, with DrSAS-64 of 10 forming either an 8-fold symmetric shallow spiral or an 8-fold symmetric ring [29], while LmSAS-6 whileinferred LmSAS-6 oligomer inferred symmetry oligomer ranges symmetry from 7.8- toranges 11.3-fold from [28 7.8-]. The to variation11.3-fold in[28]. oligomer The variation symmetry in oligomerfrom different symmetry SAS-6 from crystallographic different SAS-6 models crystallographic is summarised models in Figure is summarised3A. in Figure 3A.

Figure 3. Analysis of variation in SAS-6 oligomer symmetry. ( (AA)) Variation Variation of symmetry in SAS-6 oligomers [28–31] [28–31] inferred from crystal structures (black(black circles) or directly visualised by electron microscopy (grey bars). (B (B) )Variation Variation in in oligomer oligomer symmetry symmetry of of wild wild type type [37] [37] and engineered CrSAS-6 [31] [31] (denoted as in panel A). (C,E) Fluctuations in LmSAS-6 ( C) and CeSAS-6 (E) oligomer symmetry over a 100-ns molecular dynamics (MD) simulation.simulation. Triplicate si simulationsmulations are shown in blue, greengreen andand orange. orange. (D (,DF),F Frequency) Frequency of LmSAS-6of LmSAS-6 (D) and(D) CeSAS-6and CeSAS-6 (F) oligomer (F) oligomer symmetry symmetry derived derivedfrom 10,000 from conformations 10,000 conformations where both where NN both and NN CC interfacesand CC interfaces are varied. are varied.

Heterogeneity in symmetry was also observed in in vitro experiments where SAS-6 oligomers were reconstituted and theirtheir symmetrysymmetry directlydirectly examined.examined. For example, rotary metal shadowing electron microscopymicroscopy (EM) (EM) of CrSAS-6of CrSAS-6 oligomers oligomers revealed revealed the formation the formation of rings of with rings variable with diameters variable diameterscorresponding corresponding to 7.8 to 9.4-fold to 7.8 radialto 9.4-fold symmetry radial [31symmetry], a conclusion [31], a alsoconclusion supported also by supported atomic force by atomicmicroscopy force (AFM) microscopy studies (AFM) [37,38] ofstudies the same [37,38] oligomers of the (Figure same 4A).oligomers These experiments(Figure 4A). argued These experimentsthat symmetry argued differences that symmetry inferred differences from the SAS-6 inferred crystallographic from the SAS-6 structures crystallographic did not structures represent didartefacts, not represent but rather artefacts, genuine but protein rather flexibility genuine protein at the SAS-6 flexibility NN andat the CC SAS-6 interaction NN and interfaces CC interaction which interfacesdrive oligomerisation. which drive oligomerisation. Atomistic molecular Atomistic dynamics molecular (MD) dynamics simulations (MD) ofsimulations CrSAS-6 of performed CrSAS-6 previouslyperformed previously [37], and similar [37], and work similar on LmSAS-6 work on presented LmSAS-6 herepresented (described here (described in Appendix inA Appendix), showcase A), showcase this conformational flexibility. In this type of analysis, SAS-6 oligomers are built using a fixed model of one interface from crystallography and variable models of the second interface derived from snapshots of the interface MD trajectory. The resulting SAS-6 oligomers show

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Symmetry 2017, 9, 74 5 of 10 this conformational flexibility. In this type of analysis, SAS-6 oligomers are built using a fixed model of one interface from crystallography and variable models of the second interface derived from substantial fluctuations in symmetry; for example, CrSAS-6 oligomers built using a simulated NN snapshots of the interface MD trajectory. The resulting SAS-6 oligomers show substantial fluctuations interface range from 7- to 13-fold symmetry [37], while we noted 6- to 23-fold symmetries for in symmetry; for example, CrSAS-6 oligomers built using a simulated NN interface range from 7- to LmSAS-6 oligomers built from a simulated CC interface (Figure 3C). Furthermore, we derived 13-fold symmetry [37], while we noted 6- to 23-fold symmetries for LmSAS-6 oligomers built from 10,000 distinct oligomer models of LmSAS-6 by combining independent MD simulations of the NN a simulated CC interface (Figure3C). Furthermore, we derived 10,000 distinct oligomer models of and CC dimerization interfaces (Figure 3D), and noted that a substantial fraction (24.5 ± 2.7%) of LmSAS-6 by combining independent MD simulations of the NN and CC dimerization interfaces LmSAS-6 oligomers acquire 12-fold or larger symmetries, with a mean oligomer symmetry at (Figure3D), and noted that a substantial fraction (24.5 ± 2.7%) of LmSAS-6 oligomers acquire 12-fold approximately 10-fold. or larger symmetries, with a mean oligomer symmetry at approximately 10-fold.

