- Email: [email protected]
Organelle Size: A Cilium Length Signal Regulates IFT Cargo Loading
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system. The extraordinarily early and extensive gene expression in A. suum embryos shows that repression of zygotic gene expression is not a law, but a phenotype that requires explanation. The extended development of A. suum embryos may be permissive of transcription because of reduced conflict with genome replication or remodeling. It is notable that genome methylation is absent or much reduced in chromadorean nematodes (C. elegans and A. suum; methylation is present in enoplean nematodes such as Trichinella spiralis [13]), and thus chromatin remodeling to produce a totipotent state may not be as difficult in these species. Extensive, early zygotic gene expression might be observed in other taxa with extended embryonic division timings. Expression observed in pronuclei may be part of the production of arrested eggs. Is pronuclear gene expression also observed in other dormant eggs? Has A. suum evolved a distinct method of protection against genomic parasites? Is there a mechanistic link with chromatin diminution? The second object lesson from these data is that development evolves, and that the mechanisms and patterns of development are adapted to the life history strategies of the animals they produce. It is particularly striking that C. elegans and A. suum, which have near-identical early cell cycles and cell determination patterns (albeit with very different timings), differ so profoundly in how development is
delivered in the embryo. In C. elegans, the maternal contribution is extensive and essential. In A. suum it is not yet known which components of the maternally provided transcriptome are essential, but it is clear that many mRNAs that are essential and maternal in C. elegans are zygotic in A. suum. The production of the same (or highly similar) outcomes through different mechanisms has been termed developmental system drift: the output remains the same while the underpinning circuitry changes [14]. The extensive expression from zygotic genes from fertilisation in A. suum allows us to revisit the questions of why zygotic gene expression is silenced in many species. Which of the arguments best explain the observed patterns of maternal provisioning and zygotic silence across species? While the model species have revealed some of the answers, only by using a diverse sample of contrasting, but accessible, species can the truth, or falsity, of inferred laws be determined. References 1. van Beneden, E´. (1883). Recherches sur la Maturation de l’Oeuf, la Fecondation et la Division Cellulaire (Gand and Lipzig: Libraire Clemm). 2. Wang, J., Garrey, J., and Davis, R.E. (2014). Transcription in pronuclei and one- to four-cell embryos drives early development in a nematode. Curr. Biol. 24, 124–133. 3. Goldschmid, R. (1908). Das Nervensystem von Ascaris lumbricoides und megalocephala. Zeit. wiss. Zool. 90, 73–136. 4. Sulston, J.E., Schierenberg, E., White, J.G., and Thomson, J.N. (1983). The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev. Biol. 100, 64–119.
Organelle Size: A Cilium Length Signal Regulates IFT Cargo Loading Cilia grow by assembling structural precursors delivered to their tips by intraflagellar transport. New work on ciliary length control indicates that, during ciliary growth, cilia send a length signal to the cytoplasm that regulates cargo loading onto the constitutively trafficking intraflagellar transport machinery. Junmin Pan1 and William J. Snell2,* Almost every cell in vertebrates possesses a primary cilium that plays key sensory roles in development and homeostasis [1]. Although we are beginning to learn the cellular mechanisms for assembling this
organelle, whose structural core is the set of nine outer microtubule doublets that constitute the axoneme, our understanding of the mechanisms that regulate ciliary length has lagged behind [2]. During ciliary assembly, cells use intraflagellar transport (IFT) to deliver ciliary components from the
5. Wang, J., Mitreva, M., Berriman, M., Thorne, A., Magrini, V., Koutsovoulos, G., Kumar, S., Blaxter, M.L., and Davis, R.E. (2012). Silencing of germline-expressed genes by DNA elimination in somatic cells. Dev. Cell 23, 1072–1080. 6. Wang, J., Czech, B., Crunk, A., Wallace, A., Mitreva, M., Hannon, G.J., and Davis, R.E. (2011). Deep small RNA sequencing from the nematode Ascaris reveals conservation, functional diversification, and novel developmental profiles. Genome Res. 21, 1462–1477. 7. Harvey, E.B. (1936). Parthenogenetic merogony or cleavage without nuclei in Arbacia punctulata. Biol. Bull. 71, 101–121. 8. Stitzel, M.L., and Seydoux, G. (2007). Regulation of the oocyte-to-zygote transition. Science 316, 407–408. 9. Baroux, C., Autran, D., Gillmor, C.S., Grimanelli, D., and Grossniklaus, U. (2008). The maternal to zygotic transition in animals and plants. Cold Spring Harb. Symp. Quant. Biol. 73, 89–100. 10. Schier, A.F. (2007). The maternal-zygotic transition: death and birth of RNAs. Science 316, 406–407. 11. Lee, M.T., Bonneau, A.R., Takacs, C.M., Bazzini, A.A., DiVito, K.R., Fleming, E.S., and Giraldez, A.J. (2013). Nanog, Pou5f1 and SoxB1 activate zygotic gene expression during the maternal-to-zygotic transition. Nature 503, 360–364. 12. Schauer, I.E., and Wood, W.B. (1990). Early C. elegans embryos are transcriptionally active. Development 110, 1303–1317. 13. Gao, F., Liu, X., Wu, X.P., Wang, X.L., Gong, D., Lu, H., Xia, Y., Song, Y., Wang, J., Du, J., et al. (2012). Differential DNA methylation in discrete developmental stages of the parasitic nematode Trichinella spiralis. Genome Biol. 13, R100. 14. True, J.R., and Haag, E.S. (2001). Developmental system drift and flexibility in evolutionary trajectories. Evol. Dev. 3, 109–119.
