- Email: [email protected]
Ciliary Trafficking: CEP290 Guards a Gated Community
Current Biology Vol 20 No 21 R928
in the regulation of sexual maturity in male morphs was not entirely predictable, yet with some imagination such a role can be reconciled with Mc4r’s known functions and is thus intriguing enough to stimulate further investigation [14]. For one, Mc4r acts in the hypothalamus, thus making a link to the hormonal hypothalamus–pituitary–gonad (HPG) axis that triggers sexual maturity in vertebrates at least plausible. In fact, in female rats Mc4r activity can affect expression of certain hormones of the HPG axis, such as LH and GnRH, but its role in males — aside from Mc4r stimulation promoting erection through direct action in the penis — is not well understood [14]. Mc4r is, of course, best known for its role in energy homeostasis where it mediates the effects of leptin in the hypothalamus. When an animal is hungry, leptin levels are low and, via lowered activity of POMC neurons, the activity of neurons secreting the Mc4r antagonist AgRP is increased, resulting in lowered Mc4r signalling. Low Mc4r activity means that food intake will be increased and energy expenditure decreased. These functions appear to be largely conserved across vertebrates, and it will be interesting to see if Mc4r affects puberty onset via direct action on the HPG axis or by some indirect means related to its function in energy homeostasis. There is some indication that bourgeois and parasitic morphs vary in their energy expenditure, though if and how this is
linked to Mc4r function remains to be seen [15]. Perhaps the most fascinating question is how the traits that differ between the morphs — size, by way of puberty, and behaviour, especially mating and courtship — are connected. Are they both directly regulated by Mc4r, in a hard-wired fashion? Or is there some kind of plastic, feed-back mechanism, where for instance a smaller male adapts its behavioural strategy in response to the phenotype it has been dealt by its genetic makeup. Whether these questions will be studied in Xiphophorus or in other fish models, Mc4r offers a molecular handle on these processes and an especially relevant one at that, as it is the particular link in the system that evolution seems to have tweaked to endow male X. nigrensis with two different reproductive strategies. Hopefully, the small males will have a big future ahead of themselves. References 1. Sinervo, B., and Lively, C.M. (1996). The rock–paper–scissors game and the evolution of alternative male strategies. Nature 380, 240–243. 2. Shuster, S.M., and Wade, M.J. (1991). Equal mating success among male reproductive strategies in a marine isopod. Nature 350, 608–610. 3. Hanlon, R.T., Naud, M.-J., Shaw, P.W., and Havenhand, J.N. (2005). Transient sexual mimicry leads to fertilisation. Nature 430, 212. 4. Lampert, K.P., Schmidt, C., Fischer, P., Volff, J.-N., Hoffmann, C., Muck, J., Lohse, M.J., Ryan, M.J., and Schartl, M. (2010). Determination of onset of sexual maturation and mating behavior by melanocortin
Ciliary Trafficking: CEP290 Guards a Gated Community A recent study reveals that the large coiled-coil protein CEP290 is an integral component of the transition zone between the cell body and the cilium and functions as a gatekeeper to regulate trafficking of ciliary proteins. Ewelina Betleja and Douglas G. Cole Cilia and flagella are dedicated organelles with specialized functions that require protein and lipid compositions that differ considerably from the rest of the cell. These distinct compositions are maintained by a strict border policy that governs movement across the transition zone between the
cell body and the cilium. The transition zone is a short region of the cilium that lies between the basal body and axonemal microtubules, where the basal body triplet microtubules transition into the axonemal doublets. Although the complex ultrastructure of the transition zone was beautifully documented in early studies by Ringo [1] and Gilula and Satir [2], its protein
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receptor 4 polymorphisms. Curr. Biol. 20, 1729–1734. Oliveira, R.F. (2005). Neuroendocrine mechanisms of alternative reproductive tactics in fish. Fish Physiol. 24, 297–357. Neff, B.D. (2004). Increased performance of offspring sired by parasitic males in bluegill sunfish. Behav. Ecol. 15, 327–331. Rosenthal, G.G. (2010). Encyclopedia of animal behavior. In Swordtails and Platyfishes, volume 3, M.D. Breed and J. Moore, eds. (Oxford: Academic Press), pp. 363–367. Kallman, K.D., and Borkoski, V. (1978). A sex-linked gene controlling the onset of sexual maturity in female and male platyfish (Xiphophours maculatus), fecundity in females and adult size in males. Genetics 89, 79–119. Ryan, M.J., Hews, D.K., and Wagner, W.E.J. (1990). Sexual selection on alleles that determine body size in the swordtail Xiphophorus nigrensis. Behav. Ecol. Sociobiol. 26, 231–237. Ryan, M.J., Pease, C.M., and Morris, M.R. (1992). A genetic polymorphism in the swordtail Xiphophorus nigrensis: Testing the prediction of equal fitnesses. Am. Nat. 139, 21–31. Morris, M.R., and Ryan, M.J. (1990). Age at sexual maturity of male Xiphophorus nigrensis in nature. Copeia 1990, 747–751. Redon, R., Ishikawa, S., Fitch, K.R., Feuk, L., Perry, G.H., Andrews, T.D., Fiegler, H., Shapero, M.H., Carson, A.R., Chen, W., et al. (2006). Global variation in copy number in the human genome. Nature 444, 444–454. Zhang, F., Gu, W., Hurles, M.E., and Lupski, J.R. (2009). Copy Number variation in human health, disease, and evolution. Annu. Rev. Genomics Hum. Genet. 10, 451–481. Tao, Y.-X. (2010). The melanocortin-4 receptor: physiology, pharmacology, and pathophysiology. Endocr. Rev. 31, 506–543. Cummings, M.E., and Gelineau-Kattner, R.J. (2009). The energetic costs of alternative male reproductive strategies in Xiphophorus nigrensis. Comp. Physiol. A Neuroethol. Sens. Neural. Behav. Physiol. 195, 935–946.
