2014 Abstracts

Dzhindzhev, NS; Tzolovsky, G; Lipinszki, Z; Schneider, S; Lattao, R; Fu, J; Debski, J; Dadlez, M; Glover, DM

Plk4 Phosphorylates Ana2 to Trigger Sas6 Recruitment and Procentriole Formation.

Centrioles are 9-fold symmetrical structures at the core of centrosomes and base of cilia whose dysfunction has been linked to a wide range of inherited diseases and cancer [1]. Their duplication is regulated by a protein kinase of conserved structure, the C. elegans ZYG-1 or its Polo-like kinase 4 (Plk4) counterpart in other organisms [2-4]. Although Plk4's centriolar partners and mechanisms that regulate its stability are known, its crucial substrates for centriole duplication have never been identified. Here we show that Drosophila Plk4 phosphorylates four conserved serines in the STAN motif of the core centriole protein Ana2 to enable it to bind and recruit its Sas6 partner. Ana2 and Sas6 normally load onto both mother and daughter centrioles immediately after their disengagement toward the end of mitosis to seed procentriole formation. Nonphosphorylatable Ana2 still localizes to the centriole but can no longer recruit Sas6 and centriole duplication fails. Thus, following centriole disengagement, recruitment of Ana2 and its phosphorylation by Plk4 are the earliest known events in centriole duplication to recruit Sas6 and thereby establish the architecture of the new procentriole engaged with its parent

Current Biology Sep 24. pii: S0960-9822(14)01074-4. doi: 10.1016/j.cub.2014.08.061

Alqarni, SS; Murthy, A; Zhang, W; Przewloka, MR; Silva, AP; Watson, AA; Lejon, S; Pei, XY; Smits, AH; Kloet, SL; Wang, H; Shepherd, NE; Stokes, PH; Blobel, GA; Vermeulen, M; Glover, DM; Mackay, JP; La

Insight into the architecture of the NuRD complex: Structure of the RbAp48-MTA1 sub-complex

The Nucleosome Remodeling and Deacetylase (NuRD) complex is a widely conserved transcriptional co-regulator that harbors both nucleosome remodeling and histone deacetylase activities. It plays a critical role in the early stages of ES cell differentiation and the reprogramming of somatic to induced pluripotent stem cells. Abnormalities in several NuRD proteins are associated with cancer and ageing. We have investigated the architecture of NuRD by determining the structure of a sub-complex comprising RbAp48 and MTA1. Surprisingly, RbAp48 recognizes MTA1 using the same site that it uses to bind histone H4, showing that assembly into NuRD modulates RbAp46/48 interactions with histones. Taken together with other results, our data shows that the MTA proteins act as scaffolds for NuRD complex assembly. We further show that the RbAp48-MTA1 interaction is essential for the in vivo integration of RbAp46/48 into the NuRD complex.

J. Biol. Chem. vol. 289(32) pp. 21844-55

Lipinszki, Z; Wang, P; Grant, R; Lindon, C; Dzhindzhev, NS; D'Avino, PP; Przewloka, MR; Glover, DM; Archambault, V

Affinity Purification of Protein Complexes from Drosophila Embryos in Cell Cycle Studies

The ability to identify protein interactions is key to elucidating the molecular mechanisms of cellular processes, including mitosis and cell cycle regulation. Drosophila melanogaster, as a model system, provides powerful tools to study cell division using genetics, microscopy, and RNAi. Drosophila early embryos are highly enriched in mitotic protein complexes as their nuclei undergo 13 rounds of rapid, synchronous mitotic nuclear divisions in a syncytium during the first 2 h of development. Here, we describe simple methods for the affinity purification of protein complexes from transgenic fly embryos via protein A- and green fluorescent protein-tags fused to bait proteins of interest. This in vivo proteomics approach has allowed the identification of several known and novel mitotic protein interactions using mass spectrometry, and it expands the use of the Drosophila model in modern molecular biology.

Methods in Molecular Biology 1170:571-588

Savoian, MS; Glover, DM

Differing requirements for Augmin in male meiotic and mitotic spindle formation in Drosophila

Animal cells divide using a microtubule-based, bipolar spindle. Both somatic, mitotic cells and sperm-producing male meiotic spermatocytes use centrosome-dependent and acentrosomal spindle-forming mechanisms. Here, we characterize the largely undefined, centrosome-independent spindle formation pathway used during male meiosis. Our live and fixed cell analyses of Drosophila spermatocytes reveal that acentrosomal microtubules are nucleated at kinetochores and in the vicinity of chromatin and that together these assemble into functional spindles. Mutational studies indicate that γ-tubulin and its extra-centrosomal targeting complex, Augmin, are vital for this process. In addition, Augmin facilitates efficient spindle assembly in the presence of centrosomes. In contrast to the pronounced recruitment of Augmin on spindles in other cell types, the complex is absent from those of spermatocytes but does accumulate on kinetochores. Polo kinase facilitates this kinetochore recruitment while inhibiting Augmin's spindle association, and this in turn dictates γ-tubulin distribution and spindle density. Polo's negative regulation of Augmin in male meiosis contrasts with its requirement in loading Augmin along mitotic spindles in somatic Drosophila cells. Together our data identify a novel mechanism of acentrosomal spindle formation in spermatocytes and reveal its divergence from that used in mitotic cells.

Open Biology vol. 4(5), PMID: 24829288

Riparbelli, MG; Gottardo, M; Glover, DM; Callaini, G

Inhibition of Polo kinase by BI2536 affects centriole separation during Drosophila male meiosis

Pharmacological inhibition of Drosophila Polo kinase with BI2536 has allowed us to re-examine the requirements for Polo during Drosophila male gametogenesis. BI2536-treated spermatocytes persisted in a pro-metaphase state without dividing and had condensed chromosomes that did not separate. Centrosomes failed to recruit γ-tubulin and centrosomin (Cnn) and were not associated with microtubule arrays that were abnormal and did not form proper bipolar spindles. Centrioles, which usually separate during the anaphase of the first meiosis, remained held together in a V-shaped configuration suggesting that Polo kinase regulates the proteolysis that breaks centriole linkage to ensure their disengagement. Despite these defects spermatid differentiation proceeds, leading to axoneme formation.

Cell Cycle vol. 13(13), PMID: 24802643


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