Limits...
The homo-oligomerisation of both Sas-6 and Ana2 is required for efficient centriole assembly in flies.

Cottee MA, Muschalik N, Johnson S, Leveson J, Raff JW, Lea SM - Elife (2015)

Bottom Line: We also solve the structure of the Drosophila Sas-6 N-terminal domain to 2.9 Å revealing that it forms higher-order oligomers through canonical interactions.Point mutations that perturb Sas-6 or Ana2 homo-oligomerisation in vitro strongly perturb centriole assembly in vivo.Thus, efficient centriole duplication in flies requires the homo-oligomerisation of both Sas-6 and Ana2, and the Ana2 CCCD tetramer structure provides important information on how these proteins might cooperate to form a cartwheel structure.

View Article: PubMed Central - PubMed

Affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Sas-6 and Ana2/STIL proteins are required for centriole duplication and the homo-oligomerisation properties of Sas-6 help establish the ninefold symmetry of the central cartwheel that initiates centriole assembly. Ana2/STIL proteins are poorly conserved, but they all contain a predicted Central Coiled-Coil Domain (CCCD). Here we show that the Drosophila Ana2 CCCD forms a tetramer, and we solve its structure to 0.8 Å, revealing that it adopts an unusual parallel-coil topology. We also solve the structure of the Drosophila Sas-6 N-terminal domain to 2.9 Å revealing that it forms higher-order oligomers through canonical interactions. Point mutations that perturb Sas-6 or Ana2 homo-oligomerisation in vitro strongly perturb centriole assembly in vivo. Thus, efficient centriole duplication in flies requires the homo-oligomerisation of both Sas-6 and Ana2, and the Ana2 CCCD tetramer structure provides important information on how these proteins might cooperate to form a cartwheel structure.

No MeSH data available.


A structure/function analysis of Drosophila Ana2.(A) A schematic representation of Drosophila Ana2 highlighting the conserved domains and illustrating the GFP constructs analysed in this study. In vitro transcribed mRNA encoding each of these constructs was injected into Drosophila embryos expressing the PCM marker, RFP-Cnn; the distribution of each fusion protein was analysed in living embryos. (B) Micrographs show examples of typical centrosomes in embryos injected with the Ana2 constructs shown in (A). The localisation of the GFP-fusion protein (green) is shown on its own (left panel) and merged with RFP-Cnn (right panel). (C) Bars quantify the localisation behaviour of the various GFP-fusions. Images of 30–80 embryos were analysed for each construct. Images of each embryo were collected and then manually sorted into various categories based on the centrosomal localisation of the GFP-fusion construct (see colour table at bottom of figure). All sorting was performed blind.DOI:http://dx.doi.org/10.7554/eLife.07236.003
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4471874&req=5

fig1: A structure/function analysis of Drosophila Ana2.(A) A schematic representation of Drosophila Ana2 highlighting the conserved domains and illustrating the GFP constructs analysed in this study. In vitro transcribed mRNA encoding each of these constructs was injected into Drosophila embryos expressing the PCM marker, RFP-Cnn; the distribution of each fusion protein was analysed in living embryos. (B) Micrographs show examples of typical centrosomes in embryos injected with the Ana2 constructs shown in (A). The localisation of the GFP-fusion protein (green) is shown on its own (left panel) and merged with RFP-Cnn (right panel). (C) Bars quantify the localisation behaviour of the various GFP-fusions. Images of 30–80 embryos were analysed for each construct. Images of each embryo were collected and then manually sorted into various categories based on the centrosomal localisation of the GFP-fusion construct (see colour table at bottom of figure). All sorting was performed blind.DOI:http://dx.doi.org/10.7554/eLife.07236.003

Mentions: The Drosophila Ana2 protein contains four regions that have significant homology to Ana2/STIL proteins from other species (Figures 1A, 2A) (Cottee et al., 2013). Fly Ana2 lacks the conserved region 1 found towards the N-terminus in vertebrate STIL proteins (Figure 2A), but contains a CR2 domain that interacts with Sas-4 (Cottee et al., 2013; Hatzopoulos et al., 2013), a predicted central coiled-coiled domain (CCCD), a STAN domain (Stevens et al., 2010a) that interacts with Sas-6 (Dzhindzhev et al., 2014; Ohta et al., 2014) and a short C-terminal CR4 domain (Figure 1A) (Cottee et al., 2013). To examine the potential function of these conserved regions, we synthesised mRNAs in vitro that contained either wild type (WT) or truncated versions of Ana2 fused to either an N- or C-terminal GFP (Figure 1A). These mRNAs were injected into WT early embryos (that contain unlabelled endogenous WT Ana2 protein) expressing RFP-Centrosomin (Cnn) as a centrosomal marker (Conduit et al., 2010). The localisation of the encoded GFP-fusion protein was assessed 90–120 min after mRNA injection (Figure 1B,C).10.7554/eLife.07236.003Figure 1.A structure/function analysis of Drosophila Ana2.