Institute of Evolutionary Biology, The University of Edinburgh, Edinburgh EH9 3JT, UK. E mail: [email protected]
http://dx.doi.org/10.1016/j.cub.2013.11.051
cytoplasm to the ciliary tip [3,4]. The highly conserved IFT machinery has two microtubule motors — an anterograde kinesin-2 and a retrograde cytoplasmic dynein — and a set of associated cargo carriers called IFT particles (themselves composed of IFT-A and IFT-B complexes). The current model for growing a cilium is straightforward: the IFT complexes bind to a ciliary precursor cargo (e.g., a structural component of the axoneme) near the base of the organelle, bind to the anterograde motor, and are carried to the tip of the growing axoneme, where they release their cargo, which assembles on to the end of a growing microtubule doublet of the
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Cargo loading
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Figure 1. Length sensing controls cargo loading to regulate cilium length. Information received in the cytoplasm from an uncharacterized length signal generated during ciliary growth is converted into differential loading of cargo onto constitutively trafficking IFT complexes. Genes encoding CNK2, long flagella proteins LF1 LF5, and CALK (whose T193 phosphorylation state is proportional to cilia length) are implicated in ciliary length control in Chlamydomonas and could participate in length signaling or regulation of cargo loading. (N.B. Only one of the two cilia of Chlamydomonas is depicted.)
axoneme. Length control models before the discovery of IFT posited a length sensor as part of a length-feedback system [5], but, after the characterization of IFT, the notion emerged that cells could control ciliary length by specifying boundary conditions: assign a fixed number of IFT complexes to a cilium, have a fixed proportion of these complexes carrying cargo, and, when the round-trip for an IFT particle to the ciliary tip and back gets to be too long to keep the precursor concentration high enough at the tip, axoneme assembly comes into balance with disassembly [6]. No length sensing required. No need to
regulate cargo loading. In a recent issue of Current Biology, however, Wren et al. [7] elegantly remind us that nothing is as simple as it seems and that you can learn a lot by looking. By using live-cell imaging to track IFT particles and cargo simultaneously, Lechtreck’s group demonstrated first, that soluble axonemal precursors are indeed moved by IFT, and second, that as cilia increase in length, the proportion of IFT particles carrying precursors decreases [7]. Thus, the proportion of (constitutively trafficking) IFT complexes carrying cargo is not fixed, but instead cargo loading is regulated by a length signal that feeds
back to the cytoplasm during ciliary growth (Figure 1). Wren et al. [7] exploited one of the go-to model organisms for many questions in ciliary biology, Chlamydomonas, to dissect ciliary assembly mechanisms. Chlamydomonas cilia (also termed flagella) are easy to visualize and measure, IFT can be experimentally blocked in existing cilia by use of a conditional kinesin-2 mutant, cells can be triggered to grow new cilia by simple methods, ciliary growth can be experimentally blocked at varying organelle lengths, and the behavior of wild-type ciliary proteins introduced into the cytoplasm of ciliary mutants can be examined by imaging zygote cilia within the first hour after coalescence of mutant and wild-type gametes during fertilization. The investigators used total internal reflection fluorescence (TIRF) microscopy to image single molecules of a fluorescently tagged axonemal component, DRC4–GFP, and a fluorescently tagged IFT component, IFT20–mCherry. Photobleaching the existing tagged proteins in cilia allowed Wren et al. [7] to image newly entering proteins. In steady-state cilia, DRC4–GFP was visualized moving in either the anterograde or retrograde direction at typical IFT particle velocities (approximately 1.9 mm/sec and 3.0 mm/sec, respectively), results similar to those reported earlier in Caenorhabditis elegans for tubulin subunits [8]. Moreover, DRC4–GFP co-localized with IFT20–mCherry during anterograde transport, thereby providing robust, direct confirmation of the long-standing model that axonemal precursors travel within cilia as cargoes on IFT particles. Recent work has shown that soluble, non-ciliary proteins can move by diffusion through the ciliary barrier at the base of the cilium, that some membrane proteins can enter the cilium without IFT, and that other membrane proteins can move within cilia independently of IFT [9–13]. Thus, it was possible that diffusion could account for substantial amounts of ciliary entry of soluble, bona fide ciliary components. Direct experimental testing of the entry-by-diffusion model, however, showed that entrance of DRC4–GFP into cilia was abrogated in a temperature-sensitive kinesin-2 mutant in which anterograde IFT was conditionally inhibited. For soluble
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ciliary proteins, then, a second leg of the IFT model was confirmed: IFT carries cargo through the ciliary diffusion barrier into the organelle. The investigators did uncover one variation on IFT dogma. Only about half of the DRC4–GFP made it all the way to the ciliary tip in steady-state cilia. The other half of the cargo was released at varying sites along the length of the cilium where it became incorporated into the axoneme, while the newly cargo-bereft IFT particles continued unabated to the tip. By exploiting the coalescence of gamete cytoplasms during fertilization as a way in which to introduce DRC4–GFP into the cilia produced by drc4 mutant gametes, Wren et al. [7] also showed that the availability of free binding sites on the axoneme determined the duration of diffusion after unloading. The most important finding in this manuscript is that cargo loading is not fixed during ciliary growth, but that loading decreases with increasing length. Evidence has been mounting that cells can regulate both IFT trafficking and cargo loading and that they can sense and respond to ciliary length. For example, when ciliary shortening was experimentally triggered in Chlamydomonas, IFT trafficking in cilia increased immediately (even before shortening could be detected), but the amount of anterograde cargo trafficking in the cilia