- Email: [email protected]
Double life of centrioles: CP110 in the spotlight
Update 15 Martinon, F. et al. (2004) Identification of bacterial muramyl dipeptide as activator of the NALP3/cryopyrin inflammasome. Curr. Biol. 14, 1929–1934 16 Mariathasan, S. et al. (2006) Cryopyrin activates the inflammasome in response to toxins and ATP. Nature 440, 228–232 17 Martinon, F. et al. (2006) Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 440, 237–241 18 Pe´trilli, V. et al. (2007) Activation of the NALP3 inflammasome is triggered by low intracellular potassium concentration. Cell Death Differ. 14, 1583–1589 19 Mariathasan, S. et al. (2004) Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 430, 213–218 20 Solle, M. et al. (2001) Altered cytokine production in mice lacking P2X(7) receptors. J. Biol. Chem. 276, 125–132 21 Pelegrin, P. and Surprenant, A. (2006) Pannexin-1 mediates large pore formation and interleukin-1b release by the ATP-gated P2X7 receptor. EMBO J. 25, 5071–5082
TRENDS in Cell Biology Vol.18 No.1 22 Munding, C. et al. (2006) The estrogen-responsive B box protein: a novel enhancer of interleukin-1b secretion. Cell Death Differ. 13, 1938– 1949 23 Reed, J.C. et al. (2003) Comparative analysis of apoptosis and inflammation genes of mice and humans. Genome Res. 13 (6B), 1376–1388 24 Boyden, E.D. and Dietrich, W.F. (2006) Nalp1b controls mouse macrophage susceptibility to anthrax lethal toxin. Nat. Genet. 38, 240–244 25 Enk, A.H. and Katz, S.I. (1992) Early molecular events in the induction phase of contact sensitivity. Proc. Natl. Acad. Sci. U. S. A. 89, 1398–1402 26 Sutterwala, F.S. et al. (2006) Critical role for NALP3/CIAS1/cryopyrin in innate and adaptive immunity through its regulation of caspase-1. Immunity 24, 317–327 27 Bruey, J.M. et al. (2007) Bcl-2 and Bcl-XL regulate proinflammatory caspase-1 activation by interaction with NALP1. Cell 129, 45–56 0962-8924/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2007.10.004 Available online 20 December 2007
Research Focus
Double life of centrioles: CP110 in the spotlight Mo´nica Bettencourt-Dias and Zita Carvalho-Santos Cell Cycle Regulation Lab, Instituto Gulbenkian de Cieˆncia, Rua da Quinta Grande, 6, P-2780-156 Oeiras, Portugal
Centrioles lead an important double life: they can give rise to the centrosome or convert to basal bodies and template cilia. Little is known about the control of centriole fate. Spektor and colleagues have now identified a centriolar complex, composed of CP110 and CEP97, which inhibits centriole to basal body conversion, preventing cilia formation. This work paves the way to understanding centriole and cilia biogenesis, which are two processes misregulated in human diseases, such as cancer and polycystic kidney disease.
The centriole or basal body double life Centrioles (see Glossary) are essential for the formation of several microtubule (MT)-organizing structures, including centrosomes, cilia and flagella (reviewed in [1,2]). Two centrioles associate with pericentriolar material (PCM) to form a centrosome, which is the major microtubuleorganizing center in animal cells. In interphase or quiescent ciliated cells, at least one centriole, called the basal body, is tethered to the membrane, where it grows the axoneme, the MT-based structure of cilia and flagella. Most known animal cycling cells resorb their cilia on cell-cycle re-entry in G1 or before entry into mitosis, after which the centriole leaves the membrane and is part of the interphase or mitotic centrosome [3,4] (Figure 1). On mitotic exit, the centriole is converted to a basal body that migrates to the membrane to form cilia [4,5]. Molecular phylogeny suggests the centriole or basal body was associated with a flagellum in the early eukaryotic cell [6]. This duality of the centriole as a basal body is therefore inbuilt into the cellular and developmental program of those structures [7] (Figure 1A,B). Fertilization is a great Corresponding author: Bettencourt-Dias, M. ([email protected]).
