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The Homo-Oligomerisation of Both Sas-6 and Ana2 Is Required for Efficient Centriole Assembly in Flies

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The Homo-Oligomerisation of Both Sas-6 and Ana2 Is Required for Efficient Centriole Assembly in Flies 1 1 The homo-oligomerisation of both Sas-6 and Ana2 is required 2 for efficient centriole assembly in flies 3 4 5 Matthew A. Cottee1, Nadine Muschalik1*, Steven Johnson, Joanna Leveson, 6 Jordan W. Raff2 and Susan M. Lea2. 7 8 Sir William Dunn School of Pathology, University of Oxford, UK 9 *present address: Division of Cell Biology, MRC-Laboratory of Molecular 10 Biology, Cambridge, UK 11 12 1 These authors contributed equally to this work 13 2 corresponding authors: [email protected]; 14 [email protected] 15 2 16 17 Abstract 18 Sas-6 and Ana2/STIL proteins are required for centriole duplication and the 19 homo-oligomerisation properties of Sas-6 help establish the nine-fold 20 symmetry of the central cartwheel that initiates centriole assembly. Ana2/STIL 21 proteins are poorly conserved, but they all contain a predicted Central Coiled- 22 Coil Domain (CCCD). Here we show that the Drosophila Ana2 CCCD forms a 23 tetramer, and we solve its structure to 0.8 Å, revealing that it adopts an 24 unusual parallel-coil topology. We also solve the structure of the Drosophila 25 Sas-6 N-terminal domain to 2.9 Å revealing that it forms higher-order 26 oligomers through canonical interactions. Point mutations that perturb Sas-6 27 or Ana2 homo-oligomerisation in vitro strongly perturb centriole assembly in 28 vivo. Thus, efficient centriole duplication in flies requires the homo- 29 oligomerisation of both Sas-6 and Ana2, and the Ana2 CCCD tetramer 30 structure provides important information on how these proteins might 31 cooperate to form a cartwheel structure. 32 3 33 34 Introduction 35 Centrioles are complex microtubule (MT) based structures that are required 36 for the formation of centrosomes and cilia/flagella. These organelles have 37 many important functions in cells, and their dysfunction has been linked to a 38 plethora of human pathologies, ranging from cancer to microcephaly to 39 obesity (Bettencourt-Dias et al., 2011; Nigg and Raff, 2009). Thus, 40 understanding how these organelles assemble and function is an important 41 goal of both basic and biomedical research. 42 43 Although several hundred proteins are thought to be concentrated at 44 centrioles, only a small number appear to form a conserved “core” pathway 45 that is essential for centriole assembly (Delattre et al., 2006; Gönczy, 2012; 46 Pelletier et al., 2006). During canonical centriole duplication, the protein 47 kinase Plk4/Sak/ZYG-1 is recruited to the mother centriole by SPD-2 in worms 48 (Delattre et al., 2006; Pelletier et al., 2006; Shimanovskaya et al., 2014), by 49 Asterless (Asl) in flies (Blachon et al., 2008; Dzhindzhev et al., 2010), or by a 50 combination of the two (Cep192 and Cep152, respectively) in humans 51 (Cizmecioglu et al., 2010; Hatch et al., 2010; Kim et al., 2013; Sonnen et al., 52 2013). The protein kinase recruits STIL/Ana2/SAS-5 and Sas-6 to a single site 53 on the side of the mother centriole where they assemble with CPAP/Sas-4 54 into a cartwheel structure that helps to establish the 9-fold symmetry of the 55 centriole (Arquint et al., 2012; Dammermann et al., 2004; Delattre et al., 2004; 56 Leidel et al., 2005; Nakazawa et al., 2007; Peel et al., 2007; Stevens et al., 57 2010a; Strnad et al., 2007; Tang et al., 2011). CPAP/Sas-4 can interact with 4 58 tubulin (Hung et al., 2004) and is required to recruit the centriole MTs to the 59 outer region of the cartwheel (Pelletier et al., 2006), possibly working together 60 with Cep135/Bld10 (Hiraki et al., 2007; Lin et al., 2013)—although no 61 homologue of this protein has been identified in worms, and it does not 62 appear to be essential for centriole duplication in flies (Carvalho-Santos et al., 63 2012; Mottier-Pavie and Megraw, 2009; roque et al., 2012). 64 65 Great progress has been made recently in understanding how these proteins 66 interact and how these interactions are regulated to ensure that a new 67 centriole is only formed at the right place and at the right time. In particular, 68 the crystal structure of Sas-6 from several species has revealed how this 69 protein forms a dimer through its C-terminal coiled-coil domain (C-C) that can 70 then further homo-oligomerise through an N-terminal head group interaction 71 (N-N) to form a ring structure from which the C-C domains emanate as 72 spokes (Hilbert et al., 2013; Kitagawa et al., 2011; van Breugel et al., 2011; 73 2014). This Sas-6 ring structure can be modelled into EM tomographic 74 reconstructions of the cartwheel from Trichonympha centrioles (Guichard et 75 al., 2012; 2013), strongly suggesting that these Sas-6 rings form the basic 76 building blocks of the cartwheel. In support of this hypothesis, mutant forms of 77 Sas-6 that cannot homo-oligomerise through the N-N interaction are unable to 78 support efficient centriole duplication (Kitagawa et al., 2011; van Breugel et 79 al., 2011), although they can still target to centrioles, a function that seems to 80 rely on the C-C domain (Fong et al., 2014; Keller et al., 2014). 