actually decreased [14]. More recent reports showed that even though cilia contain a fixed amount of IFT complexes, the size and entry frequency of IFT trains (collections of IFT complexes traveling together), as well as the amount of IFT material that accumulated at the bases of the cilia, varied with length [15,16]. And, importantly, the data showing that length can be controlled by genes encoding protein kinases, including the genes disrupted in several long flagella (LF) mutants, have strongly indicated that length control is a highly regulated, complex process that depends on length signals received in the cytoplasm [17–19]. Until recently, at least two elements required in a length-feedback model had been missing, however. Evidence that the LF protein kinases, or other proteins implicated in length control (in Chlamydomonas and other systems), change their properties during growth was needed [2]. And, evidence that loading of cargo onto IFT complexes
is regulated during ciliary growth was also lacking. Recently, the first missing element was uncovered: a change in the properties of a protein coincident with changes in ciliary length. The proportion of the Chlamydomonas aurora-like protein kinase (CALK) phosphorylated on the activity-regulating residue T193 in the kinase activation loop increases during ciliary growth, and decreases as cilia shorten [20]. Although this finding provides the first biochemical marker of ciliary length, it is still unknown whether CALK is a key sensor for ciliary length or an effector of length control that responds to another sensor as part of a length-feedback system. The Lechtreck group [7] has now uncovered the second element needed for a length-feedback model: regulated cargo loading during ciliary growth. They showed that the anterograde cargo transport frequency in short, newly growing cilia was much higher than the cargo transport frequency in cilia at full length, and that this transport frequency decreased proportionately with length. The higher frequency of cargo transport in shorter flagella did not simply reflect that a given number of IFT complexes carrying a fixed proportion of cargo per complex was making more tours through the shorter cilia. Based solely on changes in IFT tour time, the IFT frequency would be expected to decrease approximately 2.4-fold when a cilium lengthens from 5 mm to 12 mm, whereas Wren et al. [7] found that the cargo frequency was actually reduced between 6- and 10-fold. Thus, the increased proportion of IFT complexes carrying cargo in half-length compared with full-length cilia contributed much more to increased cargo entry than did the shorter tour time of IFT complexes. The investigators also showed that the cytoplasmic pool of DRC4–GFP changed very little during ciliary growth and that, even when the pool was experimentally diminished, cargo frequency was unchanged, indicating that cargo availability was not a limiting factor. Importantly, the cargo transport frequency of cilia whose growth was experimentally blocked at half length for several hours was the same as that of control cilia at half length. Thus, the amount of cargo loaded onto IFT particles in the cytoplasm for entry into the cilium is tightly linked to ciliary length. These important findings highlight both new and longstanding questions.
What feature of the length of a cilium is translated into a change in a cytoplasmic molecule that can regulate cargo loading? Does phosphorylation of cargo or IFT particles by the LF protein kinases or CALK regulate loading and does IFT particle protein phosphorylation change with length? A central element of ciliary length models is the as yet untested idea (based on properties of singlet, cytoplasmic microtubules) that assembly of doublet microtubules is driven by the concentration of tubulin dimers at the ciliary tip. Is IFT loading of tubulin subunits regulated similarly to DRC4, or is the supply of tubulin to the tips constant, and is length controlled primarily by regulation of proteins that stabilize or destabilize microtubules [17,18]? It should be exciting to watch the ‘cilia watchers’ as the field of ciliary length control continues to mature. References 1. Goetz, S.C., and Anderson, K.V. (2010). The primary cilium: a signalling centre during vertebrate development. Nat. Rev. Genet. 11, 331–344. 2. Chan, Y.H., and Marshall, W.F. (2012). How cells know the size of their organelles. Science 337, 1186–1189. 3. Rosenbaum, J.L., and Witman, G.B. (2002). Intraflagellar transport. Nat. Rev. Mol. Cell Biol. 3, 813–825. 4. Scholey, J.M. (2003). Intraflagellar transport. Annu. Rev. Cell. Dev. Biol. 19, 423–443. 5. Lefebvre, P.A., Asleson, C.M., and Tam, L.-W. (1995). Control of flagellar length in Chlamydomonas. Semin. Dev. Biol. 6, 317–323. 6. Marshall, W.F., and Rosenbaum, J.L. (2001). Intraflagellar transport balances continuous turnover of outer doublet microtubules: implications for flagellar length control. J. Cell Biol. 155, 405–414. 7. Wren, K.N., Craft, J.M., Tritschler, D., Schauer, A., Patel, D.K., Smith, E.F., Porter, M.E., Kner, P., and Lechtreck, K.F. (2013). A differential cargo loading model of ciliary length regulation by IFT. Curr. Biol. 23, 2463–2471. 8. Hao, L., Thein, M., Brust-Mascher, I., Civelekoglu-Scholey, G., Lu, Y., Acar, S., Prevo, B., Shaham, S., and Scholey, J.M. (2011). Intraflagellar transport delivers tubulin isotypes to sensory cilium middle and distal segments. Nat. Cell Biol. 13, 790–798. 9. Lechtreck, K.F., Brown, J.M., Sampaio, J.L., Craft, J.M., Shevchenko, A., Evans, J.E., and Witman, G.B. (2013). Cycling of the signaling protein phospholipase D through cilia requires the BBSome only for the export phase. J. Cell Biol. 201, 249–261. 10. Belzile, O., Hernandez-Lara, C.I., Wang, Q., and Snell, W.J. (2013). Regulated membrane protein entry into flagella is facilitated by cytoplasmic microtubules and does not require IFT. Curr. Biol. 23, 1460–1465. 11. Kee, H.L., Dishinger, J.F., Blasius, T.L., Liu, C.J., Margolis, B., and Verhey, K.J. (2012). A size-exclusion permeability barrier and nucleoporins characterize a ciliary pore complex that regulates transport into cilia. Nat. Cell Biol. 14, 431–437. 12. Huang, K., Diener, D.R., Mitchell, A., Pazour, G.J., Witman, G.B., and Rosenbaum, J.L. (2007). Function and dynamics of PKD2 in Chlamydomonas. reinhardtii flagella. J. Cell Biol. 179, 501–514.