8
example of this duality. The flagellated sperm provides the basal body to the egg that lost its centrioles during oogenesis. Within the egg, the basal body of the sperm will duplicate and recruit PCM, giving rise to centrosomes, which are essential for embryonic development [8,9]. Little is known about the molecular mechanisms governing the switch from a centriole to a basal body and vice versa. Recently, Spektor and colleagues found new clues, showing that CP110 and its regulator CEP97 have an important role in inhibiting basal body fate [10]. Here, we discuss that result and its implications in the light of recent findings on centriole and cilia biogenesis. CP110 teams up with CEP97 in cycling cells CP110 is a coiled-coil protein necessary for centriole duplication [11,12]. Its presence at the distal end of growing centrioles suggests that it regulates centriole elongation [11]. In a search for CP110-binding partners, Spektor and colleagues used immunoaffinity purification of Flag-tagged CP110 protein complexes followed by mass-spectrometry sequencing and found several components of the human centrosome proteome [10,13]. Among those centrosome components was leucine-rich repeats and IQ motif-containing 2 (LRRIQ2), which the authors renamed Cep97 because of its size and localization at the centriole [10]. The CEP97– CP110 interaction does not depend on calmodulin, a molecule that binds both CP110 and CEP97 [10,14]. Furthermore, this interaction is likely to be direct, based on in vitro translation and immunoprecipitation [10]. What is the relevance of this complex? Spektor and colleagues showed that the localization of CEP97 depends strongly on CP110, suggesting recruitment by this molecule [10]. However, CEP97 is not important for centriole duplication, although it is essential for CP110 stability [10], suggesting a role in preventing its degradation at a
Update Glossary Anaphase-promoting complex/Cyclosome (APC/C): Proteolysis by ubiquitin and the 26S proteasome pathway is a fundamental mechanism for protein degradation. The APC is an ubiquitin ligase responsible for ubiquitination of proteins at the metaphase–anaphase and mitosis–G1 transitions. Two major degradation motifs have been identified in the protein sequence of APC substrates: destruction box (D-box) and another destruction motif called KEN box. Axoneme: The microtubule-based structure of cilia and flagella. It gives those structures their rigidity and ability to move. It is cylindrical and comprised of nine pairs of doublet microtubules, around a central pair of single microtubules (9C2). The central microtubules might be absent in non-motile cilia (9C0). Axoneme microtubules are very stable. Basal body: Structure found at the base of eukaryotic cilia and flagella that organizes the assembly of the axoneme. The structure of the basal body is the same as the one of the centriole. Additionally, it has a transition zone at the distal end, contiguous with the axoneme. Axoneme microtubules are templated from the distal part of the basal body. Centriole: The canonical centriole is a cylinder comprised of nine microtubule triplets. It is approximately 0.5 mm long and shows appendages at the distal ends on maturation. There are variations of this structure, in which triplets are substituted by singlets or doublets, such as in C. elegans and D. melanogaster. When the centriole is tethered to the membrane, it is called a ‘basal body’. In a centriole or basal body pair within the centrosome or tethered to the membrane, the oldest centriole or basal body is referred to as the ‘mother’, whereas the youngest is referred to as the ‘daughter’. Centrosome: The centrosome is the primary microtubule-organizing centre (MTOC) in animal cells. It regulates cell motility, adhesion and polarity in interphase and facilitates the organization of the spindle poles during mitosis. It is comprised of two centrioles surrounded by an electron-dense matrix, the pericentriolar material (PCM). Cilia and flagella: Cilia and flagella are projections from cells that either enable movement of the cell itself or facilitate movement or sensing of substances around cells. Evidence has grown for their indispensable role in a variety of cellular and developmental processes: motility, propagation of morphogenetic signals in embryogenesis and sensory perception (reviewed in [20]). The majority of the cells in vertebrates have cilia that act as sensory organelles or can move fluids around the cell. Flagella help to propel cells. Microtubule: A hollow tube, 25 nm in diameter, formed by the lateral association of 13 protofilaments. Each protofilament is a polymer of a- and b-tubulin subunits. Pericentriolar material: Fibrillar material surrounding centrioles in the centrosome that nucleates the growth of new microtubules.
phase where CP110 is not required for centriole formation. Indeed, the levels of both molecules show evidence of cellcycle regulation because they are low in quiescent cells and go up on cell-cycle entry [10,12]. Additionally, CP110 protein has destruction boxes, suggesting degradation on mitotic exit [12] (Figure 1A). CEP97 and CP110 block cilia formation The most striking observation of Spektor and colleagues was made in U2OS cells, which do not form cilia normally. They observed that, after depletion of Cep97 or CP110 by RNAi, 30% of the cells showed centrin fibers emanating from the centrioles, which were similar to cilia. These fibers showed cilia markers, such as polycystin 2 and polyglutamylated and acetylated tubulin [10]. RNAi of CP110 or CEP97 in ciliated cells led to a twofold increase in cilia formation, suggesting these proteins might prevent cilia formation normally. This result is unlikely to reflect forced quiescence because depletion of CP110 and CEP97 did not lead to cell-cycle arrest [10,12]. Moreover, overexpression of CP110 in quiescent cilia-prone 3T3 cells abrogated cilia formation. Together, these data suggest strongly that CP110 at the centrioles inhibits their ability to form cilia. Indeed, in ciliated NIH 3T3 cells, CP110 localizes to centrioles but not to the basal body at the base of the cilium [10].