Figure 4. InIn vitrovitro visualisationvisualisation ofof SAS-6SAS-6 oligomers.oligomers. (A)) AtomicAtomic forceforce microscopymicroscopy imagesimages of CrSAS-6CrSAS-6 oligomers forming rings. Left panel presents an overview of the sample; right panel magnifiesmagnifies the indicated region. Reprinted Reprinted with with permission permission from from [38]. [38]. Copyright Copyright 2014 2014 Americ Americanan Chemical Chemical Society. Society. ((B)) NegativeNegative stainstain transmission transmission electron electron microscopy microscopy images images of CeSAS-6 of CeSAS-6 protein protein spirals spirals [30]. Left[30]. panel Left showspanel shows long CeSAS-6 long CeSAS-6 double spirals;double whitespirals; arrowheads white arrowheads denote points denote at whichpoints individualat which individual strands of thestrands double of spiralsthe double separate spirals or come separate together. or come Right together. panel shows Right a magnifiedpanel shows view a ofmagnified isolated CeSAS-6view of spiralisolated strands. CeSAS-6 spiral strands.

3. SAS-6 Spirals Although C. elegans elegans centriolescentrioles show show strong strong structural structural and and functional functional conservation conservation compared compared to tothose those of ofother other species species [28,29,31] [28,29,31, ],crystallographic crystallographic and and electron electron microscopy microscopy studies studies of CeSAS-6CeSAS-6 suggested thatthat thisthis proteinprotein variant variant forms forms spiral spiral oligomers oligomers rather rather than than rings. rings. Interestingly, Interestingly, despite despite the limitedthe limited sequence sequence similarity similarity between between CeSAS-6 CeSAS-6 and itsan homologuesd its homologues [24,39 ,[24,39,40],40], both theboth overall the overall SAS-6 architectureSAS-6 architecture of a globular of a N-terminalglobular N-terminal domain succeeded domain bysucceeded a coiled coilby anda coiled the tertiary coil and structure the tertiary of the N-terminalstructure of domain the N-terminal itself were domain highly similar.itself were Rather, highly the discrepancysimilar. Rather, of SAS-6 the oligomericdiscrepancy architecture of SAS-6 betweenoligomericC. architecture elegans and between other organisms C. elegans appears and other tooriginate organisms at appears the protein to originate region connecting at the protein the N-terminalregion connecting domain the and N-terminal the coiled domain coil, known and asthe the coiled hinge coil, (Figures known1 andas the5). hinge There, (Figures in ring-forming 1 and 5). SAS-6,There, invariablyin ring-forming exists SAS-6, a small invariably amino acid exists residue, a small typically amino glycine acid orresidue, alanine. typically In contrast, glycine in theor spiralalanine. forming In contrast, CeSAS-6 in the this spiral position forming is occupiedCeSAS-6 bythis a position valine (Figure is occupied5); a valineby a valine is also (Figure found 5); at a thevaline hinge is also position found of at XRCC4,the hinge which position forms of XRCC4, spirals similar which toforms CeSAS-6 spirals [32 similar,34–36 to]. CeSAS-6 The steric [32,34– effect imposed36]. The steric by the effect valine imposed in CeSAS-6 by the moves valine the in coiled-coil CeSAS-6 helixmoves relative the coiled-coil to the N-terminal helix relative domain to the by ◦ approximatelyN-terminal domain 40 compared by approximately to other SAS-6 40° compared variants [30 to], other thereby SAS-6 driving