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17.
18.
19.
20.
Marshall, W.F. (2013). Avalanche-like behavior in ciliary import. Proc. Natl. Acad. Sci. USA 110, 3925–3930. Tam, L.W., Ranum, P.T., and Lefebvre, P.A. (2013). CDKL5 regulates flagellar length and localizes to the base of the flagella in Chlamydomonas. Mol. Biol. Cell 24, 588–600. Hilton, L.K., Gunawardane, K., Kim, J.W., Schwarz, M.C., and Quarmby, L.M. (2013). The kinases LF4 and CNK2 control ciliary length by feedback regulation of assembly and disassembly rates. Curr. Biol. 23, 2208–2214. Berman, S.A., Wilson, N.F., Haas, N.A., and Lefebvre, P.A. (2003). A novel MAP kinase regulates flagellar length in Chlamydomonas. Curr. Biol. 13, 1145–1149. Cao, M., Meng, D., Wang, L., Bei, S., Snell, W.J., and Pan, J. (2013). Activation loop
Insect Vision: Emergence of Pattern Recognition from Coarse Encoding Neurogenetic tools of Drosophila research allow unique access to the neural circuitry underpinning visually guided behaviours. New research is highlighting how particular areas in the fly’s central brain needed for pattern recognition provide a coarse visual encoding.
Animals face complex visual worlds from which they must extract the right type of information to guide behaviour appropriately. How something as small as an insect brain can achieve this given the complexity of natural environments is a fascinating question. We know how insect visual systems extract motion information for flight control [1], polarisation information for course setting [2] or a moving-target for pursuit [3]. But little is known about the visual circuitry involved when insects discriminate patterns of a specific shape, such as flowers for foraging bees [4], panoramas for navigating ants [5] or artificial patterns for tethered Drosophila [6]. In a recent paper, Seelig and Jayaraman [7] have provided descriptions of the visual receptive fields of a population of neurons in a higher brain structure called the central complex, a region known to play a key role in sensory–motor integration in many insect species [2,6,8,9]. This is a significant breakthrough as it provides a description of an entire population of specific visual cells that appear to be involved in pattern recognition [10]. Seelig and Jayaraman [7] combined two-photon calcium imaging with the neurogenetic tools available to
researchers working on the fruitfly Drosophila melanogaster to observe in vivo how populations of neurons in
1MOE Key Laboratory of Protein Science, School of Life Sciences, Tsinghua University, Beijing 100084, China. 2Department of Cell Biology, University of Texas Southwestern Medical School, Dallas, TX 75390, USA. *E-mail: [email protected]
http://dx.doi.org/10.1016/j.cub.2013.11.043
the fly’s central brain respond to visual stimuli. They targeted the ring neurons R2 and R3/R4d; these receive input from glomeruli in the lateral triangle — presumably after preprocessing in the sensory areas — and project to the ellipsoid body of the central complex [11] (Figure 1A). By presenting black and white noise patterns to Drosophila and correlating the visual stimulus with cell responses, Seelig and Jayaraman [7] were able to determine the proprieties of the cell’s
A
C Centrall Cent Central comple complex omple p x
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Antoine Wystrach*, Alex D.M. Dewar, and Paul Graham
phosphorylation of a protein kinase is a molecular marker of organelle size that dynamically reports flagellar length. Proc. Natl. Acad. Sci. USA 110, 12337–12342.
270°
Left hemisphere R2 Cells
Activation
13. Ye, F., Breslow, D.K., Koslover, E.F., Spakowitz, A.J., Nelson, W.J., and Nachury, M.V. (2013). Single molecule imaging reveals a major role for diffusion in the exploration of ciliary space by signaling receptors. eLife 2, e00654. 14. Pan, J., and Snell, W.J. (2005). Chlamydomonas. shortens its flagella by activating axonemal disassembly, stimulating IFT particle trafficking, and blocking anterograde cargo loading. Dev. Cell 9, 431–438. 15. Engel, B.D., Ludington, W.B., and Marshall, W.F. (2009). Intraflagellar transport particle size scales inversely with flagellar length: revisiting the balance-point length control model. J. Cell Biol. 187, 81–89. 16. Ludington, W.B., Wemmer, K.A., Lechtreck, K.F., Witman, G.B., and
1 0 -1 Current Biology
Figure 1. Ring neurons of Drosophila melanogaster central complex encode coarse visual information. (A) Schematic of the fly’s brain (top) and ellipsoid body of the central complex (bottom) (after [7]). The ring neurons receive input from the glomeruli of the lateral triangles and project to specific regions of the ellipsoid body. The different regions, and thus the different ring neurons play different roles in visuo-spatial behaviour. (B) Receptive fields of the 14 left hemisphere cells of R2 ring neurons that responded to visual stimuli, showing their excitatory (red) and inhibitory (blue) regions. The receptive field’s shape and location on the visual field for each cell is stereotypic and coherent across flies [7]. Thus, those depicted here have been averaged across flies. (C) The same receptive fields as B overlaid on the fly’s visual field. Note, the cells shown are all from the left brain. Each receptive field covers 90 or more of the fly’s visual field. (D) Response of a R2 cell to vertical and horizontal bars. Since the inhibitory area is to the side of the excitatory one, the neuron responds maximally to vertically oriented bars.
