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1.
EMBO Rep ; 25(1): 102-127, 2024 Jan.
Article in English | MEDLINE | ID: mdl-38200359

ABSTRACT

Centrioles are part of centrosomes and cilia, which are microtubule organising centres (MTOC) with diverse functions. Despite their stability, centrioles can disappear during differentiation, such as in oocytes, but little is known about the regulation of their structural integrity. Our previous research revealed that the pericentriolar material (PCM) that surrounds centrioles and its recruiter, Polo kinase, are downregulated in oogenesis and sufficient for maintaining both centrosome structural integrity and MTOC activity. We now show that the expression of specific components of the centriole cartwheel and wall, including ANA1/CEP295, is essential for maintaining centrosome integrity. We find that Polo kinase requires ANA1 to promote centriole stability in cultured cells and eggs. In addition, ANA1 expression prevents the loss of centrioles observed upon PCM-downregulation. However, the centrioles maintained by overexpressing and tethering ANA1 are inactive, unlike the MTOCs observed upon tethering Polo kinase. These findings demonstrate that several centriole components are needed to maintain centrosome structure. Our study also highlights that centrioles are more dynamic than previously believed, with their structural stability relying on the continuous expression of multiple components.


Subject(s)
Centrioles , Centrosome , Drosophila Proteins , Microtubule-Associated Proteins , Centrioles/metabolism , Centrosome/metabolism , Oocytes/metabolism , Oogenesis , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Animals , Drosophila melanogaster , Drosophila Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Humans
2.
Curr Biol ; 30(12): R687-R689, 2020 06 22.
Article in English | MEDLINE | ID: mdl-32574625

ABSTRACT

Pimento-Marques and Bettencourt-Dias discuss the composition, assembly and function of pericentriolar material - the proteinaceous material that surrounds the centrioles and forms the centrosome, the main microtubule organizing center found in animal cells.


Subject(s)
Centrosome/metabolism , Animals , Centrioles/metabolism , Microtubules/metabolism
3.
J Cell Sci ; 130(22): 3789-3800, 2017 Nov 15.
Article in English | MEDLINE | ID: mdl-29142065

ABSTRACT

Centrosomes and cilia are present in organisms from all branches of the eukaryotic tree of life. These structures are composed of microtubules and various other proteins, and are required for a plethora of cell processes such as structuring the cytoskeleton, sensing the environment, and motility. Deregulation of centrosome and cilium components leads to a wide range of diseases, some of which are incompatible with life. Centrosomes and cilia are thought to be very stable and can persist over long periods of time. However, these structures can disappear in certain developmental stages and diseases. Moreover, some centrosome and cilia components are quite dynamic. While a large body of knowledge has been produced regarding the biogenesis of these structures, little is known about how they are maintained. In this Review, we propose the existence of specific centrosome and cilia maintenance programs, which are regulated during development and homeostasis, and when deregulated can lead to disease.


Subject(s)
Centrosome/physiology , Cilia/physiology , Animals , Centrosome/ultrastructure , Cilia/ultrastructure , Homeostasis , Humans , Microtubules/metabolism , Protein Stability , Regeneration
4.
Curr Biol ; 23(22): 2245-2254, 2013 Nov 18.
Article in English | MEDLINE | ID: mdl-24184099

ABSTRACT

Polo-like kinase 4 (PLK4) is a major player in centriole biogenesis: in its absence centrioles fail to form, while in excess leads to centriole amplification. The SCF-Slimb/ßTrCP-E3 ubiquitin ligase controls PLK4 levels through recognition of a conserved phosphodegron. SCF-Slimb/ßTrCP substrate binding and targeting for degradation is normally regulated by phosphorylation cascades, controlling complex processes, such as circadian clocks and morphogenesis. Here, we show that PLK4 is a suicide kinase, autophosphorylating in residues that are critical for SCF-Slimb/ßTrCP binding. We demonstrate a multisite trans-autophosphorylation mechanism, likely to ensure that both a threshold of PLK4 concentration is attained and a sequence of events is observed before PLK4 can autodestruct. First, we show that PLK4 trans-autophosphorylates other PLK4 molecules on both Ser293 and Thr297 within the degron and that these residues contribute differently for PLK4 degradation, the first being critical and the second maximizing auto-destruction. Second, PLK4 trans-autophosphorylates a phospho-cluster outside the degron, which regulates Thr297 phosphorylation, PLK4 degradation, and centriole number. Finally, we show the importance of PLK4-Slimb/ßTrCP regulation as it operates in both soma and germline. As ßTrCP, PLK4, and centriole number are deregulated in several cancers, our work provides novel links between centriole number control and tumorigenesis.