variants successive [30], CeSAS-6thereby driving dimers outsuccessive of the horizontal CeSAS-6 planedimers and out into of the a steep horizontal spiral of plane ~30nm and pitch. into a Such steep CeSAS-6 spiral of spirals ~30 nm were pitch. observed Such toCeSAS-6 intertwine spiralsin vitro were(Figure observed4B); thus,to intertwine it was proposed in vitro that (Figure in C. 4B); elegans thus,centrioles it was proposed two CeSAS-6 that spirals in C. mayelegans form centrioles a central two scaffold CeSAS-6 analogous spirals may to the form cartwheel a central [30 scaffold]. analogous to the cartwheel [30]. Similar to SAS-6 from otherother species,species, oligomers of CeSAS-6CeSAS-6 inferredinferred fromfrom crystallographiccrystallographic structures of the NN andand CCCC interfacesinterfaces showshow substantialsubstantial variation in symmetrysymmetry ranging from approximately 9-9- to to 11-fold 11-fold per per turn turn of an of intertwined an intertwined spiral spiral (Figure (Figure3A), thus 3A), suggesting thus suggesting the presence the ofpresence conformational of conformational flexibility flexibility in the NN in and the CCNN interfaces. and CC interfaces. Indeed, direct Indeed, visualization direct visualization of CeSAS-6 of spiralCeSAS-6 oligomers spiral byoligomers negative by stain negative EM demonstrated stain EM andemonstrated even broader an symmetry even broader distribution symmetry from 7-distribution to 11-fold; from it is therefore 7- to 11-fold; possible it thatis therefore the structural possible variation that seenthe instructural crystallographic variation structures seen in underestimatescrystallographic thestructures true degree underestimates of interface the flexibility. true degree MD of simulations interface flexibility. of the CeSAS-6 MD simulations CC interface of resultedthe CeSAS-6 in spiral CC interface SAS-6 oligomersresulted in of spiral 7- to SAS-6 16-fold oligomers symmetry of 7- (Figure to 16-fold3E; [ symmetry37]); more (Figure strikingly, 3E; when[37]); more combining strikingly, simulations when combining of the NN andsimulations CC interfaces, of the CeSAS-6 NN and oligomerCC interfaces, symmetries CeSAS-6 ranged oligomer from symmetries ranged from 6.4- to 16-fold, with a mean symmetry of 8.8-fold (Figure 3F). This indicates that, despite the steric clash at the hinge region imposed on CeSAS-6, the protein remains flexile and able to accommodate a range of oligomer symmetries.

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6.4- to 16-fold, with a mean symmetry of 8.8-fold (Figure3F). This indicates that, despite the steric clash at the hinge region imposed on CeSAS-6, the protein remains flexile and able to accommodate aSymmetry range of2017 oligomer, 9, 74 symmetries. 6 of 10

Figure 5. Comparison of SAS-6 variants. (A) Multiple sequence alignment of SAS-6 variants with the Figure 5. Comparison of SAS-6 variants. (A) Multiple sequence alignment of SAS-6 variants with the hinge residue highlighted in green. (B) Overlay of LmSAS-6 (dark blue) and CeSAS-6 (light blue) with hinge residue highlighted in green. (B) Overlay of LmSAS-6 (dark blue) and CeSAS-6 (light blue) the hinge indicated by the red circle. A zoomed-in view of the hinge region is shown in panel (C), with the hinge indicated by the red circle. A zoomed-in view of the hinge region is shown in panel (C), with the hinge valine of CeSAS-6 shown in the green sphere representation. with the hinge valine of CeSAS-6 shown in the green sphere representation.