system. The extraordinarily early and extensive gene expression in A. suum embryos shows that repression of zygotic gene expression is not a law, but a phenotype that requires explanation. The extended development of A. suum embryos may be permissive of transcription because of reduced conflict with genome replication or remodeling. It is notable that genome methylation is absent or much reduced in chromadorean nematodes (C. elegans and A. suum; methylation is present in enoplean nematodes such as Trichinella spiralis [13]), and thus chromatin remodeling to produce a totipotent state may not be as difficult in these species. Extensive, early zygotic gene expression might be observed in other taxa with extended embryonic division timings. Expression observed in pronuclei may be part of the production of arrested eggs. Is pronuclear gene expression also observed in other dormant eggs? Has A. suum evolved a distinct method of protection against genomic parasites? Is there a mechanistic link with chromatin diminution? The second object lesson from these data is that development evolves, and that the mechanisms and patterns of development are adapted to the life history strategies of the animals they produce. It is particularly striking that C. elegans and A. suum, which have near-identical early cell cycles and cell determination patterns (albeit with very different timings), differ so profoundly in how development is
delivered in the embryo. In C. elegans, the maternal contribution is extensive and essential. In A. suum it is not yet known which components of the maternally provided transcriptome are essential, but it is clear that many mRNAs that are essential and maternal in C. elegans are zygotic in A. suum. The production of the same (or highly similar) outcomes through different mechanisms has been termed developmental system drift: the output remains the same while the underpinning circuitry changes [14]. The extensive expression from zygotic genes from fertilisation in A. suum allows us to revisit the questions of why zygotic gene expression is silenced in many species. Which of the arguments best explain the observed patterns of maternal provisioning and zygotic silence across species? While the model species have revealed some of the answers, only by using a diverse sample of contrasting, but accessible, species can the truth, or falsity, of inferred laws be determined. References 1. van Beneden, E´. (1883). Recherches sur la Maturation de l’Oeuf, la Fecondation et la Division Cellulaire (Gand and Lipzig: Libraire Clemm). 2. Wang, J., Garrey, J., and Davis, R.E. (2014). Transcription in pronuclei and one- to four-cell embryos drives early development in a nematode. Curr. Biol. 24, 124–133. 3. Goldschmid, R. (1908). Das Nervensystem von Ascaris lumbricoides und megalocephala. Zeit. wiss. Zool. 90, 73–136. 4. Sulston, J.E., Schierenberg, E., White, J.G., and Thomson, J.N. (1983). The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev. Biol. 100, 64–119.
Organelle Size: A Cilium Length Signal Regulates IFT Cargo Loading Cilia grow by assembling structural precursors delivered to their tips by intraflagellar transport. New work on ciliary length control indicates that, during ciliary growth, cilia send a length signal to the cytoplasm that regulates cargo loading onto the constitutively trafficking intraflagellar transport machinery. Junmin Pan1 and William J. Snell2,* Almost every cell in vertebrates possesses a primary cilium that plays key sensory roles in development and homeostasis [1]. Although we are beginning to learn the cellular mechanisms for assembling this
organelle, whose structural core is the set of nine outer microtubule doublets that constitute the axoneme, our understanding of the mechanisms that regulate ciliary length has lagged behind [2]. During ciliary assembly, cells use intraflagellar transport (IFT) to deliver ciliary components from the
5. Wang, J., Mitreva, M., Berriman, M., Thorne, A., Magrini, V., Koutsovoulos, G., Kumar, S., Blaxter, M.L., and Davis, R.E. (2012). Silencing of germline-expressed genes by DNA elimination in somatic cells. Dev. Cell 23, 1072–1080. 6. Wang, J., Czech, B., Crunk, A., Wallace, A., Mitreva, M., Hannon, G.J., and Davis, R.E. (2011). Deep small RNA sequencing from the nematode Ascaris reveals conservation, functional diversification, and novel developmental profiles. Genome Res. 21, 1462–1477. 7. Harvey, E.B. (1936). Parthenogenetic merogony or cleavage without nuclei in Arbacia punctulata. Biol. Bull. 71, 101–121. 8. Stitzel, M.L., and Seydoux, G. (2007). Regulation of the oocyte-to-zygote transition. Science 316, 407–408. 9. Baroux, C., Autran, D., Gillmor, C.S., Grimanelli, D., and Grossniklaus, U. (2008). The maternal to zygotic transition in animals and plants. Cold Spring Harb. Symp. Quant. Biol. 73, 89–100. 10. Schier, A.F. (2007). The maternal-zygotic transition: death and birth of RNAs. Science 316, 406–407. 11. Lee, M.T., Bonneau, A.R., Takacs, C.M., Bazzini, A.A., DiVito, K.R., Fleming, E.S., and Giraldez, A.J. (2013). Nanog, Pou5f1 and SoxB1 activate zygotic gene expression during the maternal-to-zygotic transition. Nature 503, 360–364. 12. Schauer, I.E., and Wood, W.B. (1990). Early C. elegans embryos are transcriptionally active. Development 110, 1303–1317. 13. Gao, F., Liu, X., Wu, X.P., Wang, X.L., Gong, D., Lu, H., Xia, Y., Song, Y., Wang, J., Du, J., et al. (2012). Differential DNA methylation in discrete developmental stages of the parasitic nematode Trichinella spiralis. Genome Biol. 13, R100. 14. True, J.R., and Haag, E.S. (2001). Developmental system drift and flexibility in evolutionary trajectories. Evol. Dev. 3, 109–119.
