Drosophila SPD-2 Is an Essential Centriole Component Required for PCM Recruitment and Astral-Microtubule Nucleation

Abstract

SPD-2 is a C. elegans centriolar protein required for both centriole duplication and pericentriolar material (PCM) recruitment [1-4]. SPD-2 is conserved in Drosophila (DSpd-2) and is a component of the fly centriole [5-7]. The analysis of a P element-induced hypomorphic mutation has shown that DSpd-2 is primarily required for PCM recruitment at the sperm centriole but is dispensable for both centriole duplication and aster formation [5]. Here we show that null mutations carrying early stop codons in the DSpd-2 coding sequence suppress astral microtubule (MT) nucleation in both neuroblasts (NBs) and spermatocytes. These mutations also disrupt proper Miranda localization in dividing NBs, as previously observed in mutants lacking astral MTs [8-10]. Spermatocyte analysis revealed that DSpd-2 is enriched at both the centrioles and the PCM and is required for the maintenance of cohesion between the two centrioles but not for centriole duplication. We found that DSpd-2 localization at the centrosome requires the wild-type activity of Asl but is independent of the function of D-PLP, Cnn, gamma-tubulin, DGrip91, and D-TACC. Conversely, DSpd-2 mutants displayed normal centrosomal accumulations of Asl and D-PLP, strongly reduced amounts of Cnn, gamma-tubulin, and DGrip91, and diffuse localization of D-TACC. These results indicate that DSpd-2 functions in a very early step of the PCM recruitment pathway.

The Centrosome and Its Duplication Cycle

“…This echoes the finding of the dependency of ZYG-1 recruitment on SPD-2 in C. elegans. However, in Drosophila, Spd-2 barely plays any role in centriole assembly but is instead required for PCM recruitment on mitotic entry (Dix and Raff 2007;Giansanti et al 2008). In Planarians where the canonical centriole duplication pathway was abandoned during evolution and centrioles are only assembled in terminally differentiating ciliated cells through an acentriolar pathway, Spd-2/Cep192 along with CNN/CDK5RAP2 and Nek2 are all absent from the genome (Azimzadeh et al 2012).…”

DrosophilaBld10 Is a Centriolar Protein That Regulates Centriole, Basal Body, and Motile Cilium Assembly

“…Slides were incubated overnight at 4°C with the following primary antibodies diluted in PBS: mouse anti-␥-tubulin clone GTU88 (Sigma; 1:1000), mouse anti-␣-tubulin antibody (clone DM1A, Sigma, 1:1000), rabbit affinity-purified anti-Bld10 UT530 or UT530 antibodies (1:1000), anti-Cnn (1:1000), rabbit anti-SPD-2 (1:1000; Giansanti et al, 2008); mouse anti-green fluorescent protein (GFP; Invitrogen, Carlsbad, CA, 1:250). Secondary antibody conjugates to Alexa 488 or 546 (Invitrogen) were used at 1:400 dilution.…”

“…Mutations in genes encoding centriolar and basal body proteins often lead to defects in the biogenesis of centrioles and in the assembly of functional centrosomes at mitosis, loss of locomotion due to impaired basal bodies of ciliated neurons, and immotile sperm due to axoneme dysfunction Martinez-Campos et al, 2004;Bettencourt-Dias et al, 2005;Basto et al, 2006;Rodrigues-Martins et al, 2007;Varmark et al, 2007;Blachon et al, 2008;Giansanti et al, 2008).…”

“…In flies, mutations in these genes abolish centrosome and cilium/flagellum assembly (except mutations in ana1 and ana2, which have not been described yet). Additional centriole/ basal body proteins including Spd-2, Drosophila pericentrin-like protein (D-PLP)/CP309, and uncoordinated (UNC) function in pericentriolar material (PCM) recruitment to centrosomes and/or the assembly of cilia and flagella Kawaguchi and Zheng, 2004;Martinez-Campos et al, 2004;Dix and Raff, 2007;Giansanti et al, 2008). Identification of the complete set of centriole components is necessary to define their individual and cooperative roles in the assembly and function of centrioles and cilia.…”

50 ways to build a spindle: the complexity of microtubule generation during mitosis

“…In flies, where the pathway of centrosome maturation has been more extensively studied, the Pericentrin-like protein (D-PLP) shares this role with centrosomin (Cnn; Martinez-Campos et al 2004). The localisation of both these PCM proteins is dependent upon the presence of the core centriolar component D-Spd2 (Dix and Raff 2007;Giansanti et al 2008), whilst the phosphorylation of Cnn by the mitotic kinase, Polo, correlates with centrosome maturation (Dobbelaere et al 2008), suggesting a hierarchical pathway in which γ-tubulin is targeted and maintained at the PCM in a stepwise fashion.…”