Subject(s)
Centrioles/metabolism , Drosophila Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Animals , Animals, Genetically Modified , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Drosophila/genetics , Drosophila/metabolism , Drosophila Proteins/genetics , Female , Gene Expression Regulation , Male , Molecular Sequence Data , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Serine/metabolism , Threonine/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
5.
Development ; 139(3): 503-13, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22223679

ABSTRACT

Epithelial cells mostly orient the spindle along the plane of the epithelium (planar orientation) for mitosis to produce two identical daughter cells. The correct orientation of the spindle relies on the interaction between cortical polarity components and astral microtubules. Recent studies in mammalian tissue culture cells suggest that the apically localised atypical protein kinase C (aPKC) is important for the planar orientation of the mitotic spindle in dividing epithelial cells. Yet, in chicken neuroepithelial cells, aPKC is not required in vivo for spindle orientation, and it has been proposed that the polarization cues vary between different epithelial cell types and/or developmental processes. In order to investigate whether Drosophila aPKC is required for spindle orientation during symmetric division of epithelial cells, we took advantage of a previously isolated temperature-sensitive allele of aPKC. We showed that Drosophila aPKC is required in vivo for spindle planar orientation and apical exclusion of Pins (Raps). This suggests that the cortical cues necessary for spindle orientation are not only conserved between Drosophila and mammalian cells, but are also similar to those required for spindle apicobasal orientation during asymmetric cell division.


Subject(s)
Cell Division , Drosophila Proteins/metabolism , Drosophila melanogaster/enzymology , Protein Kinase C/metabolism , Spindle Apparatus/enzymology , Animals , Cell Cycle Proteins , Cell Polarity , Drosophila Proteins/genetics , Drosophila melanogaster/cytology , Epithelial Cells/metabolism , Female , Guanine Nucleotide Dissociation Inhibitors/metabolism , Male , Mutation , Protein Kinase C/genetics
6.
PLoS Genet ; 7(7): e1002169, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21750686

ABSTRACT

N-terminal acetylation (N-Ac) is a highly abundant eukaryotic protein modification. Proteomics revealed a significant increase in the occurrence of N-Ac from lower to higher eukaryotes, but evidence explaining the underlying molecular mechanism(s) is currently lacking. We first analysed protein N-termini and their acetylation degrees, suggesting that evolution of substrates is not a major cause for the evolutionary shift in N-Ac. Further, we investigated the presence of putative N-terminal acetyltransferases (NATs) in higher eukaryotes. The purified recombinant human and Drosophila homologues of a novel NAT candidate was subjected to in vitro peptide library acetylation assays. This provided evidence for its NAT activity targeting Met-Lys- and other Met-starting protein N-termini, and the enzyme was termed Naa60p and its activity NatF. Its in vivo activity was investigated by ectopically expressing human Naa60p in yeast followed by N-terminal COFRADIC analyses. hNaa60p acetylated distinct Met-starting yeast protein N-termini and increased general acetylation levels, thereby altering yeast in vivo acetylation patterns towards those of higher eukaryotes. Further, its activity in human cells was verified by overexpression and knockdown of hNAA60 followed by N-terminal COFRADIC. NatF's cellular impact was demonstrated in Drosophila cells where NAA60 knockdown induced chromosomal segregation defects. In summary, our study revealed a novel major protein modifier contributing to the evolution of N-Ac, redundancy among NATs, and an essential regulator of normal chromosome segregation. With the characterization of NatF, the co-translational N-Ac machinery appears complete since all the major substrate groups in eukaryotes are accounted for.


Subject(s)
Acetyltransferases , Chromosome Segregation/physiology , Drosophila Proteins/metabolism , Fungal Proteins/metabolism , Protein Processing, Post-Translational , Proteomics/methods , Acetylation , Acetyltransferases/genetics , Acetyltransferases/metabolism , Animals , Drosophila Proteins/genetics , Drosophila melanogaster/enzymology , Drosophila melanogaster/genetics , Evolution, Molecular , Fungal Proteins/genetics , Humans , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Substrate Specificity
7.
Dev Biol ; 323(2): 197-206, 2008 Nov 15.
Article in English | MEDLINE | ID: mdl-18801358

ABSTRACT

During mitosis different types of cells can have differential requirements for chromosome segregation. We isolated two new alleles of the separation anxiety gene (san). san was previously described in both Drosophila and in humans to be required for centromeric sister chromatid cohesion (Hou et al., 2007; Williams et al., 2003). Our work confirms and expands the observation that san is required in vivo for normal mitosis of different types of somatic cells. In addition, we suggest that san is also important for the correct resolution of chromosomes, implying a more general function of this acetyltransferase. Surprisingly, during oogenesis we cannot detect mitotic defects in germ line cells mutant for san. We hypothesize the female germ line stem cells have differential requirements for mitotic sister chromatid cohesion.


Subject(s)
Acetyltransferases/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/cytology , Drosophila melanogaster/enzymology , Germ Cells/cytology , Germ Cells/enzymology , Mitosis , Alleles , Animals , Blastoderm/cytology , Blastoderm/enzymology , Chromosome Segregation , Chromosomes/enzymology , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Genes, Insect , Larva/cytology , Larva/enzymology , Neurons/cytology , Neurons/enzymology , Oogenesis , Sister Chromatid Exchange , Zygote/cytology , Zygote/enzymology
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