Institute of Evolutionary Biology, The University of Edinburgh, Edinburgh EH9 3JT, UK. E mail: [email protected]
http://dx.doi.org/10.1016/j.cub.2013.11.051
cytoplasm to the ciliary tip [3,4]. The highly conserved IFT machinery has two microtubule motors — an anterograde kinesin-2 and a retrograde cytoplasmic dynein — and a set of associated cargo carriers called IFT particles (themselves composed of IFT-A and IFT-B complexes). The current model for growing a cilium is straightforward: the IFT complexes bind to a ciliary precursor cargo (e.g., a structural component of the axoneme) near the base of the organelle, bind to the anterograde motor, and are carried to the tip of the growing axoneme, where they release their cargo, which assembles on to the end of a growing microtubule doublet of the
Current Biology Vol 24 No 2 R76
Cargo IFT particle
T193P/total CALK
Cargo loading
Cargo loading
Cargo loading
Length signal
Length signal
Length signal
Current Biology
Figure 1. Length sensing controls cargo loading to regulate cilium length. Information received in the cytoplasm from an uncharacterized length signal generated during ciliary growth is converted into differential loading of cargo onto constitutively trafficking IFT complexes. Genes encoding CNK2, long flagella proteins LF1 LF5, and CALK (whose T193 phosphorylation state is proportional to cilia length) are implicated in ciliary length control in Chlamydomonas and could participate in length signaling or regulation of cargo loading. (N.B. Only one of the two cilia of Chlamydomonas is depicted.)
axoneme. Length control models before the discovery of IFT posited a length sensor as part of a length-feedback system [5], but, after the characterization of IFT, the notion emerged that cells could control ciliary length by specifying boundary conditions: assign a fixed number of IFT complexes to a cilium, have a fixed proportion of these complexes carrying cargo, and, when the round-trip for an IFT particle to the ciliary tip and back gets to be too long to keep the precursor concentration high enough at the tip, axoneme assembly comes into balance with disassembly [6]. No length sensing required. No need to
regulate cargo loading. In a recent issue of Current Biology, however, Wren et al. [7] elegantly remind us that nothing is as simple as it seems and that you can learn a lot by looking. By using live-cell imaging to track IFT particles and cargo simultaneously, Lechtreck’s group demonstrated first, that soluble axonemal precursors are indeed moved by IFT, and second, that as cilia increase in length, the proportion of IFT particles carrying precursors decreases [7]. Thus, the proportion of (constitutively trafficking) IFT complexes carrying cargo is not fixed, but instead cargo loading is regulated by a length signal that feeds
back to the cytoplasm during ciliary growth (Figure 1). Wren et al. [7] exploited one of the go-to model organisms for many questions in ciliary biology, Chlamydomonas, to dissect ciliary assembly mechanisms. Chlamydomonas cilia (also termed flagella) are easy to visualize and measure, IFT can be experimentally blocked in existing cilia by use of a conditional kinesin-2 mutant, cells can be triggered to grow new cilia by simple methods, ciliary growth can be experimentally blocked at varying organelle lengths, and the behavior of wild-type ciliary proteins introduced into the cytoplasm of ciliary mutants can be examined by imaging zygote cilia within the first hour after coalescence of mutant and wild-type gametes during fertilization. The investigators used total internal reflection fluorescence (TIRF) microscopy to image single molecules of a fluorescently tagged axonemal component, DRC4–GFP, and a fluorescently tagged IFT component, IFT20–mCherry. Photobleaching the existing tagged proteins in cilia allowed Wren et al. [7] to image newly entering proteins. In steady-state cilia, DRC4–GFP was visualized moving in either the anterograde or retrograde direction at typical IFT particle velocities (approximately 1.9 mm/sec and 3.0 mm/sec, respectively), results similar to those reported earlier in Caenorhabditis elegans for tubulin subunits [8]. Moreover, DRC4–GFP co-localized with IFT20–mCherry during anterograde transport, thereby providing robust, direct confirmation of the long-standing model that axonemal precursors travel within cilia as cargoes on IFT particles. Recent work has shown that soluble, non-ciliary proteins can move by diffusion through the ciliary barrier at the base of the cilium, that some membrane proteins can enter the cilium without IFT, and that other membrane proteins can move within cilia independently of IFT [9–13]. Thus, it was possible that diffusion could account for substantial amounts of ciliary entry of soluble, bona fide ciliary components. Direct experimental testing of the entry-by-diffusion model, however, showed that entrance of DRC4–GFP into cilia was abrogated in a temperature-sensitive kinesin-2 mutant in which anterograde IFT was conditionally inhibited. For soluble
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ciliary proteins, then, a second leg of the IFT model was confirmed: IFT carries cargo through the ciliary diffusion barrier into the organelle. The investigators did uncover one variation on IFT dogma. Only about half of the DRC4–GFP made it all the way to the ciliary tip in steady-state cilia. The other half of the cargo was released at varying sites along the length of the cilium where it became incorporated into the axoneme, while the newly cargo-bereft IFT particles continued unabated to the tip. By exploiting the coalescence of gamete cytoplasms during fertilization as a way in which to introduce DRC4–GFP into the cilia produced by drc4 mutant gametes, Wren et al. [7] also showed that the availability of free binding sites on the axoneme determined the duration of diffusion after unloading. The most important finding in this manuscript is that cargo loading is not fixed during ciliary growth, but that loading decreases with increasing length. Evidence has been mounting that cells can regulate both IFT trafficking and cargo loading and that they can sense and respond to ciliary length. For example, when ciliary shortening was experimentally triggered in Chlamydomonas, IFT trafficking in cilia increased immediately (even before shortening could be detected), but the amount of anterograde cargo