uncoordinated: Biological Overview | References
Gene name - uncoordinated
Cytological map position-19F1-19F1
Function - ciliogenesis
Symbol - unc
FlyBase ID: FBgn0003950
Genetic map position - X: 20,982,440..20,987,534 [-]
Classification - coiled-coil protein
Cellular location - cytoplasmic
|Recent literature||Gottardo, M., Callaini, G. and Riparbelli, M. G. (2016). Does Unc-GFP uncover ciliary structures in the rhabdomeric eye of Drosophila?.J Cell Sci 129(14):2726-31. PubMed ID: 27235419
The Uncoordinated (Unc) gene product, a potential ortholog of orofaciodigital syndrome 1 (Ofd1), is involved in the assembly of the ciliary axoneme in Drosophila and it is, therefore, constrained to cell types that have ciliary structures, namely type 1 sensory neurons and male germ cells. This study shows that evenly spaced Unc-GFP spots are present in the eye imaginal discs of third instar larvae. These spots are restricted to the R8 photoreceptor cell of each ommatidium in association with mother centrioles. This finding is unexpected since the Drosophila eye is of rhabdomeric type and should lack ciliary structures (Gottardo, 2016).
uncoordinated (unc) mutants of Drosophila, which lack transduction in ciliated mechanosensory neurons (see Schematic diagram of type I mechanosensory organs in Eberl, 2000), do not produce motile sperm. Both sensory and spermatogenesis defects are associated with disrupted ciliary structures: mutant sensory neurons have truncated cilia, and sensory neurons and spermatids show defects in axoneme ultrastructure. unc encodes a novel protein with coiled-coil segments and a LisH motif, and is expressed in type I sensory neurons and in the male germline, the only ciliogenic cells in the fly. A functional UNC-GFP fusion protein specifically localizes to both basal bodies in differentiating sensory neurons. In premeiotic spermatocytes it localizes to all four centrioles in early G2, remaining associated with them through meiosis and as they become the basal bodies for the elongating spermatid flagella. UNC is thus specifically required for normal ciliogenesis. Its localization is an early marker for the centriole-basal body transition, a central but enigmatic event in eukaryotic cell differentiation (Baker, 2004).
In many eukaryotic cells, the transition from cell proliferation to differentiation is marked by the formation of a cilium: a cellular appendage with a radially symmetric axoneme cytoskeleton, templated on a similarly symmetric basal body. It has long been known that basal bodies are identical with and derived from the centrioles that nucleate mitotic centrosomes, but the mechanisms by which a centriole is converted into a basal body remain obscure. Much work has focused on the role of centrioles and centriole-associated proteins that participate in centrosome replication and reconfiguration during the mitotic cell cycle. Less attention has been paid to the mechanisms by which a mitotic centriole is reconfigured as a ciliogenic basal body in differentiating cells. A molecular understanding of this process is nevertheless important because it may be a crucial step in the transition from cell proliferation to differentiation. Some disorders of ciliated sensory organs such as retinal photoreceptors are the result of defective ciliary differentiation or transport. More generally, several lines of evidence now indicate that the less overtly specialized primary cilia on mammalian cells have diverse functions in developmental signaling. For example, ciliary or basal body defects may underlie the diverse symptoms, including situs inversus, epithelial cysts, retinal dystrophy, skeletal abnormalities and obesity, that are associated with polycystic kidney disease and Bardet-Biedl syndrome (Baker, 2004 and references therein).
Interconversion of centrioles and basal bodies occurs in eukaryotes ranging from algae to mammals. A centriole may move to the cell periphery and form a basal body in G1 of interphase, or in quiescent (G0) or differentiating cells. In dividing cells, such basal bodies revert to a centriolar role prior to mitosis when the cilia are resorbed and centrosomes assemble. In many animal zygotes, the sperm flagellar basal body recruits maternal pericentriolar material to form the initial centrosome. Centrioles are conservatively replicated once per cell cycle and segregate so that each daughter cell inherits an older and a younger centriole; in mammalian somatic cells only the older centriole nucleates a cilium. The older centriole is also distinguished from the younger by distal and subdistal appendages, by its ability to anchor microtubules, and by its more stable location in the cell (Piel, 2000). A newly assembled mammalian centriole matures over two cell cycles in a stepwise process that includes the acquisition of appendage structures and component proteins such as ninein, cenexin/odf2 and epsilon-tubulin (Baker, 2004).