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Intriguingly, depletion of CP110 or CEP97 or two other CP110 interactors (calmodulin and centrin) leads to mitotic-spindle abnormalities and an increase in cytokinetic failure [10,14]. In certain human cell lines, the mother centriole moves towards the mid-body at the end of telophase and this movement coincides with abscission, the event that separates the two cells [15,16]. It is possible that the absence of those molecules impairs the function of the centriole or creates artifacts in cytokinesis, which might or might not be associated with cilia formation. Alternatively, those phenotypes might reflect a centriole-independent function of those molecules, which is yet to be explored. To be or not to be a basal body: decisions in a centriole’s life If CP110 truly prevents centrioles from forming cilia, the question that naturally arises is how does it do so? Does it prevent centrioles from recruiting molecules required for appendage and cilia formation (Figure 1A,B), hence preventing movement to or attachment to the cytoplasmic membrane? Or is CP110 involved in inhibiting the first steps in axoneme formation? Given that overexpression of CP110 inhibits normal cilia formation, the probable explanation is that it prevents centriole or basal body movement or attachment to the membrane (Figure 1B). Centrioles and basal bodies show clearly distinct features: they nucleate and anchor different MT (centrosomal versus axonemal MT); additionally basal bodies show a transition zone at the distal tip. A crucial step to elucidate CP110 activity will be to better describe the consequences of its depletion and overexpression: what happens to the ultrastructure of the centriole or basal body and the composition of the MTs it nucleates? Where is the centriole or basal body localized in the cell in relation to the centrosome and the plasma membrane? Understanding the regulation of CP110 is crucial to shedding light on the cell-cycle regulation of cilia formation. First, what targets it to the centriole? CP110 interacts with centrin [12,14], an early centriole marker, which, like CP110, also localizes to the distal part of centrioles [11,17]. Although centrin is at the right place and time to recruit CP110, CP110 localization is not dependent on centrin [11]. Second, what removes CP110 from the mother centriole to enable cilia formation in G0/G1? Spektor and colleagues suggest that CEP97 is recruited by CP110 and stabilizes it [10]. One possibility is that CP110 is degraded in the mother centriole at mitotic exit because it has destruction motifs for the APC/C [12] (Figure 1B). Further experiments are required to investigate the role of the APC/C and CEP97 in CP110 stability. In any case, removal of CP110 cannot be the only trigger for cilia formation because, in the majority of cells, only one of the centrioles formed cilia after CP110 depletion [10]. It is possible that other molecules required for cilliogenesis, such as the outer dense fibre 2 (ODF2), which associates exclusively with the mother centrioles, confer the selectively for this centriole to form cilia [1,2,18] (Figure 1A,B). Finally, what happens on mitotic entry (Figure 1F)? Does the basal body incorporate CP110 de novo to prevent cilia formation? Analysis of CP110 localization throughout the cell cycle should be informative. 9
Update
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Figure 1. Model of the regulation of cilia formation by CP110 in cycling and quiescent cells. (A) When a cell enters a new cycle it only has one centrosome, composed of a mother and a daughter centriole. The mother centriole matures on mitotic entry, recruiting molecules that enable it to form appendages, such as ODF2 (red triangle) [18,19]. It is possible that upon mitotic exit, CEP97 (orange circle) is degraded or removed from centrioles leading to destabilization of CP110 at the mother centriole (blue square). It has been suggested that CP110 might be degraded by the APC/C [12]. (B) In G1, the absence of CP110 and presence of ODF2 specifically in the mother centriole enables it to become a basal body, tethering at the membrane where it forms an axoneme [10,17]. (C) If nutrients are removed, cells can exit the cell cycle and become quiescent (G0). At this stage, the cilium can grow further [20]. (D) On serum stimulation, cells re-enter the cell cycle and the cilia are reabsorbed. (E) The centrioles or basal bodies duplicate in S phase. CP110 is necessary in the daughter centrioles for their biogenesis [11,12]. (F) On centrosome maturation at the G2/M transition, the cilium is reabsorbed and centrioles move to the center to participate in mitotic-spindle assembly [3]. The mitotic kinase Aurora A (pink oval) activates tubulin deacetylase, hence promoting ciliary disassembly and internalization of the basal body [4].