Florian Maderspacher is Current Biology’s Senior Reviews Editor. E-mail: florian.maderspacher@ current-biology.com DOI: 10.1016/j.cub.2010.10.004
composition has largely remained elusive. Interest in this region, however, is increasing as researchers examine the transport of specific ciliary materials. Intraflagellar transport (IFT) particles powered by kinesin-2 and cytoplasmic dynein 1b, for example, must travel through the transition zone to enter and exit the organelle. Distinct from the transition zone are the transitional fibers that connect the nine basal body triplet microtubules to the plasma membrane. Given the accumulation of IFT complexes at the distal end of these structures, it has been suggested that the transitional fibers serve as a docking site or staging area for IFT particle formation prior to entry into the organelle [3].
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By analogy with the nuclear pore complex, transitional fibers could serve as structural components of a ciliary pore complex that control the movement of particles between the cytoplasmic and ciliary compartments [4]. Any particles, however, that make it past the transitional fibers must then pass through the constricted transition zone, a region where the ciliary membrane is contracted and in tight association with the microtubule doublets. This region of the organelle is routinely identified in electron micrographs by its unique appearance. Between the transition zone doublet microtubules and the membrane are a series of fibrous proteins that give rise to wedge-shaped connectors observed in longitudinal sections (Figure 1A) and Y-shaped connectors observed in cross-sections (Figure 1B). The relationship between these two types of connectors is not clear and, until recently, the proteins responsible for forming these structures have remained unknown. A recent study by Craige and co-workers [5] elegantly exploits the combined strengths of Chlamydomonas cell biology, biochemistry and genetics to reveal CEP290 as an integral component of the transition zone in the region where the wedge- and Y-shaped connectors are found. Conserved in ciliated organisms, CEP290 has been associated with multiple ciliopathies, including nephronophthisis and Joubert syndrome [6,7]. Previous immunolocalization studies using other model organisms established CEP290 as a centrosomal protein that could also be found near the base of primary and connecting cilia [8–11], but the high-resolution immuno-gold labeling by Craige and co-workers [5] now reveals that Chlamydomonas CEP290 is localized specifically between the outer doublet microtubules and membrane of the transition zone (Figure 2A). Of special note is the differential localization of distinct domains of the large CEP290 protein identified using gold-conjugated secondary antibodies to primary antibodies directed against an internal epitope (HA-tagged; 6 nm gold) and a carboxy-terminal epitope (12 nm gold) within CEP290 (Figure 2B,C). The internal domain of CEP290 appears to be closely associated with the Y-connectors, whereas the carboxyl terminus is more dispersed
with a bias toward the proximal end of the transition zone. Importantly, the wedge- and Y-shaped connectors are mostly absent in the cep290 mutant cells, implicating CEP290 as an integral component of these structures [5]. Consistent with this idea is the important observation that the ciliary membrane is no longer tightly associated with the doublet microtubules within the cep290 transition zone. Biochemical analysis of isolated cep290 cilia reveals abnormal accumulations of certain proteins, such as IFT complex B and components of the BBSome (a complex of Bardet-Biedl Syndrome (BBS) proteins), with concomitant reductions of other proteins, such as IFT complex A and the membrane-associated polycystin-2. Thus, CEP290 and the transition zone connectors appear to function as gatekeepers that allow specific proteins to pass, thereby regulating protein content of the organelle (Figure 2D). A gatekeeping function that regulates the transport of multiple cargos could explain why different mutations in the CEP290 gene give rise to a wide spectrum of clinically and genetically heterogeneous disorders, ranging from isolated blindness to complex syndromes that affect multiple organs. While its protein product was initially identified biochemically as a component of the human centrosome and Chlamydomonas centriole [12,13], CEP290 was determined to be a causative disease gene for Joubert syndrome and related disorders [9,10]. Soon after, Leber congenital amaurosis (LCA), Meckel-Gru¨ber syndrome (MKS) and BBS expanded the list of partially overlapping yet distinct disorders caused by CEP290 mutations [14–16]. In order to keep track of the growing list of patient mutations and corresponding phenotypes, Coppieters and co-workers developed a CEP290-specific database (http:// medgen.ugent.be/cep290base) [17]. To explain the clinical variability observed with CEP290 and associated proteins, it has been suggested that modifier genes could directly or indirectly affect CEP290 function. Alternatively, some mutations may yield differences in the expression or behavior of CEP290. It is the latter suggestion that is particularly intriguing in light of the newly implicated
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Current Biology
Figure 1. Ultrastructural features of the Chlamydomonas transition zone. (A) Longitudinal section of the constricted transition zone (TZ) reveals wedge-shaped connectors (W) and the characteristic H-shaped internal structure. (B) Cross-sections through the transition zone reveal an internal star-shaped structure and the Y-connectors (Y) that span from doublet microtubules to the ciliary membrane. Reprinted with permission from the Rockefeller University Press [5].