By contrast, centrioles in embryos and most somatic cells of Drosophila are rudimentary: they are composed of microtubule doublets rather than triplets, lack most of the specialized accessory structures characteristic of mature vertebrate centrioles, and do not form primary cilia. The only cilia in the fly are found in the peripheral nervous system (PNS) and in the male germline. In type I sensory neurons, a cilium extends from the more distal of two basal bodies at the tip of the single sensory process. This cilium is the probable site of sensory transduction, and is highly modified for this role in mechanosensory neurons. In mature spermatocytes, two pairs of centrioles with microtubule triplets migrate to the cell periphery, where their differentiated distal ends protrude from the cell. During meiosis, they segregate to the meiotic spindle poles without further replication, so that a single centriole associates with each postmeiotic haploid nucleus and becomes the basal body of the spermatid flagellum. At fertilization, the entire sperm enters the acentrosomal oocyte, where the basal body recruits maternal components to form the zygotic centrosome (Baker, 2004).
Thus, mutations disrupting ciliogenesis in Drosophila are expected specifically to affect sensory neurons and spermatids. The uncoordinated (unc) gene product, which appears to be required to construct normal cilia in these cell types. Previously, unc mutants were found to be defective in transduction by ciliated mechanosensory neurons (Eberl, 2000; Kernan, 1994). This study shows that unc mutant males are also defective in spermatogenesis: spermatid nuclei are detached from basal bodies and flagellar axonemes are disrupted. Axonemal defects are also found in the ciliated endings of mutant sensory neurons. unc encodes a large, novel, partly coiled-coil protein that is expressed specifically in ciliated sensory neurons and in male germline cells. Its localization in spermatocytes suggests an early role in reconfiguring centrioles as basal bodies (Baker, 2004).
Mutations affecting ciliogenesis have been identified in eukaryotes ranging from algae to mammals. Their phenotypes reflect the functions that cilia perform in each organism, and range from motility and signaling defects in Chlamydomonas, to situs inversus, skeletal defects, male-sterility and cyst formation in mammals. The unc mutant phenotypes reveal the results of a general failure of ciliogenesis in Drosophila: a combination of sensory and spermatogenesis defects reflecting a requirement for normal ciliary differentiation in type I sensory neurons and in spermatids. Though similarly based on axonemal structures, sensory cilia and sperm flagella differ in form and molecular composition. Many male-sterile mutations of Drosophila specifically affect spermatid axonemes. For example, loss of axonemal dynein subunits encoded on the Y chromosome leads to male-sterility with immotile spermatids, but neither XO males nor, of course, normal XX females show sensory defects. Conversely, Drosophila mutants defective in intraflagellar transport, a conserved protein complex that is required to assemble and maintain axonemal structures in other systems, lack normal sensory cilia but, surprisingly, produce functional sperm. touch-insensitive-larvaB (tilB) mutants (Eberl, 2000) combine sperm axoneme defects with sensory defects specific to chordotonal organs. unc, by contrast, functions in all ciliated sensory neurons, as well as spermatids, suggesting that it affects a core ciliogenesis function (Baker, 2004).
unc is expressed only in postmitotic cells that are forming or will form cilia, and it is localized to centrioles in these cells. The stability and persistence of UNC-GFP labeling on the centrioles, from early spermatocytes through meiosis, indicate that it associates more closely with the centriole than do γ-tubulin and centrosomin, which show a more diffuse and cell cycle-dependent association. However, UNC is probably not an integral component of the centriole microtubules, as its later redistribution in maturing spermatids, like that of γ-tubulin, appears to reflect the transformation of an accessory structure, the centriole adjunct, from a sheath to a doughnut-shaped collar. UNC-GFP is cytoplasmic in dividing gonial cells or when ectopically expressed in early embryos, while it forms large, apparently artefactual aggregates when overexpressed in differentiated somatic cells. Therefore, specific modifications of the centrioles or of UNC itself must regulate its normal localization in neurons and spermatocytes (Baker, 2004).