trafficking in the cilia actually decreased [14]. More recent reports showed that even though cilia contain a fixed amount of IFT complexes, the size and entry frequency of IFT trains (collections of IFT complexes traveling together), as well as the amount of IFT material that accumulated at the bases of the cilia, varied with length [15,16]. And, importantly, the data showing that length can be controlled by genes encoding protein kinases, including the genes disrupted in several long flagella (LF) mutants, have strongly indicated that length control is a highly regulated, complex process that depends on length signals received in the cytoplasm [17–19]. Until recently, at least two elements required in a length-feedback model had been missing, however. Evidence that the LF protein kinases, or other proteins implicated in length control (in Chlamydomonas and other systems), change their properties during growth was needed [2]. And, evidence that loading of cargo onto IFT complexes
is regulated during ciliary growth was also lacking. Recently, the first missing element was uncovered: a change in the properties of a protein coincident with changes in ciliary length. The proportion of the Chlamydomonas aurora-like protein kinase (CALK) phosphorylated on the activity-regulating residue T193 in the kinase activation loop increases during ciliary growth, and decreases as cilia shorten [20]. Although this finding provides the first biochemical marker of ciliary length, it is still unknown whether CALK is a key sensor for ciliary length or an effector of length control that responds to another sensor as part of a length-feedback system. The Lechtreck group [7] has now uncovered the second element needed for a length-feedback model: regulated cargo loading during ciliary growth. They showed that the anterograde cargo transport frequency in short, newly growing cilia was much higher than the cargo transport frequency in cilia at full length, and that this transport frequency decreased proportionately with length. The higher frequency of cargo transport in shorter flagella did not simply reflect that a given number of IFT complexes carrying a fixed proportion of cargo per complex was making more tours through the shorter cilia. Based solely on changes in IFT tour time, the IFT frequency would be expected to decrease approximately 2.4-fold when a cilium lengthens from 5 mm to 12 mm, whereas Wren et al. [7] found that the cargo frequency was actually reduced between 6- and 10-fold. Thus, the increased proportion of IFT complexes carrying cargo in half-length compared with full-length cilia contributed much more to increased cargo entry than did the shorter tour time of IFT complexes. The investigators also showed that the cytoplasmic pool of DRC4–GFP changed very little during ciliary growth and that, even when the pool was experimentally diminished, cargo frequency was unchanged, indicating that cargo availability was not a limiting factor. Importantly, the cargo transport frequency of cilia whose growth was experimentally blocked at half length for several hours was the same as that of control cilia at half length. Thus, the amount of cargo loaded onto IFT particles in the cytoplasm for entry into the cilium is tightly linked to ciliary length. These important findings highlight both new and longstanding questions.
What feature of the length of a cilium is translated into a change in a cytoplasmic molecule that can regulate cargo loading? Does phosphorylation of cargo or IFT particles by the LF protein kinases or CALK regulate loading and does IFT particle protein phosphorylation change with length? A central element of ciliary length models is the as yet untested idea (based on properties of singlet, cytoplasmic microtubules) that assembly of doublet microtubules is driven by the concentration of tubulin dimers at the ciliary tip. Is IFT loading of tubulin subunits regulated similarly to DRC4, or is the supply of tubulin to the tips constant, and is length controlled primarily by regulation of proteins that stabilize or destabilize microtubules [17,18]? It should be exciting to watch the ‘cilia watchers’ as the field of ciliary length control continues to mature. References 1. Goetz, S.C., and Anderson, K.V. (2010). The primary cilium: a signalling centre during vertebrate development. Nat. Rev. Genet. 11, 331–344. 2. Chan, Y.H., and Marshall, W.F. (2012). How cells know the size of their organelles. Science 337, 1186–1189. 3. Rosenbaum, J.L., and Witman, G.B. (2002). Intraflagellar transport. Nat. Rev. Mol. Cell Biol. 3, 813–825. 4. Scholey, J.M. (2003). Intraflagellar transport. Annu. Rev. Cell. Dev. Biol. 19, 423–443. 5. Lefebvre, P.A., Asleson, C.M., and Tam, L.-W. (1995). Control of flagellar length in Chlamydomonas. Semin. Dev. Biol. 6, 317–323. 6. Marshall, W.F., and Rosenbaum, J.L. (2001). Intraflagellar transport balances continuous turnover of outer doublet microtubules: implications for flagellar length control. J. Cell Biol. 155, 405–414. 7. Wren, K.N., Craft, J.M., Tritschler, D., Schauer, A., Patel, D.K., Smith, E.F., Porter, M.E., Kner, P., and Lechtreck, K.F. (2013). A differential cargo loading model of ciliary length regulation by IFT. Curr. Biol. 23, 2463–2471. 8. Hao, L., Thein, M., Brust-Mascher, I., Civelekoglu-Scholey, G., Lu, Y., Acar, S., Prevo, B., Shaham, S., and Scholey, J.M. (2011). Intraflagellar transport delivers tubulin isotypes to sensory cilium middle and distal segments. Nat. Cell Biol. 13, 790–798. 9. Lechtreck, K.F., Brown, J.M., Sampaio, J.L., Craft, J.M., Shevchenko, A., Evans, J.E., and Witman, G.B. (2013). Cycling of the signaling protein phospholipase D through cilia requires the BBSome only for the export phase. J. Cell Biol. 201, 249–261. 10. Belzile, O., Hernandez-Lara, C.I., Wang, Q., and Snell, W.J. (2013). Regulated membrane protein entry into flagella is facilitated by cytoplasmic microtubules and does not require IFT. Curr. Biol. 23, 1460–1465. 11. Kee, H.L., Dishinger, J.F., Blasius, T.L., Liu, C.J., Margolis, B., and Verhey, K.J. (2012). A size-exclusion permeability barrier and nucleoporins characterize a ciliary pore complex that regulates transport into cilia. Nat. Cell Biol. 14, 431–437. 12. Huang, K., Diener, D.R., Mitchell, A., Pazour, G.J., Witman, G.B., and Rosenbaum, J.L. (2007). Function and dynamics of PKD2 in Chlamydomonas. reinhardtii flagella. J. Cell Biol. 179, 501–514.