gatekeeping role. If a large protein like CEP290, for example, is responsible for the transition zone transport of numerous cargos, mutations affecting one domain of CEP290 may limit disruption to a subset of CEP290-mediated transport events. Such selective disruptions could give rise to the distinct and overlapping phenotypes observed in human patients. Indeed, the loss of CEP290 in Chlamydomonas resulted in a ciliary accumulation of BBS4 and a ciliary reduction in polycystin-2 [5]. The concomitant elevation and depletion of two different membrane-associated proteins suggest that some non-overlapping processes are
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Figure 2. Immunogold localization of CEP290 to the Chlamydomonas transition zone. (A) The locations of 34 gold particles from w30 sections are indicated by black dots superimposed on the electron micrograph. For simplicity, the black dots are depicted on only one side of the transition zone; no bias for either side of the transition zone was observed. TZ, transition zone; PC, proximal cylinder; DC, distal cylinder; BB, basal body. (B) cep290::CEP290-HA cytoskeletons were double-labeled with antibodies to a carboxy-terminal (C-term) peptide of CEP290 (12 nm gold; black dots) and an internal HA tag (6 nm gold; red dots). The locations of particles from several longitudinal sections are represented by black and red dots superimposed on a single electron micrograph. Bars represent 100 nm. (C) Schematic of HA-tagged CEP290 depicting the locations of the HA and carboxy-terminal epitopes. (D) Diagram depicting ciliary transport at the base of the organelle. IFT complexes dock onto the transitional fibers near the basal bodies prior to passage through the transition zone. The location of CEP290 and the wedge- and Y-shaped connectors are ideal for their function as gatekeepers of the organelle. Reprinted with permission from the Rockefeller University Press [5].
involved in their ciliary transport. It seems reasonable, then, that certain mutations of CEP290 could selectively disrupt the transport of one but not both of these disease-related proteins. Additional results by Craige et al. [5] suggest intriguing potential for translational therapies. Using dikaryon rescue experiments that combine mutant and wild-type cells, these researchers showed that CEP290 is a dynamic ciliary component capable of shuttling between the cytoplasm and CEP290-defective transition zones in a manner that rescues transport into the organelle. This result makes CEP290 a strong candidate for gene therapy. Mutations in CEP290, for example, account for w20% of LCA cases, a ciliopathy that causes retinal degeneration resulting in blindness [14,18]. Somatic gene therapy to replace a defective LCA-causing gene, RPE65, is already showing promise for affected patients [19,20]. In conclusion, the Craige et al. studies [5] provide key evidence that CEP290
sits at the membrane-to-microtubule connectors at the transition zone of cilia and flagella where it functions to mediate transport into, and probably out of, the organelle. It seems likely that associated disease-causing nephronophthisis proteins [6] will work together with CEP290 to control what passes through the ciliary gate. Future efforts will surely focus on the continued identification and genetic dissection of the proteins that form the connectors and other machinery found in the ciliary transition zones. References 1. Ringo, D.L. (1967). Flagellar motion and fine structure of the flagellar apparatus in Chlamydomonas. J. Cell Biol. 33, 543–571. 2. Gilula, N.B., and Satir, P. (1972). The ciliary necklace. A ciliary membrane specialization. J. Cell Biol. 53, 494–509. 3. Deane, J.A., Cole, D.G., Seeley, E.S., Diener, D.R., and Rosenbaum, J.L. (2001). Localization of intraflagellar transport protein IFT52 identifies basal body transitional fibers as the docking site for IFT particles. Curr. Biol. 11, 1586–1590. 4. Rosenbaum, J.L., and Witman, G.B. (2002). Intraflagellar transport. Nat. Rev. Mol. Cell Biol. 3, 813–825.
5. Craige, B., Tsao, C.-C., Diener, D.R., Hou, Y., Lechtreck, K.-F., Rosenbaum, J.L., and Witman, G.B. (2010). CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content. J. Cell Biol. 190, 927–940. 6. Hildebrandt, F., and Zhou, W. (2007). Nephronophthisis-associated ciliopathies. J. Am. Soc. Nephrol. 18, 1855–1871. 7. Valente, E.M., Brancati, F., and Dallapiccola, B. (2008). Genotypes and phenotypes of Joubert syndrome and related disorders. Eur. J. Med. Genet. 51, 1–23. 8. Chang, B., Khanna, H., Hawes, N., Jimeno, D., He, S., Lillo, C., Parapuram, S.K., Cheng, H., Scott, A., Hurd, R.E., et al. (2006). In-frame deletion in a novel centrosomal/ciliary protein CEP290/NPHP6 perturbs its interaction with RPGR and results in early-onset retinal degeneration in the rd16 mouse. Hum. Mol. Genet. 15, 1847–1857. 9. Sayer, J.A., Otto, E.A., O’Toole, J.F., Nurnberg, G., Kennedy, M.A., Becker, C., Hennies, H.C., Helou, J., Attanasio, M., Fausett, B.V., et al. (2006). The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4. Nat. Genet. 38, 674–681. 10. Valente, E.M., Silhavy, J.L., Brancati, F., Barrano, G., Krishnaswami, S.R., Castori, M., Lancaster, M.A., Boltshauser, E., Boccone, L., Al-Gazali, L., et al. (2006). Mutations in CEP290, which encodes a centrosomal protein, cause pleiotropic forms of Joubert syndrome. Nat. Genet. 38, 623–625. 11. Tsang, W.Y., Bossard, C., Khanna, H., Pera¨nen, J., Swaroop, A., Malhotra, V., and