4. Discussion Crystallographic, biophysical and computational analysis of the SAS-6 NN and CCCC interfacesinterfaces suggest thatthat these these exhibit exhibit substantial substant flexibility,ial flexibility, which which in turn in leads turn to leads heterogeneity to heterogeneity of SAS-6 oligomers.of SAS-6 Thus,oligomers. SAS-6, Thus, if acting SAS-6, alone, if acting would alone, have would given have rise togiven highly rise variable to highly cartwheels variable cartwheels and centrioles; and however,centrioles; centriolehowever, symmetry centriole symmetry is strictly is controlled strictly controlledin vivo which in vivo suggests which suggests that additional that additional factors orfactors proteins or proteins beyond beyond SAS-6 influenceSAS-6 influence the radial the radial organisation organisation of this of organelle. this organelle. This conclusionThis conclusion was reinforcedwas reinforced by a veryby a recentvery recent study study where where CrSAS-6 CrSAS-6 was subjected was subjected to rational to rational engineering engineering [37]. There, [37]. aminoThere, acidamino variants acid variants of the NNof the interface NN interf producedace produced CrSAS-6 CrSAS-6 oligomers oligomers with symmetries with symmetries ranging fromranging 5- tofrom 10-fold 5- to as 10-fold judged as by judged crystallography, by crystallography, negative stainnegative EM andstain AFM EM and (Figure AFM3B). (Figure However, 3B). centriolesHowever, incentriolesC. reinhardtii in C.cells reinhardtii incorporating cells incorporating these CrSAS-6 these variants CrSAS-6 almost variants always retainedalmost always 9-fold symmetry;retained 9-fold the sole symmetry; exception the was sole a CrSAS-6exception variant was a givingCrSAS-6 rise variant to 5-fold giving symmetric rise to oligomers5-fold symmetricin vitro thatoligomers produced in vitro a 2:1 that ratio produced of 9- and a 2:1 8-fold ratio symmetric of 9- and 8-fold centrioles symmetricin vivo .centrioles Thus, it isin clearvivo. thatThus, while it is SAS-6clear that plays while a central SAS-6 role plays in determining a central role centriole in determining symmetry, thecentriole organelle symmetry, architecture the mustorganelle also bearchitecture dependent must on other also be factors. dependent We note, on forother example, factors. thatWe nullnote, mutants for example, of C. reinhardtiithat null mutants bld10, a geneof C. orthologousreinhardtii bld10 to, humana orthologouscep135, generate to human centrioles cep135 with, generate variable centrioles numbers wi ofth variable microtubule numbers blades, of indicatingmicrotubule that blades, Bld10 indicating protein may that also Bld10 contribute protein tomay centriolar also contribute symmetry to centriolar [41]. symmetry [41]. How maymay SAS-6,SAS-6, Bld10/Cep135Bld10/Cep135 and and microtubules work together to determine centriolecentriole symmetry? AA likely likely relevant relevant observation observation is thatis that cartwheel cartwheel diameter, diameter, primarily primarily determined determined by the by length the oflength the SAS-6of the coiled SAS-6 coil coiled plus Bld10/Cep135, coil plus Bld10/Cep135, instructs the diameterinstructs andthe hencediameter the circumferenceand hence the of centrioles.circumference This, of in centrioles. turn, governs This, the in number turn, govern of microtubules the number blades of that microtubule can be accommodated blades that aroundcan be theaccommodated perimeter of around the centriole the perimeter central core. of the On centriol the othere central hand, the core. size On of microtubulethe other hand, blades the andsize the of geometrymicrotubule of the blades linkers and connecting the geometry them also of definethe li ankers preferred connecting circumference them foralso centrioles. define a Thus, preferred strict organellecircumference 9-fold for symmetry centrioles. may Thus, emerge strict as a organell result ofe coinciding 9-fold symmetry but independent may emerge weak as circumference a result of preferencescoinciding but derived independent by SAS-6 weak and Bld10/Cep135, circumference andpreferences microtubules derived at the by centriole SAS-6 and outer Bld10/Cep135, wall. In this manner,and microtubules different SAS-6at the and centriole Bld10/Cep135 outer wall. variants In this in organisms,manner, different or expression SAS-6 ofand different Bld10/Cep135 protein isoformsvariants in in organisms, different cell or types,expression could of lead different to different protein cartwheel isoforms diameters in different and cell hence types, shift could organelle lead to symmetrydifferent cartwheel towards diameters different modal and hence points. shift Although organelle speculative, symmetry towards such molecular different processes modal points. may accountAlthough for speculative, observations such in Acerentomon molecularcentrioles, processes which may possessaccount 9-fold for observations symmetry in somaticin Acerentomon cells but centrioles, which possess 9-fold symmetry in somatic cells but 14-fold symmetry in spermatocytes with a corresponding enlargement of diameter [42]. Alternatively, different SAS-6 and Bld10/Cep135 isoforms could be accommodated in centrioles without varying symmetry by changing the size of microtubule blades at the outer organelle wall in a compensatory manner. We note, for example, that D. melanogaster germ line centrioles possess a triplet of microtubules in each organelle blade, while centrioles in somatic cells have microtubule doublets [43].