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Marshall, W.F. (2013). Avalanche-like behavior in ciliary import. Proc. Natl. Acad. Sci. USA 110, 3925–3930. Tam, L.W., Ranum, P.T., and Lefebvre, P.A. (2013). CDKL5 regulates flagellar length and localizes to the base of the flagella in Chlamydomonas. Mol. Biol. Cell 24, 588–600. Hilton, L.K., Gunawardane, K., Kim, J.W., Schwarz, M.C., and Quarmby, L.M. (2013). The kinases LF4 and CNK2 control ciliary length by feedback regulation of assembly and disassembly rates. Curr. Biol. 23, 2208–2214. Berman, S.A., Wilson, N.F., Haas, N.A., and Lefebvre, P.A. (2003). A novel MAP kinase regulates flagellar length in Chlamydomonas. Curr. Biol. 13, 1145–1149. Cao, M., Meng, D., Wang, L., Bei, S., Snell, W.J., and Pan, J. (2013). Activation loop
Insect Vision: Emergence of Pattern Recognition from Coarse Encoding Neurogenetic tools of Drosophila research allow unique access to the neural circuitry underpinning visually guided behaviours. New research is highlighting how particular areas in the fly’s central brain needed for pattern recognition provide a coarse visual encoding.
Animals face complex visual worlds from which they must extract the right type of information to guide behaviour appropriately. How something as small as an insect brain can achieve this given the complexity of natural environments is a fascinating question. We know how insect visual systems extract motion information for flight control [1], polarisation information for course setting [2] or a moving-target for pursuit [3]. But little is known about the visual circuitry involved when insects discriminate patterns of a specific shape, such as flowers for foraging bees [4], panoramas for navigating ants [5] or artificial patterns for tethered Drosophila [6]. In a recent paper, Seelig and Jayaraman [7] have provided descriptions of the visual receptive fields of a population of neurons in a higher brain structure called the central complex, a region known to play a key role in sensory–motor integration in many insect species [2,6,8,9]. This is a significant breakthrough as it provides a description of an entire population of specific visual cells that appear to be involved in pattern recognition [10]. Seelig and Jayaraman [7] combined two-photon calcium imaging with the neurogenetic tools available to
researchers working on the fruitfly Drosophila melanogaster to observe in vivo how populations of neurons in
1MOE Key Laboratory of Protein Science, School of Life Sciences, Tsinghua University, Beijing 100084, China. 2Department of Cell Biology, University of Texas Southwestern Medical School, Dallas, TX 75390, USA. *E-mail: [email protected]
http://dx.doi.org/10.1016/j.cub.2013.11.043
the fly’s central brain respond to visual stimuli. They targeted the ring neurons R2 and R3/R4d; these receive input from glomeruli in the lateral triangle — presumably after preprocessing in the sensory areas — and project to the ellipsoid body of the central complex [11] (Figure 1A). By presenting black and white noise patterns to Drosophila and correlating the visual stimulus with cell responses, Seelig and Jayaraman [7] were able to determine the proprieties of the cell’s
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Antoine Wystrach*, Alex D.M. Dewar, and Paul Graham
phosphorylation of a protein kinase is a molecular marker of organelle size that dynamically reports flagellar length. Proc. Natl. Acad. Sci. USA 110, 12337–12342.
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13. Ye, F., Breslow, D.K., Koslover, E.F., Spakowitz, A.J., Nelson, W.J., and Nachury, M.V. (2013). Single molecule imaging reveals a major role for diffusion in the exploration of ciliary space by signaling receptors. eLife 2, e00654. 14. Pan, J., and Snell, W.J. (2005). Chlamydomonas. shortens its flagella by activating axonemal disassembly, stimulating IFT particle trafficking, and blocking anterograde cargo loading. Dev. Cell 9, 431–438. 15. Engel, B.D., Ludington, W.B., and Marshall, W.F. (2009). Intraflagellar transport particle size scales inversely with flagellar length: revisiting the balance-point length control model. J. Cell Biol. 187, 81–89. 16. Ludington, W.B., Wemmer, K.A., Lechtreck, K.F., Witman, G.B., and
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Figure 1. Ring neurons of Drosophila melanogaster central complex encode coarse visual information. (A) Schematic of the fly’s brain (top) and ellipsoid body of the central complex (bottom) (after [7]). The ring neurons receive input from the glomeruli of the lateral triangles and project to specific regions of the ellipsoid body. The different regions, and thus the different ring neurons play different roles in visuo-spatial behaviour. (B) Receptive fields of the 14 left hemisphere cells of R2 ring neurons that responded to visual stimuli, showing their excitatory (red) and inhibitory (blue) regions. The receptive field’s shape and location on the visual field for each cell is stereotypic and coherent across flies [7]. Thus, those depicted here have been averaged across flies. (C) The same receptive fields as B overlaid on the fly’s visual field. Note, the cells shown are all from the left brain. Each receptive field covers 90 or more of the fly’s visual field. (D) Response of a R2 cell to vertical and horizontal bars. Since the inhibitory area is to the side of the excitatory one, the neuron responds maximally to vertically oriented bars.