Recently, it was shown that the mitotic kinase Aurora A acts on the conversion of the basal body to centriole by activating tubulin deacetylase, hence promoting ciliary disassembly and internalization of the basal body, both at G1/S and mitotic entry [4] (Figure 1F). These studies show that research into the centriole to basal body interconversion has just started. Aurora A and CP110 have entered center stage in this exciting new field that promises to unravel the secrets of this mysterious and ubiquitously important structure: the cilia. Acknowledgements We thank A Rodrigues-Martins, J Pereira-Leal, J Lamego and L Sau´de for criticisms and comments on this manuscript. Work in the MBD laboratory is funded by Fundac¸a˜o Calouste Gulbenkian and Fundac¸a˜o para a Cieˆncia e Tecnologia.
References 1 Bornens, M. (2002) Centrosome composition and microtubule anchoring mechanisms. Curr. Opin. Cell Biol. 14, 25–34 2 Bettencourt-Dias, M. and Glover, D.M. (2007) Centrosome biogenesis and function: centrosomics brings new understanding. Nat. Rev. Mol. Cell Biol. 8, 451–463 3 Rieder, C.L. et al. (1979) The resorption of primary cilia during mitosis in a vertebrate (PtK1) cell line. J. Ultrastruct. Res. 68, 173–185 4 Pugacheva, E.N. et al. (2007) HEF1-dependent Aurora A activation induces disassembly of the primary cilium. Cell 129, 1351–1363 5 Dutcher, S.K. (2004) Dissection of basal body and centriole function in the unicellular green alga Chlamydomonas reinhardtii. In Centrosomes in Development and Disease (Nigg, E.A., ed.), Wiley-VCH
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6 Cavalier-Smith, T. (2002) The phagotrophic origin of eukaryotes and phylogenetic classification of Protozoa. Int. J. Syst. Evol. Microbiol. 52, 297–354 7 Azimzadeh, J. and Bornens, M. (2004) The centrosome in evolution. In Centrosomes in Development and Disease (Nigg, E.A., ed.), pp. 93–122, Wiley-VCH 8 Stevens, N.R. et al. (2007) From stem cell to embryo without centrioles. Curr. Biol. 17, 1498–1503 9 Pelletier, L. et al. (2004) The C. elegans centrosome during early embryonic development. In Centrosomes in Development and Disease (Nigg, E.A., ed.), Wiley-VCH 10 Spektor, A. et al. (2007) Cep97 and CP110 suppress a cilia assembly program. Cell 130, 678–690 11 Kleylein-Sohn, J. et al. (2007) Plk4-induced centriole biogenesis in human cells. Dev. Cell 13, 190–202 12 Chen, Z. et al. (2002) CP110, a cell cycle-dependent CDK substrate, regulates centrosome duplication in human cells. Dev. Cell 3, 339– 350 13 Andersen, J.S. et al. (2003) Proteomic characterization of the human centrosome by protein correlation profiling. Nature 426, 570–574 14 Tsang, W.Y. et al. (2006) CP110 cooperates with two calcium-binding proteins to regulate cytokinesis and genome stability. Mol. Biol. Cell 17, 3423–3434 15 Piel, M. et al. (2000) The respective contributions of the mother and daughter centrioles to centrosome activity and behavior in vertebrate cells. J. Cell Biol. 149, 317–330 16 Piel, M. et al. (2001) Centrosome-dependent exit of cytokinesis in animal cells. Science 291, 1550–1553 17 Laoukili, J. et al. (2000) Differential expression and cellular distribution of centrin isoforms during human ciliated cell differentiation in vitro. J. Cell Sci. 113, 1355–1364
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18 Ishikawa, H. et al. (2005) Odf2-deficient mother centrioles lack distal/ subdistal appendages and the ability to generate primary cilia. Nat. Cell Biol. 7, 517–524 19 Lange, B.M. and Gull, K. (1995) A molecular marker for centriole maturation in the mammalian cell cycle. J. Cell Biol. 130, 919–927
20 Badano, J.L. et al. (2005) The centrosome in human genetic disease. Nat. Rev. Genet. 6, 194–205 0962-8924/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2007.11.002
Corrigendum
Corrigendum: Shedding light on Merlin’s wizardry Trends in Cell Biology 17 (2007), 222–229
In this article by Tomoyo Okadaa, Liru Youa and Filippo G. Giancotti, published in the May 2007 issue of TCB there was an incorrect statement in the second to last sentence of the abstract regarding the relationship between the proteins Merlin, Expanded and Hippo. The authors would like to clarify that in Drosophila, Merlin functions together with the band 4.1 protein Expanded to promote the endocytosis of many signaling receptors, limiting their accumulation at the plasma membrane, and to activate the Hippo signaling pathway. The corrected abstract should read as follows: Inactivation of the tumor suppressor Merlin, encoded by the NF2 (Neurofibromatosis type 2) gene, contributes to malignant conversion in many cell types. Merlin is an Ezrin–Radixin–Moesin protein and localizes underneath the plasma membrane at cell–cell junctions and other actin-rich sites. Recent studies indicate that Merlin mediates contact inhibition of proliferation by blocking