Symmetry 2017, 9, 74 7 of 10 Symmetry 2017, 9, 74 7 of 10 Further molecular mechanisms may of course influence centriole symmetry; for example, work in human cells has suggested that pre-existing centrioles may template newly assembled organelles 14-fold symmetry in spermatocytes with a corresponding enlargement of diameter [42]. Alternatively, through possible interactions of new SAS-6 cartwheels with the mother centriole lumen [44,45]. different SAS-6 and Bld10/Cep135 isoforms could be accommodated in centrioles without varying However, it is worth noting that the lumen of human centrioles becomes vacant, and hence available symmetry by changing the size of microtubule blades at the outer organelle wall in a compensatory to template new cartwheels, only due to SAS-6 degradation during the G1 phase [1]. Other manner. We note, for example, that D. melanogaster germ line centrioles possess a triplet of microtubules organisms, such as C. reinhardtii and C. elegans, do not degrade SAS-6 in the same manner; hence, in each organelle blade, while centrioles in somatic cells have microtubule doublets [43]. template-based assembly of new SAS-6 cartwheels cannot account for strict centriole symmetry in Further molecular mechanisms may of course influence centriole symmetry; for example, work these species. in human cells has suggested that pre-existing centrioles may template newly assembled organelles Cryo-electron tomography of Trichonympha sp. [46,47] and C. reinhardtii [48] centrioles revealed through possible interactions of new SAS-6 cartwheels with the mother centriole lumen [44,45]. that the spokes of successive SAS-6 rings merge as they radiate outwards, leading to stacking of However, it is worth noting that the lumen of human centrioles becomes vacant, and hence available SAS-6 rings with a periodicity of approximately 8.5 nm at the central hub and 17 nm at the end of to template new cartwheels, only due to SAS-6 degradation during the G1 phase [1]. Other organisms, spokes (Figure 6A). SAS-6 stacking periodicity aligns with the axial periodicity of microtubules at such as C. reinhardtii and C. elegans, do not degrade SAS-6 in the same manner; hence, template-based the outer centriole wall. C. elegans centrioles, in contrast, likely use a different mechanism to reach assembly of new SAS-6 cartwheels cannot account for strict centriole symmetry in these species. the same periodicity. In EM of fixed C. elegans embryos an electron lucent centriole centre was Cryo-electron tomography of Trichonympha sp. [46,47] and C. reinhardtii [48] centrioles revealed observed with no evidence of aligned, or merged, cartwheel spokes [49,50], which was interpreted as that the spokes of successive SAS-6 rings merge as they radiate outwards, leading to stacking of SAS-6 possible evidence for a centriole cartwheel formed by CeSAS-6 spirals rather than stacked rings. rings with a periodicity of approximately 8.5 nm at the central hub and 17 nm at the end of spokes Reconstructions of such CeSAS-6 spirals from the crystallographic structures show successive (Figure6A). SAS-6 stacking periodicity aligns with the axial periodicity of microtubules at the outer spokes with 16 nm axial periodicity, thereby aligning with microtubules, but offset by a ~20° angle centriole wall. C. elegans centrioles, in contrast, likely use a different mechanism to reach the same [30]. In this manner, CeSAS-6 spokes do not align to give rise to strong electron scattering (Figure periodicity. In EM of fixed C. elegans embryos an electron lucent centriole centre was observed with no 6B). Thus, we surmise that both spiral and ring SAS-6 arrangements may be present in centrioles, as evidence of aligned, or merged, cartwheel spokes [49,50], which was interpreted as possible evidence they both provide contact points with the microtubule wall at equivalent spacing and hence can be for a centriole cartwheel formed by CeSAS-6 spirals rather than stacked rings. Reconstructions of accommodated with no additional alterations of the centriole architecture. such CeSAS-6 spirals from the crystallographic structures show successive spokes with 16 nm axial In summary, the conformational flexibility observed in SAS-6 oligomers suggests it is unlikely periodicity, thereby aligning with microtubules, but offset by a ~20◦ angle [30]. In this manner, this protein alone dictates centriole symmetry. Rather, SAS-6 and other protein factors, including CeSAS-6 spokes do not align to give rise to strong electron scattering (Figure6B). Thus, we surmise Bld10/Cep135 and microtubules, collaborate to strictly define the radial appearance of this organelle. In that both spiral and ring SAS-6 arrangements may be present in centrioles, as they both provide contact contrast, different SAS-6 oligomeric architectures, spirals and rings, can be accommodated equally well points with the microtubule wall at equivalent spacing and hence can be accommodated with no within a cylindrical microtubule wall, and thus may be both present in centrioles from different species. additional alterations of the centriole architecture.