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Dynlacht, B.D. (2008). CP110 suppresses primary cilia formation through its interaction with CEP290, a protein deficient in human ciliary disease. Dev. Cell 15, 187–197. Andersen, J.S., Wilkinson, C.J., Mayor, T., Mortensen, P., Nigg, E.A., and Mann, M. (2003). Proteomic characterization of the human centrosome by protein correlation profiling. Nature 426, 570–574. Keller, L.C., Romijn, E.P., Zamora, I., Yates, J.R., III, and Marshall, W.F. (2005). Proteomic analysis of isolated Chlamydomonas centrioles reveals orthologs of ciliary-disease genes. Curr. Biol. 15, 1090–1098. den Hollander, A.I., Koenekoop, R.K., Yzer, S., Lopez, I., Arends, M.L., Voesenek, K.E., Zonneveld, M.N., Strom, T.M., Meitinger, T., Brunner, H.G., et al. (2006). Mutations in the CEP290 (NPHP6) gene are a frequent cause of Leber congenital amaurosis. Am. J. Hum. Genet. 79, 556–561. Baala, L., Audollent, S., Martinovic, J., Ozilou, C., Babron, M.C.,
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Sivanandamoorthy, S., Saunier, S., Salomon, R., Gonzales, M., Rattenberry, E., et al. (2007). Pleiotropic effects of CEP290 (NPHP6) mutations extend to Meckel syndrome. Am. J. Hum. Genet. 81, 170–179. Leitch, C.C., Zaghloul, N.A., Davis, E.E., Stoetzel, C., Diaz-Font, A., Rix, S., Alfadhel, M., Lewis, R.A., Eyaid, W., Banin, E., et al. (2008). Hypomorphic mutations in syndromic encephalocele genes are associated with Bardet-Biedl syndrome. Nat. Genet. 40, 443–448. Coppieters, F., Lefever, S., Leroy, B.P., and De Baere, E. (2010). CEP290, a gene with many faces: mutation overview and presentation of CEP290base. Hum. Mutat. 31, 1097–1108. Sundaresan, P., Vijayalakshmi, P., Thompson, S., Ko, A.C., Fingert, J.H., and Stone, E.M. (2009). Mutations that are a common cause of Leber congenital amaurosis in northern America are rare in southern India. Mol. Vis. 15, 1781–1787. Bainbridge, J.W., Smith, A.J., Barker, S.S., Robbie, S., Henderson, R., Balaggan, K.,
Communal Breeding: Clever Defense Against Cheats High levels of conspecific brood parasitism are found in a communally breeding bird, with implications for the evolutionary links between brood parasitism and communal breeding. It also uncovers a novel egg recognition mechanism hosts use to foil brood parasites. Bruce E. Lyon1 and Daizaburo Shizuka2 Avian breeding systems often reflect a mix of cooperation and conflict over allocation of the costs and benefits of parental care [1–4]. This interesting juxtaposition of cooperation and conflict is particularly evident in the communally breeding birds, where two or more females lay eggs in the same nest and typically cooperate to raise the offspring. Beneath the veneer of group cooperation often lurks severe competition among females within the breeding group to maximize their share of reproduction [5,6]. The resolution to these conflicts results in communal breeding systems that range from nearly egalitarian — in terms of shared costs and benefits — to those that border on parasitism [5,6]. Conflicts over the costs and benefits of parental care are taken to the extreme in another breeding system in which one female lays eggs in another female’s nest but fails to provide any subsequent parental investment — brood parasitism. Both of these strategies — communal breeding and brood parasitism — are
widespread in birds, although usually they do not co-occur in the same species. Common threads between these two breeding systems include multiple females laying eggs in a single nest and the egg tossing behavior used to control whose eggs then remain in the nest [6,7]. The difference has to do with who pays for the subsequent cost of parental investment: do all females share the cost, or do some cheat on investment? While theory suggests potential evolutionary links between brood parasitism and some forms of communal breeding [1,8,9], these ideas have been difficult to test empirically. A recent study in Current Biology by Christina Riehl [10] adds a new beam to the proposed bridge between parasitism and communal breeding. Riehl demonstrates for the first time high levels of conspecific brood parasitism in an obligate communal breeder and also reveals a novel mechanism that birds use to foil many instances of brood parasitism. A brief description of the strange reproductive antics of anis and their relatives is necessary to put the new discoveries into context. The Old World cuckoos are famous for their brood parasitic habits but the four species
Viswanathan, A., Holder, G.E., Stockman, A., Tyler, N., et al. (2008). Effect of gene therapy on visual function in Leber’s congenital amaurosis. N. Engl. J. Med. 358, 2231–2239. 20. Maguire, A.M., Simonelli, F., Pierce, E.A., Pugh, E.N., Jr., Mingozzi, F., Bennicelli, J., Banfi, S., Marshall, K.A., Testa, F., Surace, E.M., et al. (2008). Safety and efficacy of gene transfer for Leber’s congenital amaurosis. N. Engl. J. Med. 358, 2240–2248.