What does UNC do? Centrosome organization and replication, the functions of centrioles in dividing cells, are not affected in unc mutants: γ-tubulin and centrosomin are still recruited to the meiotic centrosomes, bipolar spindles form and meiosis proceeds normally. Abnormalities in unc mutants are restricted to axonemal structures, implying a specific defect in basal body function. Basal bodies are present and correctly located in mutant sensory neurons, but show ultrastructural defects and fail to template normal axonemes. Similar defects are caused by mutations affecting intraflagellar transport (IFT) in Chlamydomonas, nematodes and mammals. IFT is required for the assembly and maintenance of flagella and cilia, and involves transport of a protein complex along axonemes by kinesin II (reviewed by Rosenbaum, 2002). However, UNC is not part of the core IFT mechanism. IFT particle proteins, unlike UNC, are well-conserved across eukaryotic phyla, including Drosophila. Drosophila IFT complexes are present along sensory cilia (Han, 2003), while UNC-GFP appears only at the basal body. Finally, IFT is not needed to assemble Drosophila sperm flagella: mutants lacking the Drosophila homolog of the IFT88 protein (Han, 2003), or with defects in the kinesin II accessory protein (Sarpal, 2003) have defective sensory cilia but normal sperm (Baker, 2004).
UNC could, however, recruit IFT particles or other axoneme components to the basal body for export to cilia. Its sequence is consistent with such a role. The coiled-coil segments that are its main structural motifs are a common feature of multiprotein complexes, including centrosome and spindle pole body proteins. The several separate segments in UNC may enable it to bring together multiple coiled-coil protein partners, while the aggregates produced by UNC overexpression suggest that it may self-associate. UNC shares a conserved, partial lissencephaly homology (LisH) motif with a number of proteins involved in microtubule organization (Emes, 2001), but the specific function of this motif is unknown. In humans, the gene mutated in oral-facial-digital syndrome (OFD1), a probable ciliary disorder, encodes a protein with a similar arrangement of a LisH domain and coiled-coil segments, which is localized to centrosomes (Ferrante, 2001; Romio, 2003). OFD1 may therefore have a basal body function similar to that of UNC. However, OFD1 failed to rescue unc mutant sensory defects when expressed from a transgene in Drosophila sensory neurons (Baker, 2004).
Centrioles in Drosophila embryos and most somatic cells are composed of doublet microtubules, lack appendages and do not form cilia. Their relatively simple structure, and the absence of structural distinctions between mother and daughter centrioles suggest that they are 'neotenous' -- i.e., they reproduce without undergoing the final stages of maturation (Callaini, 1997). This is consistent with the absence of some otherwise conserved centrosomal proteins from Drosophila and other ecdysozoa. For example, δ- and ε -tubulins, which are conserved from mammals to Chlamydomonas and required for basal body assembly and ciliogenesis, are absent from the sequenced Drosophila, Anopheles and Caenorhabditis genomes. Ninein and cenexin, proteins that identify the ciliogenic centriole in mammals, are also absent from Drosophila. However, Drosophila spermatocyte centrioles are complex, with triplet microtubules and, differentiated distal segments that are, in effect, short primary cilia (Gonzalez, 1998). Other insect spermatocytes have more elongated flagella associated with centrioles before and during meiosis, a feature that first established the identity of centrioles with basal bodies. Spermatocyte and sensory neuron centrioles may be the only centrioles in the fly comparable with mature ciliogenic centrioles in vertebrates, and UNC may substitute for one or more of the proteins that distinguish the ciliogenic centriole in other systems (Baker, 2004).
The cilium on a mammalian monociliated cell forms in G1, specifically on the older of the two centrioles in a cell. By contrast, all four centrioles in Drosophila spermatocytes appear to be equivalent by the time they differentiate: all migrate to the cell periphery and form ciliary extensions. UNC first localizes to spermatocyte centrioles after they have duplicated, and binds equally to all four centrioles. This may reflect a difference between vertebrate and invertebrate ciliogenesis. Alternatively, it may reveal a general uncoupling of centriole duplication and maturation from the cell division cycle in the extended G2 phase that precedes meiosis. In sensory neurons, UNC-GFP is localized to both the proximal and distal basal bodies, both of which are located at the base of the cilium and aligned with its axis. It will be of interest to determine when UNC is first expressed and localized in the neuronal cell lineage (Baker, 2004).