recruitment of Rac to the plasma membrane. In mitogenstimulated cells, p21-activated kinase phosphorylates Ser518 in the C-terminus of Merlin, inactivating the growth suppressive function of the protein. Furthermore, the myosin phosphatase MYPT1–PP1d, has been identified as a direct activator of Merlin and its inhibition has been linked to malignant transformation. Finally, studies in the fruit fly Drosophila melanogaster have revealed that Merlin functions together with the band 4.1 protein Expanded to promote the endocytosis of many signaling receptors, limiting their accumulation at the plasma membrane, and to activate the Hippo signaling pathway. Here, we review these recent findings and their relevance to the tumor suppressor function of Merlin. The authors and Trends in Cell Biology apologize for this error. 0962-8924/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2007.11.001 Available online 26 November 2007
DOI of original article: 10.1016/j.tcb.2007.03.006
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TRENDS in Cell Biology Vol.18 No.1 22 Munding, C. et al. (2006) The estrogen-responsive B box protein: a novel enhancer of interleukin-1b secretion. Cell Death Differ. 13, 1938– 1949 23 Reed, J.C. et al. (2003) Comparative analysis of apoptosis and inflammation genes of mice and humans. Genome Res. 13 (6B), 1376–1388 24 Boyden, E.D. and Dietrich, W.F. (2006) Nalp1b controls mouse macrophage susceptibility to anthrax lethal toxin. Nat. Genet. 38, 240–244 25 Enk, A.H. and Katz, S.I. (1992) Early molecular events in the induction phase of contact sensitivity. Proc. Natl. Acad. Sci. U. S. A. 89, 1398–1402 26 Sutterwala, F.S. et al. (2006) Critical role for NALP3/CIAS1/cryopyrin in innate and adaptive immunity through its regulation of caspase-1. Immunity 24, 317–327 27 Bruey, J.M. et al. (2007) Bcl-2 and Bcl-XL regulate proinflammatory caspase-1 activation by interaction with NALP1. Cell 129, 45–56 0962-8924/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2007.10.004 Available online 20 December 2007
Research Focus
Double life of centrioles: CP110 in the spotlight Mo´nica Bettencourt-Dias and Zita Carvalho-Santos Cell Cycle Regulation Lab, Instituto Gulbenkian de Cieˆncia, Rua da Quinta Grande, 6, P-2780-156 Oeiras, Portugal
Centrioles lead an important double life: they can give rise to the centrosome or convert to basal bodies and template cilia. Little is known about the control of centriole fate. Spektor and colleagues have now identified a centriolar complex, composed of CP110 and CEP97, which inhibits centriole to basal body conversion, preventing cilia formation. This work paves the way to understanding centriole and cilia biogenesis, which are two processes misregulated in human diseases, such as cancer and polycystic kidney disease.
The centriole or basal body double life Centrioles (see Glossary) are essential for the formation of several microtubule (MT)-organizing structures, including centrosomes, cilia and flagella (reviewed in [1,2]). Two centrioles associate with pericentriolar material (PCM) to form a centrosome, which is the major microtubuleorganizing center in animal cells. In interphase or quiescent ciliated cells, at least one centriole, called the basal body, is tethered to the membrane, where it grows the axoneme, the MT-based structure of cilia and flagella. Most known animal cycling cells resorb their cilia on cell-cycle re-entry in G1 or before entry into mitosis, after which the centriole leaves the membrane and is part of the interphase or mitotic centrosome [3,4] (Figure 1). On mitotic exit, the centriole is converted to a basal body that migrates to the membrane to form cilia [4,5]. Molecular phylogeny suggests the centriole or basal body was associated with a flagellum in the early eukaryotic cell [6]. This duality of the centriole as a basal body is therefore inbuilt into the cellular and developmental program of those structures [7] (Figure 1A,B). Fertilization is a great Corresponding author: Bettencourt-Dias, M. ([email protected]).