Department of Microbiology, Molecular Biology and Biochemistry, MMBB LSS142, University of Idaho, Moscow, ID 83844-3052, USA. E-mail: [email protected], [email protected] DOI: 10.1016/j.cub.2010.09.058
of non-parasitic New World cuckoo in the subfamily Crotophaginae — three species of ani (Crotophaga spp.) and the guira cuckoo (Guira guira; Figure 1) — have become textbook examples for their communal breeding habits. The four species vary in subtle ways, but Riehl’s observations of greater anis (Crotophaga major) capture the essential details of communal breeding in this group [11]. Breeding groups typically comprise two or more pairs of birds that join together to cooperatively rear offspring in the same nest. An intriguing aspect of communal breeding — both in the Crotophagine cuckoos and in some of the other communal breeders as well [6] — is that nesting females remove eggs of other group members to increase their share of the group’s reproductive output. Females simply eject eggs from the nest until they themselves have started to lay eggs. This egg removal synchronizes laying among females and, although the egg-tossing females often end up with a few more eggs in the clutch, reproductive skew tends to be fairly low [5]. Riehl [10] has now shown that the females outside of the group also try to get in on the game through conspecific brood parasitism — they lay eggs in nests without contributing to later parental care. To document the occurrence of brood parasitism, Riehl obtained maternal DNA by swabbing the surface of freshly laid eggs [12]. A maternal genetic signature (as opposed to genotyping the parasitic offspring themselves) makes
in the regulation of sexual maturity in male morphs was not entirely predictable, yet with some imagination such a role can be reconciled with Mc4r’s known functions and is thus intriguing enough to stimulate further investigation [14]. For one, Mc4r acts in the hypothalamus, thus making a link to the hormonal hypothalamus–pituitary–gonad (HPG) axis that triggers sexual maturity in vertebrates at least plausible. In fact, in female rats Mc4r activity can affect expression of certain hormones of the HPG axis, such as LH and GnRH, but its role in males — aside from Mc4r stimulation promoting erection through direct action in the penis — is not well understood [14]. Mc4r is, of course, best known for its role in energy homeostasis where it mediates the effects of leptin in the hypothalamus. When an animal is hungry, leptin levels are low and, via lowered activity of POMC neurons, the activity of neurons secreting the Mc4r antagonist AgRP is increased, resulting in lowered Mc4r signalling. Low Mc4r activity means that food intake will be increased and energy expenditure decreased. These functions appear to be largely conserved across vertebrates, and it will be interesting to see if Mc4r affects puberty onset via direct action on the HPG axis or by some indirect means related to its function in energy homeostasis. There is some indication that bourgeois and parasitic morphs vary in their energy expenditure, though if and how this is
linked to Mc4r function remains to be seen [15]. Perhaps the most fascinating question is how the traits that differ between the morphs — size, by way of puberty, and behaviour, especially mating and courtship — are connected. Are they both directly regulated by Mc4r, in a hard-wired fashion? Or is there some kind of plastic, feed-back mechanism, where for instance a smaller male adapts its behavioural strategy in response to the phenotype it has been dealt by its genetic makeup. Whether these questions will be studied in Xiphophorus or in other fish models, Mc4r offers a molecular handle on these processes and an especially relevant one at that, as it is the particular link in the system that evolution seems to have tweaked to endow male X. nigrensis with two different reproductive strategies. Hopefully, the small males will have a big future ahead of themselves. References 1. Sinervo, B., and Lively, C.M. (1996). The rock–paper–scissors game and the evolution of alternative male strategies. Nature 380, 240–243. 2. Shuster, S.M., and Wade, M.J. (1991). Equal mating success among male reproductive strategies in a marine isopod. Nature 350, 608–610. 3. Hanlon, R.T., Naud, M.-J., Shaw, P.W., and Havenhand, J.N. (2005). Transient sexual mimicry leads to fertilisation. Nature 430, 212. 4. Lampert, K.P., Schmidt, C., Fischer, P., Volff, J.-N., Hoffmann, C., Muck, J., Lohse, M.J., Ryan, M.J., and Schartl, M. (2010). Determination of onset of sexual maturation and mating behavior by melanocortin
Ciliary Trafficking: CEP290 Guards a Gated Community A recent study reveals that the large coiled-coil protein CEP290 is an integral component of the transition zone between the cell body and the cilium and functions as a gatekeeper to regulate trafficking of ciliary proteins. Ewelina Betleja and Douglas G. Cole Cilia and flagella are dedicated organelles with specialized functions that require protein and lipid compositions that differ considerably from the rest of the cell. These distinct compositions are maintained by a strict border policy that governs movement across the transition zone between the
cell body and the cilium. The transition zone is a short region of the cilium that lies between the basal body and axonemal microtubules, where the basal body triplet microtubules transition into the axonemal doublets. Although the complex ultrastructure of the transition zone was beautifully documented in early studies by Ringo [1] and Gilula and Satir [2], its protein
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receptor 4 polymorphisms. Curr. Biol. 20, 1729–1734. Oliveira, R.F. (2005). Neuroendocrine mechanisms of alternative reproductive tactics in fish. Fish Physiol. 24, 297–357. Neff, B.D. (2004). Increased performance of offspring sired by parasitic males in bluegill sunfish. Behav. Ecol. 15, 327–331. Rosenthal, G.G. (2010). Encyclopedia of animal behavior. In Swordtails and Platyfishes, volume 3, M.D. Breed and J. Moore, eds. (Oxford: Academic Press), pp. 363–367. Kallman, K.D., and Borkoski, V. (1978). A sex-linked gene controlling the onset of sexual maturity in female and male platyfish (Xiphophours maculatus), fecundity in females and adult size in males. Genetics 89, 79–119. Ryan, M.J., Hews, D.K., and Wagner, W.E.J. (1990). Sexual selection on alleles that determine body size in the swordtail Xiphophorus nigrensis. Behav. Ecol. Sociobiol. 26, 231–237. Ryan, M.J., Pease, C.M., and Morris, M.R. (1992). A genetic polymorphism in the swordtail Xiphophorus nigrensis: Testing the prediction of equal fitnesses. Am. Nat. 139, 21–31. Morris, M.R., and Ryan, M.J. (1990). Age at sexual maturity of male Xiphophorus nigrensis in nature. Copeia 1990, 747–751. Redon, R., Ishikawa, S., Fitch, K.R., Feuk, L., Perry, G.H., Andrews, T.D., Fiegler, H., Shapero, M.H., Carson, A.R., Chen, W., et al. (2006). Global variation in copy number in the human genome. Nature 444, 444–454. Zhang, F., Gu, W., Hurles, M.E., and Lupski, J.R. (2009). Copy Number variation in human health, disease, and evolution. Annu. Rev. Genomics Hum. Genet. 10, 451–481. Tao, Y.-X. (2010). The melanocortin-4 receptor: physiology, pharmacology, and pathophysiology. Endocr. Rev. 31, 506–543. Cummings, M.E., and Gelineau-Kattner, R.J. (2009). The energetic costs of alternative male reproductive strategies in Xiphophorus nigrensis. Comp. Physiol. A Neuroethol. Sens. Neural. Behav. Physiol. 195, 935–946.