In summary, UNC is required for ciliogenesis, and its localization is an early marker for the conversion of a mitotic centriole into a ciliogenic basal body. Finding the proteins that interact with UNC to localize it on centrioles and mediate its function will be a key to understanding this remarkable transformation, and how it is regulated during entry into meiosis and neuronal differentiation (Baker, 2004).
To test the effects of mechanosensory mutations on hearing in Drosophila, sound-evoked potentials were recorded originating from ciliated sensory neurons in Johnston's organ, the chordotonal organ that is the sensory element of the fly's antennal ear. Electrodes inserted close to the antennal nerve were used to record extracellular compound potentials evoked by near-field sound stimuli. Sound-evoked potentials are absent in atonal mutant flies, which lack Johnston's organ. Mutations in many genes involved in mechanotransduction by tactile bristles also eliminate or reduce the Johnston's organ response, indicating that related transduction mechanisms operate in each type of mechanosensory organ. In addition, the sound-evoked response is affected by two mutations that do not affect bristle mechanotransduction, beethoven (btv) and touch-insensitive-larvaB (tilB). btv shows defects in the ciliary dilation, an elaboration of the axoneme that is characteristic of chordotonal cilia. tilB, which also causes male sterility, shows structural defects in sperm flagellar axonemes. This suggests that in addition to the shared transduction mechanism, axonemal integrity and possibly ciliary motility are required for signal amplification or transduction by chordotonal sensory neurons (Eberl, 2000).
Behavioral screens for mechanosensory defects have identified a set of mutations that affect mechanotransduction in es organs (Kernan, 1994). These include recessive mutations in the uncoordinated (unc) and uncoordinated-like (uncl) genes on the X chromosome and in several no mechanoreceptor potential (nomp) or reduced mechanoreceptor potential (remp) genes on the second chromosome. All of these mutations cause a distinctive type of uncoordination, in which legs are frequently crossed and wings are held up or out. In the most severe cases, mutant adult flies are incapable of walking or righting themselves; however, they still show vigorous, albeit uncoordinated, activity spontaneously or in response to light or harsh mechanical stimuli, indicating that general neuromuscular excitability is retained. Mechanosensory receptor potentials can be recorded directly from single bristle neurons as changes in transepithelial potential evoked by mechanical stimuli. In unc, uncl, and nomp mutants, mechanoreceptor potentials are reduced or absent in thoracic macrochaete bristles (Kernan, 1994). The global touch insensitivity and severe uncoordination characteristic of this class of mutation suggest that smaller tactile and proprioceptive hair-plate bristles are also affected (Eberl, 2000).
To determine whether these mutations also affect transduction by the antennal chordotonal organ, attempts were made to record sound-evoked potentials from the antennal nerves of representative mutant genotypes, using a standard pulse stimulus. Four mutants (unc, uncl, nompA, and nompB) were tested in which bristle mechanoreceptor potentials are invariably absent, and seven mutants (nompC, nompE, nompF, nompI, nompJ, rempA, and rempD) were tested in which the bristle mechanoreceptor potential amplitudes are usually absent or are reduced. The results were striking: all four genotypes that eliminate bristle receptor potentials also eliminated the sound-evoked response. Among the mutations that reduce or variably eliminate bristle receptor potentials, nompF and rempA showed no sound-evoked response, whereas nompE, nompI, and nompJ reduced the response to near-background levels. nompC and rempD showed less severe reductions in response amplitude. Five of the mutants (nompE, nompF, nompI, nompJ, and rempD) are each represented by homozygotes for a single allele; for these genotypes, the possibility of different phenotypes being caused by multiple linked mutations cannot be excluded (Eberl, 2000).