8
example of this duality. The flagellated sperm provides the basal body to the egg that lost its centrioles during oogenesis. Within the egg, the basal body of the sperm will duplicate and recruit PCM, giving rise to centrosomes, which are essential for embryonic development [8,9]. Little is known about the molecular mechanisms governing the switch from a centriole to a basal body and vice versa. Recently, Spektor and colleagues found new clues, showing that CP110 and its regulator CEP97 have an important role in inhibiting basal body fate [10]. Here, we discuss that result and its implications in the light of recent findings on centriole and cilia biogenesis. CP110 teams up with CEP97 in cycling cells CP110 is a coiled-coil protein necessary for centriole duplication [11,12]. Its presence at the distal end of growing centrioles suggests that it regulates centriole elongation [11]. In a search for CP110-binding partners, Spektor and colleagues used immunoaffinity purification of Flag-tagged CP110 protein complexes followed by mass-spectrometry sequencing and found several components of the human centrosome proteome [10,13]. Among those centrosome components was leucine-rich repeats and IQ motif-containing 2 (LRRIQ2), which the authors renamed Cep97 because of its size and localization at the centriole [10]. The CEP97– CP110 interaction does not depend on calmodulin, a molecule that binds both CP110 and CEP97 [10,14]. Furthermore, this interaction is likely to be direct, based on in vitro translation and immunoprecipitation [10]. What is the relevance of this complex? Spektor and colleagues showed that the localization of CEP97 depends strongly on CP110, suggesting recruitment by this molecule [10]. However, CEP97 is not important for centriole duplication, although it is essential for CP110 stability [10], suggesting a role in preventing its degradation at a
Update Glossary Anaphase-promoting complex/Cyclosome (APC/C): Proteolysis by ubiquitin and the 26S proteasome pathway is a fundamental mechanism for protein degradation. The APC is an ubiquitin ligase responsible for ubiquitination of proteins at the metaphase–anaphase and mitosis–G1 transitions. Two major degradation motifs have been identified in the protein sequence of APC substrates: destruction box (D-box) and another destruction motif called KEN box. Axoneme: The microtubule-based structure of cilia and flagella. It gives those structures their rigidity and ability to move. It is cylindrical and comprised of nine pairs of doublet microtubules, around a central pair of single microtubules (9C2). The central microtubules might be absent in non-motile cilia (9C0). Axoneme microtubules are very stable. Basal body: Structure found at the base of eukaryotic cilia and flagella that organizes the assembly of the axoneme. The structure of the basal body is the same as the one of the centriole. Additionally, it has a transition zone at the distal end, contiguous with the axoneme. Axoneme microtubules are templated from the distal part of the basal body. Centriole: The canonical centriole is a cylinder comprised of nine microtubule triplets. It is approximately 0.5 mm long and shows appendages at the distal ends on maturation. There are variations of this structure, in which triplets are substituted by singlets or doublets, such as in C. elegans and D. melanogaster. When the centriole is tethered to the membrane, it is called a ‘basal body’. In a centriole or basal body pair within the centrosome or tethered to the membrane, the oldest centriole or basal body is referred to as the ‘mother’, whereas the youngest is referred to as the ‘daughter’. Centrosome: The centrosome is the primary microtubule-organizing centre (MTOC) in animal cells. It regulates cell motility, adhesion and polarity in interphase and facilitates the organization of the spindle poles during mitosis. It is comprised of two centrioles surrounded by an electron-dense matrix, the pericentriolar material (PCM). Cilia and flagella: Cilia and flagella are projections from cells that either enable movement of the cell itself or facilitate movement or sensing of substances around cells. Evidence has grown for their indispensable role in a variety of cellular and developmental processes: motility, propagation of morphogenetic signals in embryogenesis and sensory perception (reviewed in [20]). The majority of the cells in vertebrates have cilia that act as sensory organelles or can move fluids around the cell. Flagella help to propel cells. Microtubule: A hollow tube, 25 nm in diameter, formed by the lateral association of 13 protofilaments. Each protofilament is a polymer of a- and b-tubulin subunits. Pericentriolar material: Fibrillar material surrounding centrioles in the centrosome that nucleates the growth of new microtubules.