Florian Maderspacher is Current Biology’s Senior Reviews Editor. E-mail: florian.maderspacher@ current-biology.com DOI: 10.1016/j.cub.2010.10.004
composition has largely remained elusive. Interest in this region, however, is increasing as researchers examine the transport of specific ciliary materials. Intraflagellar transport (IFT) particles powered by kinesin-2 and cytoplasmic dynein 1b, for example, must travel through the transition zone to enter and exit the organelle. Distinct from the transition zone are the transitional fibers that connect the nine basal body triplet microtubules to the plasma membrane. Given the accumulation of IFT complexes at the distal end of these structures, it has been suggested that the transitional fibers serve as a docking site or staging area for IFT particle formation prior to entry into the organelle [3].
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By analogy with the nuclear pore complex, transitional fibers could serve as structural components of a ciliary pore complex that control the movement of particles between the cytoplasmic and ciliary compartments [4]. Any particles, however, that make it past the transitional fibers must then pass through the constricted transition zone, a region where the ciliary membrane is contracted and in tight association with the microtubule doublets. This region of the organelle is routinely identified in electron micrographs by its unique appearance. Between the transition zone doublet microtubules and the membrane are a series of fibrous proteins that give rise to wedge-shaped connectors observed in longitudinal sections (Figure 1A) and Y-shaped connectors observed in cross-sections (Figure 1B). The relationship between these two types of connectors is not clear and, until recently, the proteins responsible for forming these structures have remained unknown. A recent study by Craige and co-workers [5] elegantly exploits the combined strengths of Chlamydomonas cell biology, biochemistry and genetics to reveal CEP290 as an integral component of the transition zone in the region where the wedge- and Y-shaped connectors are found. Conserved in ciliated organisms, CEP290 has been associated with multiple ciliopathies, including nephronophthisis and Joubert syndrome [6,7]. Previous immunolocalization studies using other model organisms established CEP290 as a centrosomal protein that could also be found near the base of primary and connecting cilia [8–11], but the high-resolution immuno-gold labeling by Craige and co-workers [5] now reveals that Chlamydomonas CEP290 is localized specifically between the outer doublet microtubules and membrane of the transition zone (Figure 2A). Of special note is the differential localization of distinct domains of the large CEP290 protein identified using gold-conjugated secondary antibodies to primary antibodies directed against an internal epitope (HA-tagged; 6 nm gold) and a carboxy-terminal epitope (12 nm gold) within CEP290 (Figure 2B,C). The internal domain of CEP290 appears to be closely associated with the Y-connectors, whereas the carboxyl terminus is more dispersed
with a bias toward the proximal end of the transition zone. Importantly, the wedge- and Y-shaped connectors are mostly absent in the cep290 mutant cells, implicating CEP290 as an integral component of these structures [5]. Consistent with this idea is the important observation that the ciliary membrane is no longer tightly associated with the doublet microtubules within the cep290 transition zone. Biochemical analysis of isolated cep290 cilia reveals abnormal accumulations of certain proteins, such as IFT complex B and components of the BBSome (a complex of Bardet-Biedl Syndrome (BBS) proteins), with concomitant reductions of other proteins, such as IFT complex A and the membrane-associated polycystin-2. Thus, CEP290 and the transition zone connectors appear to function as gatekeepers that allow specific proteins to pass, thereby regulating protein content of the organelle (Figure 2D). A gatekeeping function that regulates the transport of multiple cargos could explain why different mutations in the CEP290 gene give rise to a wide spectrum of clinically and genetically heterogeneous disorders, ranging from isolated blindness to complex syndromes that affect multiple organs. While its protein product was initially identified biochemically as a component of the human centrosome and Chlamydomonas centriole [12,13], CEP290 was determined to be a causative disease gene for Joubert syndrome and related disorders [9,10]. Soon after, Leber congenital amaurosis (LCA), Meckel-Gru¨ber syndrome (MKS) and BBS expanded the list of partially overlapping yet distinct disorders caused by CEP290 mutations [14–16]. In order to keep track of the growing list of patient mutations and corresponding phenotypes, Coppieters and co-workers developed a CEP290-specific database (http:// medgen.ugent.be/cep290base) [17]. To explain the clinical variability observed with CEP290 and associated proteins, it has been suggested that modifier genes could directly or indirectly affect CEP290 function. Alternatively, some mutations may yield differences in the expression or behavior of CEP290. It is the latter suggestion that is particularly intriguing in light of the newly implicated
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Figure 1. Ultrastructural features of the Chlamydomonas transition zone. (A) Longitudinal section of the constricted transition zone (TZ) reveals wedge-shaped connectors (W) and the characteristic H-shaped internal structure. (B) Cross-sections through the transition zone reveal an internal star-shaped structure and the Y-connectors (Y) that span from doublet microtubules to the ciliary membrane. Reprinted with permission from the Rockefeller University Press [5].