Figure 6. SAS-6 oligomer structural comparison. ( (AA)) A A CeSAS-6 CeSAS-6 spiral spiral oligomer oligomer is is shown in blue alongside aa stack stack of of LmSAS-6 LmSAS-6 cartwheels cartwheels in green. in green The. distance The distance between between equivalent equivalent spokes is spokes indicated; is indicated;(B) A tilted (B view) A tilted of a CeSAS-6 view of spirala CeSAS-6 as in panelspiral A as with in pa consecutivenel A with spokeconsecutive ends indicated; spoke ends the indicated; rotational theoffset rotational between offset spokes betw is evident;een spokes (C) Top is evident; view of panel(C) Top A with view a CeSAS-6of panel spiralA with and a CeSAS-6 LmSAS-6 spiral cartwheel and LmSAS-6stack overlaid, cartwheel showing stack the overlaid, spoke distribution showing fromthe sp theoke alternative distribution SAS-6 from oligomeric the alternative structures. SAS-6 oligomeric structures.

Symmetry 2017, 9, 74 8 of 10

In summary, the conformational flexibility observed in SAS-6 oligomers suggests it is unlikely this protein alone dictates centriole symmetry. Rather, SAS-6 and other protein factors, including Bld10/Cep135 and microtubules, collaborate to strictly define the radial appearance of this organelle. In contrast, different SAS-6 oligomeric architectures, spirals and rings, can be accommodated equally well within a cylindrical microtubule wall, and thus may be both present in centrioles from different species.

Acknowledgments: This work was supported by the Medical Research Council project grant MR/N009274/1 to IV. Costs related to open access publishing were provided by the RCUK Open Access Block Grant to Oxford. Author Contributions: Jodie E. Ford analysed literature and performed MD simulations, Phillip J. Stansfeld supervised the MD simulations. Jodie E. Ford and Ioannis Vakonakis wrote the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

Appendix A SAS-6 dimer loops were modelled using modeller v9.12 and the resulting models used to initiate 100-ns long MD simulations in simple point charge (SPC) water using the OPLS-AA/L forcefield at 310 K and Na and Cl ions added to neutalise charge. Pressure was maintained using the Parrinello-Rahman barostat [51] and temperature was maintained using the V-rescale thermostat [52]. Simulations were run and analysed in GROMACS v4.6 [53] with a 2-fs timestep and frames were recorded every 10 ps. Trajectories were visualised in PyMOL v1.7.x [54] and Chimera v1.10.2 [55]. (PDB) codes used in MD simulations: 4CKN, 4CKM [28], 4GFC, 4G79 [30].

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