The homo-oligomerisation of both Sas-6 and Ana2 is required for efficient centriole assembly in flies.

Cottee MA, Muschalik N, Johnson S, Leveson J, Raff JW, Lea SM - Elife (2015)

A structure/function analysis of Drosophila Ana2.(A) A schematic representation of Drosophila Ana2 highlighting the conserved domains and illustrating the GFP constructs analysed in this study. In vitro transcribed mRNA encoding each of these constructs was injected into Drosophila embryos expressing the PCM marker, RFP-Cnn; the distribution of each fusion protein was analysed in living embryos. (B) Micrographs show examples of typical centrosomes in embryos injected with the Ana2 constructs shown in (A). The localisation of the GFP-fusion protein (green) is shown on its own (left panel) and merged with RFP-Cnn (right panel). (C) Bars quantify the localisation behaviour of the various GFP-fusions. Images of 30–80 embryos were analysed for each construct. Images of each embryo were collected and then manually sorted into various categories based on the centrosomal localisation of the GFP-fusion construct (see colour table at bottom of figure). All sorting was performed blind.DOI:http://dx.doi.org/10.7554/eLife.07236.003
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4471874&req=5

fig1: A structure/function analysis of Drosophila Ana2.(A) A schematic representation of Drosophila Ana2 highlighting the conserved domains and illustrating the GFP constructs analysed in this study. In vitro transcribed mRNA encoding each of these constructs was injected into Drosophila embryos expressing the PCM marker, RFP-Cnn; the distribution of each fusion protein was analysed in living embryos. (B) Micrographs show examples of typical centrosomes in embryos injected with the Ana2 constructs shown in (A). The localisation of the GFP-fusion protein (green) is shown on its own (left panel) and merged with RFP-Cnn (right panel). (C) Bars quantify the localisation behaviour of the various GFP-fusions. Images of 30–80 embryos were analysed for each construct. Images of each embryo were collected and then manually sorted into various categories based on the centrosomal localisation of the GFP-fusion construct (see colour table at bottom of figure). All sorting was performed blind.DOI:http://dx.doi.org/10.7554/eLife.07236.003
Mentions: The Drosophila Ana2 protein contains four regions that have significant homology to Ana2/STIL proteins from other species (Figures 1A, 2A) (Cottee et al., 2013). Fly Ana2 lacks the conserved region 1 found towards the N-terminus in vertebrate STIL proteins (Figure 2A), but contains a CR2 domain that interacts with Sas-4 (Cottee et al., 2013; Hatzopoulos et al., 2013), a predicted central coiled-coiled domain (CCCD), a STAN domain (Stevens et al., 2010a) that interacts with Sas-6 (Dzhindzhev et al., 2014; Ohta et al., 2014) and a short C-terminal CR4 domain (Figure 1A) (Cottee et al., 2013). To examine the potential function of these conserved regions, we synthesised mRNAs in vitro that contained either wild type (WT) or truncated versions of Ana2 fused to either an N- or C-terminal GFP (Figure 1A). These mRNAs were injected into WT early embryos (that contain unlabelled endogenous WT Ana2 protein) expressing RFP-Centrosomin (Cnn) as a centrosomal marker (Conduit et al., 2010). The localisation of the encoded GFP-fusion protein was assessed 90–120 min after mRNA injection (Figure 1B,C).10.7554/eLife.07236.003Figure 1.A structure/function analysis of Drosophila Ana2.

Bottom Line: We also solve the structure of the Drosophila Sas-6 N-terminal domain to 2.9 Å revealing that it forms higher-order oligomers through canonical interactions.Point mutations that perturb Sas-6 or Ana2 homo-oligomerisation in vitro strongly perturb centriole assembly in vivo.Thus, efficient centriole duplication in flies requires the homo-oligomerisation of both Sas-6 and Ana2, and the Ana2 CCCD tetramer structure provides important information on how these proteins might cooperate to form a cartwheel structure.

View Article: PubMed Central - PubMed

Affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Sas-6 and Ana2/STIL proteins are required for centriole duplication and the homo-oligomerisation properties of Sas-6 help establish the ninefold symmetry of the central cartwheel that initiates centriole assembly. Ana2/STIL proteins are poorly conserved, but they all contain a predicted Central Coiled-Coil Domain (CCCD). Here we show that the Drosophila Ana2 CCCD forms a tetramer, and we solve its structure to 0.8 Å, revealing that it adopts an unusual parallel-coil topology. We also solve the structure of the Drosophila Sas-6 N-terminal domain to 2.9 Å revealing that it forms higher-order oligomers through canonical interactions. Point mutations that perturb Sas-6 or Ana2 homo-oligomerisation in vitro strongly perturb centriole assembly in vivo. Thus, efficient centriole duplication in flies requires the homo-oligomerisation of both Sas-6 and Ana2, and the Ana2 CCCD tetramer structure provides important information on how these proteins might cooperate to form a cartwheel structure.

No MeSH data available.