gatekeeping role. If a large protein like CEP290, for example, is responsible for the transition zone transport of numerous cargos, mutations affecting one domain of CEP290 may limit disruption to a subset of CEP290-mediated transport events. Such selective disruptions could give rise to the distinct and overlapping phenotypes observed in human patients. Indeed, the loss of CEP290 in Chlamydomonas resulted in a ciliary accumulation of BBS4 and a ciliary reduction in polycystin-2 [5]. The concomitant elevation and depletion of two different membrane-associated proteins suggest that some non-overlapping processes are
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Figure 2. Immunogold localization of CEP290 to the Chlamydomonas transition zone. (A) The locations of 34 gold particles from w30 sections are indicated by black dots superimposed on the electron micrograph. For simplicity, the black dots are depicted on only one side of the transition zone; no bias for either side of the transition zone was observed. TZ, transition zone; PC, proximal cylinder; DC, distal cylinder; BB, basal body. (B) cep290::CEP290-HA cytoskeletons were double-labeled with antibodies to a carboxy-terminal (C-term) peptide of CEP290 (12 nm gold; black dots) and an internal HA tag (6 nm gold; red dots). The locations of particles from several longitudinal sections are represented by black and red dots superimposed on a single electron micrograph. Bars represent 100 nm. (C) Schematic of HA-tagged CEP290 depicting the locations of the HA and carboxy-terminal epitopes. (D) Diagram depicting ciliary transport at the base of the organelle. IFT complexes dock onto the transitional fibers near the basal bodies prior to passage through the transition zone. The location of CEP290 and the wedge- and Y-shaped connectors are ideal for their function as gatekeepers of the organelle. Reprinted with permission from the Rockefeller University Press [5].
involved in their ciliary transport. It seems reasonable, then, that certain mutations of CEP290 could selectively disrupt the transport of one but not both of these disease-related proteins. Additional results by Craige et al. [5] suggest intriguing potential for translational therapies. Using dikaryon rescue experiments that combine mutant and wild-type cells, these researchers showed that CEP290 is a dynamic ciliary component capable of shuttling between the cytoplasm and CEP290-defective transition zones in a manner that rescues transport into the organelle. This result makes CEP290 a strong candidate for gene therapy. Mutations in CEP290, for example, account for w20% of LCA cases, a ciliopathy that causes retinal degeneration resulting in blindness [14,18]. Somatic gene therapy to replace a defective LCA-causing gene, RPE65, is already showing promise for affected patients [19,20]. In conclusion, the Craige et al. studies [5] provide key evidence that CEP290
sits at the membrane-to-microtubule connectors at the transition zone of cilia and flagella where it functions to mediate transport into, and probably out of, the organelle. It seems likely that associated disease-causing nephronophthisis proteins [6] will work together with CEP290 to control what passes through the ciliary gate. Future efforts will surely focus on the continued identification and genetic dissection of the proteins that form the connectors and other machinery found in the ciliary transition zones. References 1. Ringo, D.L. (1967). Flagellar motion and fine structure of the flagellar apparatus in Chlamydomonas. J. Cell Biol. 33, 543–571. 2. Gilula, N.B., and Satir, P. (1972). The ciliary necklace. A ciliary membrane specialization. J. Cell Biol. 53, 494–509. 3. Deane, J.A., Cole, D.G., Seeley, E.S., Diener, D.R., and Rosenbaum, J.L. (2001). Localization of intraflagellar transport protein IFT52 identifies basal body transitional fibers as the docking site for IFT particles. Curr. Biol. 11, 1586–1590. 4. Rosenbaum, J.L., and Witman, G.B. (2002). Intraflagellar transport. Nat. Rev. Mol. Cell Biol. 3, 813–825.
5. Craige, B., Tsao, C.-C., Diener, D.R., Hou, Y., Lechtreck, K.-F., Rosenbaum, J.L., and Witman, G.B. (2010). CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content. J. Cell Biol. 190, 927–940. 6. Hildebrandt, F., and Zhou, W. (2007). Nephronophthisis-associated ciliopathies. J. Am. Soc. Nephrol. 18, 1855–1871. 7. Valente, E.M., Brancati, F., and Dallapiccola, B. (2008). Genotypes and phenotypes of Joubert syndrome and related disorders. Eur. J. Med. Genet. 51, 1–23. 8. Chang, B., Khanna, H., Hawes, N., Jimeno, D., He, S., Lillo, C., Parapuram, S.K., Cheng, H., Scott, A., Hurd, R.E., et al. (2006). In-frame deletion in a novel centrosomal/ciliary protein CEP290/NPHP6 perturbs its interaction with RPGR and results in early-onset retinal degeneration in the rd16 mouse. Hum. Mol. Genet. 15, 1847–1857. 9. Sayer, J.A., Otto, E.A., O’Toole, J.F., Nurnberg, G., Kennedy, M.A., Becker, C., Hennies, H.C., Helou, J., Attanasio, M., Fausett, B.V., et al. (2006). The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4. Nat. Genet. 38, 674–681. 10. Valente, E.M., Silhavy, J.L., Brancati, F., Barrano, G., Krishnaswami, S.R., Castori, M., Lancaster, M.A., Boltshauser, E., Boccone, L., Al-Gazali, L., et al. (2006). Mutations in CEP290, which encodes a centrosomal protein, cause pleiotropic forms of Joubert syndrome. Nat. Genet. 38, 623–625. 11. Tsang, W.Y., Bossard, C., Khanna, H., Pera¨nen, J., Swaroop, A., Malhotra, V., and
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Dynlacht, B.D. (2008). CP110 suppresses primary cilia formation through its interaction with CEP290, a protein deficient in human ciliary disease. Dev. Cell 15, 187–197. Andersen, J.S., Wilkinson, C.J., Mayor, T., Mortensen, P., Nigg, E.A., and Mann, M. (2003). Proteomic characterization of the human centrosome by protein correlation profiling. Nature 426, 570–574. Keller, L.C., Romijn, E.P., Zamora, I., Yates, J.R., III, and Marshall, W.F. (2005). Proteomic analysis of isolated Chlamydomonas centrioles reveals orthologs of ciliary-disease genes. Curr. Biol. 15, 1090–1098. den Hollander, A.I., Koenekoop, R.K., Yzer, S., Lopez, I., Arends, M.L., Voesenek, K.E., Zonneveld, M.N., Strom, T.M., Meitinger, T., Brunner, H.G., et al. (2006). Mutations in the CEP290 (NPHP6) gene are a frequent cause of Leber congenital amaurosis. Am. J. Hum. Genet. 79, 556–561. Baala, L., Audollent, S., Martinovic, J., Ozilou, C., Babron, M.C.,