phase where CP110 is not required for centriole formation. Indeed, the levels of both molecules show evidence of cellcycle regulation because they are low in quiescent cells and go up on cell-cycle entry [10,12]. Additionally, CP110 protein has destruction boxes, suggesting degradation on mitotic exit [12] (Figure 1A). CEP97 and CP110 block cilia formation The most striking observation of Spektor and colleagues was made in U2OS cells, which do not form cilia normally. They observed that, after depletion of Cep97 or CP110 by RNAi, 30% of the cells showed centrin fibers emanating from the centrioles, which were similar to cilia. These fibers showed cilia markers, such as polycystin 2 and polyglutamylated and acetylated tubulin [10]. RNAi of CP110 or CEP97 in ciliated cells led to a twofold increase in cilia formation, suggesting these proteins might prevent cilia formation normally. This result is unlikely to reflect forced quiescence because depletion of CP110 and CEP97 did not lead to cell-cycle arrest [10,12]. Moreover, overexpression of CP110 in quiescent cilia-prone 3T3 cells abrogated cilia formation. Together, these data suggest strongly that CP110 at the centrioles inhibits their ability to form cilia. Indeed, in ciliated NIH 3T3 cells, CP110 localizes to centrioles but not to the basal body at the base of the cilium [10].
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Intriguingly, depletion of CP110 or CEP97 or two other CP110 interactors (calmodulin and centrin) leads to mitotic-spindle abnormalities and an increase in cytokinetic failure [10,14]. In certain human cell lines, the mother centriole moves towards the mid-body at the end of telophase and this movement coincides with abscission, the event that separates the two cells [15,16]. It is possible that the absence of those molecules impairs the function of the centriole or creates artifacts in cytokinesis, which might or might not be associated with cilia formation. Alternatively, those phenotypes might reflect a centriole-independent function of those molecules, which is yet to be explored. To be or not to be a basal body: decisions in a centriole’s life If CP110 truly prevents centrioles from forming cilia, the question that naturally arises is how does it do so? Does it prevent centrioles from recruiting molecules required for appendage and cilia formation (Figure 1A,B), hence preventing movement to or attachment to the cytoplasmic membrane? Or is CP110 involved in inhibiting the first steps in axoneme formation? Given that overexpression of CP110 inhibits normal cilia formation, the probable explanation is that it prevents centriole or basal body movement or attachment to the membrane (Figure 1B). Centrioles and basal bodies show clearly distinct features: they nucleate and anchor different MT (centrosomal versus axonemal MT); additionally basal bodies show a transition zone at the distal tip. A crucial step to elucidate CP110 activity will be to better describe the consequences of its depletion and overexpression: what happens to the ultrastructure of the centriole or basal body and the composition of the MTs it nucleates? Where is the centriole or basal body localized in the cell in relation to the centrosome and the plasma membrane? Understanding the regulation of CP110 is crucial to shedding light on the cell-cycle regulation of cilia formation. First, what targets it to the centriole? CP110 interacts with centrin [12,14], an early centriole marker, which, like CP110, also localizes to the distal part of centrioles [11,17]. Although centrin is at the right place and time to recruit CP110, CP110 localization is not dependent on centrin [11]. Second, what removes CP110 from the mother centriole to enable cilia formation in G0/G1? Spektor and colleagues suggest that CEP97 is recruited by CP110 and stabilizes it [10]. One possibility is that CP110 is degraded in the mother centriole at mitotic exit because it has destruction motifs for the APC/C [12] (Figure 1B). Further experiments are required to investigate the role of the APC/C and CEP97 in CP110 stability. In any case, removal of CP110 cannot be the only trigger for cilia formation because, in the majority of cells, only one of the centrioles formed cilia after CP110 depletion [10]. It is possible that other molecules required for cilliogenesis, such as the outer dense fibre 2 (ODF2), which associates exclusively with the mother centrioles, confer the selectively for this centriole to form cilia [1,2,18] (Figure 1A,B). Finally, what happens on mitotic entry (Figure 1F)? Does the basal body incorporate CP110 de novo to prevent cilia formation? Analysis of CP110 localization throughout the cell cycle should be informative. 9
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Figure 1. Model of the regulation of cilia formation by CP110 in cycling and quiescent cells. (A) When a cell enters a new cycle it only has one centrosome, composed of a mother and a daughter centriole. The mother centriole matures on mitotic entry, recruiting molecules that enable it to form appendages, such as ODF2 (red triangle) [18,19]. It is possible that upon mitotic exit, CEP97 (orange circle) is degraded or removed from centrioles leading to destabilization of CP110 at the mother centriole (blue square). It has been suggested that CP110 might be degraded by the APC/C [12]. (B) In G1, the absence of CP110 and presence of ODF2 specifically in the mother centriole enables it to become a basal body, tethering at the membrane where it forms an axoneme [10,17]. (C) If nutrients are removed, cells can exit the cell cycle and become quiescent (G0). At this stage, the cilium can grow further [20]. (D) On serum stimulation, cells re-enter the cell cycle and the cilia are reabsorbed. (E) The centrioles or basal bodies duplicate in S phase. CP110 is necessary in the daughter centrioles for their biogenesis [11,12]. (F) On centrosome maturation at the G2/M transition, the cilium is reabsorbed and centrioles move to the center to participate in mitotic-spindle assembly [3]. The mitotic kinase Aurora A (pink oval) activates tubulin deacetylase, hence promoting ciliary disassembly and internalization of the basal body [4].