81 5 82 A crystal structure of the interface between Ana2/STIL and Sas-4/CPAP has 83 also recently been solved (Cottee et al., 2013; Hatzopoulos et al., 2013), as 84 has the interaction interface between Plk4 and both Cep192/SPD-2 and 85 Cep152/Asl (Park et al., 2014); mutations that perturb these interactions in 86 vitro perturb centriole duplication in vivo, indicating that these interactions are 87 also essential for centriole duplication. More recently, it has been shown that 88 Plk4 can recruit STIL to centrioles in human cells (Ohta et al., 2014) and that 89 Plk4/Sak can phosphorylate the conserved STIL/Ana2 (STAN) domain in 90 STIL/Ana2 proteins in humans and flies, thereby promoting the interaction of 91 the STAN domain with Sas-6 (Dzhindzhev et al., 2014; Ohta et al., 2014). 92 Mutant forms of STIL/Ana2 that could not be phosphorylated strongly 93 perturbed Sas-6 recruitment to centrioles and centriole duplication. Together, 94 these studies have shed important light on the molecular mechanisms of 95 centriole assembly, but many important questions remain. 96 97 In particular, it has been proposed that the homo-oligomerisation properties of 98 Sas-6 establish the 9-fold symmetry of the centriole (Kitagawa et al., 2011), 99 and, remarkably, a 9-fold symmetric ring structure is formed in crystallo by 100 Leishmania major Sas-6 (van Breugel et al., 2014). However, although Sas-6 101 oligomers appear to have a propensity towards 9-fold symmetry, Sas-6 102 proteins spontaneously assemble into oligomers of varying stoichiometry in 103 vitro (Kitagawa et al., 2011; van Breugel et al., 2011), suggesting that the 104 homo-oligomerisation properties of Sas-6 alone may be insufficient to enforce 105 the rigorous 9-fold symmetry that is observed in centrioles from virtually all 106 species (Cottee et al., 2011). Additionally, recent Cryo-EM analysis suggests 6 107 that the basic building block of the cartwheel stack is not a single ring and 108 spoke structure, but rather a pair of rings that sit on top of one another: these 109 rings do not make direct contact with each other, but are joined in the more 110 peripheral regions through their spokes (Guichard et al., 2012; 2013). Our 111 current knowledge of Sas-6 self-association cannot explain this important 112 feature of the cartwheel structure. 113 114 We previously showed that overexpressed Sas-6 can form higher-order 115 aggregates in Drosophila spermatocytes, but these aggregates only adopt a 116 cartwheel-like structure when Ana2 is also overexpressed (Stevens et al., 117 2010b), and the STIL/Ana2 protein family is essential for the proper 118 recruitment of Sas-6 to centrioles (Dzhindzhev et al., 2014; Ohta et al., 2014). 119 We reasoned therefore, that Ana2 was likely to also play an important part in 120 determining the structure of the central cartwheel. We set out to investigate 121 the potential structural features of Ana2 that might be important for centriole 122 assembly. 7 123 Results 124 The CCCD is required for the centriolar targeting of Ana2 125 The Drosophila Ana2 protein contains 4 regions that have significant 126 homology to Ana2/STIL proteins from other species (Figure 1A, 2A) (Cottee et 127 al., 2013). Fly Ana2 lacks the conserved region 1 (CR1) found towards the N- 128 terminus in vertebrate STIL proteins (Figure 2A), but contains a CR2 domain 129 that interacts with Sas-4 (Cottee et al., 2013; Hatzopoulos et al., 2013), a 130 predicted central coiled-coiled domain (CCCD), a STAN domain (Stevens et 131 al., 2010a) that interacts with Sas-6 (Dzhindzhev et al., 2014; Ohta et al., 132 2014) and a short C-terminal CR4 domain (Figure 1A) (Cottee et al., 2013). 133 To examine the potential function of these conserved regions, we synthesised 134 mRNAs in vitro that contained either wild type (WT) or truncated versions of 135 Ana2 fused to either an N- or C-terminal GFP (Figure 1A). These mRNAs 136 were injected into WT early embryos (that contain unlabelled endogenous WT 137 Ana2 protein) expressing RFP-Centrosomin (Cnn) as a centrosomal marker 138 (Conduit et al., 2010). The localisation of the encoded GFP-fusion protein was 139 assessed 90-120mins after mRNA injection (Figure 1B,C). 140 141 Both N- and C-terminal GFP fusions of full length Ana2 (constructs 1 and 6) 142 showed a strong, compact, localisation to centrioles, as did fusions lacking 143 either CR2 or CR4 (constructs 2 and 7), suggesting that these domains are 144 not involved in Ana2 centriolar targeting.
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