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Sivanandamoorthy, S., Saunier, S., Salomon, R., Gonzales, M., Rattenberry, E., et al. (2007). Pleiotropic effects of CEP290 (NPHP6) mutations extend to Meckel syndrome. Am. J. Hum. Genet. 81, 170–179. Leitch, C.C., Zaghloul, N.A., Davis, E.E., Stoetzel, C., Diaz-Font, A., Rix, S., Alfadhel, M., Lewis, R.A., Eyaid, W., Banin, E., et al. (2008). Hypomorphic mutations in syndromic encephalocele genes are associated with Bardet-Biedl syndrome. Nat. Genet. 40, 443–448. Coppieters, F., Lefever, S., Leroy, B.P., and De Baere, E. (2010). CEP290, a gene with many faces: mutation overview and presentation of CEP290base. Hum. Mutat. 31, 1097–1108. Sundaresan, P., Vijayalakshmi, P., Thompson, S., Ko, A.C., Fingert, J.H., and Stone, E.M. (2009). Mutations that are a common cause of Leber congenital amaurosis in northern America are rare in southern India. Mol. Vis. 15, 1781–1787. Bainbridge, J.W., Smith, A.J., Barker, S.S., Robbie, S., Henderson, R., Balaggan, K.,
Communal Breeding: Clever Defense Against Cheats High levels of conspecific brood parasitism are found in a communally breeding bird, with implications for the evolutionary links between brood parasitism and communal breeding. It also uncovers a novel egg recognition mechanism hosts use to foil brood parasites. Bruce E. Lyon1 and Daizaburo Shizuka2 Avian breeding systems often reflect a mix of cooperation and conflict over allocation of the costs and benefits of parental care [1–4]. This interesting juxtaposition of cooperation and conflict is particularly evident in the communally breeding birds, where two or more females lay eggs in the same nest and typically cooperate to raise the offspring. Beneath the veneer of group cooperation often lurks severe competition among females within the breeding group to maximize their share of reproduction [5,6]. The resolution to these conflicts results in communal breeding systems that range from nearly egalitarian — in terms of shared costs and benefits — to those that border on parasitism [5,6]. Conflicts over the costs and benefits of parental care are taken to the extreme in another breeding system in which one female lays eggs in another female’s nest but fails to provide any subsequent parental investment — brood parasitism. Both of these strategies — communal breeding and brood parasitism — are
widespread in birds, although usually they do not co-occur in the same species. Common threads between these two breeding systems include multiple females laying eggs in a single nest and the egg tossing behavior used to control whose eggs then remain in the nest [6,7]. The difference has to do with who pays for the subsequent cost of parental investment: do all females share the cost, or do some cheat on investment? While theory suggests potential evolutionary links between brood parasitism and some forms of communal breeding [1,8,9], these ideas have been difficult to test empirically. A recent study in Current Biology by Christina Riehl [10] adds a new beam to the proposed bridge between parasitism and communal breeding. Riehl demonstrates for the first time high levels of conspecific brood parasitism in an obligate communal breeder and also reveals a novel mechanism that birds use to foil many instances of brood parasitism. A brief description of the strange reproductive antics of anis and their relatives is necessary to put the new discoveries into context. The Old World cuckoos are famous for their brood parasitic habits but the four species
Viswanathan, A., Holder, G.E., Stockman, A., Tyler, N., et al. (2008). Effect of gene therapy on visual function in Leber’s congenital amaurosis. N. Engl. J. Med. 358, 2231–2239. 20. Maguire, A.M., Simonelli, F., Pierce, E.A., Pugh, E.N., Jr., Mingozzi, F., Bennicelli, J., Banfi, S., Marshall, K.A., Testa, F., Surace, E.M., et al. (2008). Safety and efficacy of gene transfer for Leber’s congenital amaurosis. N. Engl. J. Med. 358, 2240–2248.
Department of Microbiology, Molecular Biology and Biochemistry, MMBB LSS142, University of Idaho, Moscow, ID 83844-3052, USA. E-mail: [email protected], [email protected] DOI: 10.1016/j.cub.2010.09.058
of non-parasitic New World cuckoo in the subfamily Crotophaginae — three species of ani (Crotophaga spp.) and the guira cuckoo (Guira guira; Figure 1) — have become textbook examples for their communal breeding habits. The four species vary in subtle ways, but Riehl’s observations of greater anis (Crotophaga major) capture the essential details of communal breeding in this group [11]. Breeding groups typically comprise two or more pairs of birds that join together to cooperatively rear offspring in the same nest. An intriguing aspect of communal breeding — both in the Crotophagine cuckoos and in some of the other communal breeders as well [6] — is that nesting females remove eggs of other group members to increase their share of the group’s reproductive output. Females simply eject eggs from the nest until they themselves have started to lay eggs. This egg removal synchronizes laying among females and, although the egg-tossing females often end up with a few more eggs in the clutch, reproductive skew tends to be fairly low [5]. Riehl [10] has now shown that the females outside of the group also try to get in on the game through conspecific brood parasitism — they lay eggs in nests without contributing to later parental care. To document the occurrence of brood parasitism, Riehl obtained maternal DNA by swabbing the surface of freshly laid eggs [12]. A maternal genetic signature (as opposed to genotyping the parasitic offspring themselves) makes