These mutants are all severely uncoordinated and consequently do not survive long as adult flies; all recordings were performed within 1-2 d of eclosion. To test whether a general debilitation affected the sound-evoked response, a mutagenized line, 5-68, was also tested that shows a similar degree of uncoordination but that has normal bristle electrophysiology. The sound-evoked potential amplitudes from this line were in the normal range. To check for more general defects in sensory cell excitability, electroretinograms (ERGs) were also recorded from flies mutant for unc, uncl, and each of the nomp and remp genes. The ERG records a sustained electrical response of retinal photoreceptors to a light stimulus; transient components at the stimulus onset and offset reflect synaptic activity in the optic lamina. All mutants tested except rempD showed electroretinogram responses indistinguishable from wild-type controls, indicating that phototransduction and associated synaptic activity are normal in these mutants. rempD mutants had reduced ERG amplitudes and off-transients: in this one case, a more general defect may affect several sensory modes (Eberl, 2000).
Baker, J. D., Adhikarakunnathu, S. and Kernan, M. J. (2004). Mechanosensory-defective, male-sterile unc mutants identify a novel basal body protein required for ciliogenesis in Drosophila. Development 131(14): 3411-22. PubMed ID: 15226257
Callaini, G., Riparbelli, M. G. and Dallai, R. (1999). Centrosome inheritance in insects: fertilization and parthenogenesis. Biol. Cell 91: 355-366. PubMed ID: 11407409
Eberl, D. F., Hardy, R. W. and Kernan, M. J. (2000). Genetically similar transduction mechanisms for touch and hearing in Drosophila. J. Neurosci. 20(16): 5981-8. PubMed ID: 10934246
Emes, R. D. and Ponting, C. P. (2001). A new sequence motif linking lissencephaly, Treacher Collins and oral-facial-digital type 1 syndromes, microtubule dynamics and cell migration. Hum. Mol. Genet. 10(24): 2813-20. PubMed ID: 11734546
Ferrante, M. I., et al. (2001). Identification of the gene for oral-facial-digital type I syndrome. Am. J. Hum. Genet. 68(3): 569-76. PubMed ID: 11179005
Gonzalez, C., Tavosanis, G. and Mollinari, C. (1998). Centrosomes and microtubule organisation during Drosophila development. J. Cell Sci. 111: 2697-2706. PubMed ID: 9718363
Gottardo, M., Callaini, G. and Riparbelli, M. G. (2016). Does Unc-GFP uncover ciliary structures in the rhabdomeric eye of Drosophila? J Cell Sci 129(14):2726-31. PubMed ID: 27235419
Han, Y.-G., Kwok, B. H. and Kernan, M. J. (2003). Intraflagellar transport is required in Drosophila to differentiate sensory cilia but not sperm. Curr. Biol. 13: 1679-1686. PubMed ID: 14521833
Kernan, M., Cowan, D. and Zuker, C. (1994). Genetic dissection of mechanosensory transduction: mechanoreception-defective mutations of Drosophila. Neuron 12: 1195-1206. PubMed ID: 8011334
Piel, M., Meyer, P., Khodjakov, A., Rieder, C. L. and Bornens, M. (2000). The respective contributions of the mother and daughter centrioles to centrosome activity and behavior in vertebrate cells. J. Cell Biol. 149: 317-330. PubMed ID: 10769025
Romio, L., et al. (2003). OFD1, the gene mutated in oral-facial-digital syndrome type 1, is expressed in the metanephros and in human embryonic renal mesenchymal cells. J. Am. Soc. Nephrol. 14: 680-689. PubMed ID: 12595504
Rosenbaum, J. L. and Witman, G. B. (2002). Intraflagellar transport. Nat. Rev. Mol. Cell Biol. 3: 813-825. PubMed ID: 12415299
Sarpal, R., Todi, S. V., Sivan-Loukianova, E., Shirolikar, S., Subramanian, N., Raff, E. C., Erickson, J. W., Ray, K. and Eberl, D. F. (2003). Drosophila KAP interacts with the kinesin II motor subunit KLP64D to assemble chordotonal sensory cilia, but not sperm tails. Curr. Biol. 13: 1687-1696. PubMed ID: 14521834
date revised: 17 January 2008
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