Recently, it was shown that the mitotic kinase Aurora A acts on the conversion of the basal body to centriole by activating tubulin deacetylase, hence promoting ciliary disassembly and internalization of the basal body, both at G1/S and mitotic entry [4] (Figure 1F). These studies show that research into the centriole to basal body interconversion has just started. Aurora A and CP110 have entered center stage in this exciting new field that promises to unravel the secrets of this mysterious and ubiquitously important structure: the cilia. Acknowledgements We thank A Rodrigues-Martins, J Pereira-Leal, J Lamego and L Sau´de for criticisms and comments on this manuscript. Work in the MBD laboratory is funded by Fundac¸a˜o Calouste Gulbenkian and Fundac¸a˜o para a Cieˆncia e Tecnologia.
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18 Ishikawa, H. et al. (2005) Odf2-deficient mother centrioles lack distal/ subdistal appendages and the ability to generate primary cilia. Nat. Cell Biol. 7, 517–524 19 Lange, B.M. and Gull, K. (1995) A molecular marker for centriole maturation in the mammalian cell cycle. J. Cell Biol. 130, 919–927
20 Badano, J.L. et al. (2005) The centrosome in human genetic disease. Nat. Rev. Genet. 6, 194–205 0962-8924/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2007.11.002
Corrigendum
Corrigendum: Shedding light on Merlin’s wizardry Trends in Cell Biology 17 (2007), 222–229
In this article by Tomoyo Okadaa, Liru Youa and Filippo G. Giancotti, published in the May 2007 issue of TCB there was an incorrect statement in the second to last sentence of the abstract regarding the relationship between the proteins Merlin, Expanded and Hippo. The authors would like to clarify that in Drosophila, Merlin functions together with the band 4.1 protein Expanded to promote the endocytosis of many signaling receptors, limiting their accumulation at the plasma membrane, and to activate the Hippo signaling pathway. The corrected abstract should read as follows: Inactivation of the tumor suppressor Merlin, encoded by the NF2 (Neurofibromatosis type 2) gene, contributes to malignant conversion in many cell types. Merlin is an Ezrin–Radixin–Moesin protein and localizes underneath the plasma membrane at cell–cell junctions and other actin-rich sites. Recent studies indicate that Merlin mediates contact inhibition of proliferation by blocking
recruitment of Rac to the plasma membrane. In mitogenstimulated cells, p21-activated kinase phosphorylates Ser518 in the C-terminus of Merlin, inactivating the growth suppressive function of the protein. Furthermore, the myosin phosphatase MYPT1–PP1d, has been identified as a direct activator of Merlin and its inhibition has been linked to malignant transformation. Finally, studies in the fruit fly Drosophila melanogaster have revealed that Merlin functions together with the band 4.1 protein Expanded to promote the endocytosis of many signaling receptors, limiting their accumulation at the plasma membrane, and to activate the Hippo signaling pathway. Here, we review these recent findings and their relevance to the tumor suppressor function of Merlin. The authors and Trends in Cell Biology apologize for this error. 0962-8924/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2007.11.001 Available online 26 November 2007
DOI of original article: 10.1016/j.tcb.2007.03.006
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