Current Biology Vol 25 No 4 R148
dopamine neurons suggest that tonic versus burst firing would result in differences in the relative occupancy of receptors with different affinities [18]. In support, pharmacological experiments in zebrafish have demonstrated that dopamine can shorten the duration of drug-evoked episodic swimming bouts in spinalized larvae, something that is attributed to the progressive innervation of the spinal cord by DDNs [7]. However, this observation is difficult to reconcile with the fact that early ablation of DDNs produced no effects on the patterning of spontaneous, real swimming. While the authors suggest possible explanations for this discrepancy, including potential off-target effects of the pharmacological manipulations, it remains to be seen exactly what DDN bursting contributes to zebrafish locomotion. In this sense, the work by Jay et al. [1] achieves the goal of all high quality studies, in that it generates more questions than it answers. The description of different modes of firing not only helps put pharmacological observations in a proper context, but also provides a framework for investigating how dynamic changes in dopamine levels in the spinal cord and elsewhere may exert differential effects on locomotor behavior. Are different dopamine receptor subtypes located on the same or different spinal circuit elements? How about targets in the brainstem or the periphery? Does the transition from tonic to burst firing orchestrate a common behavioral goal via these distributed targets? If so, what is this behavior? The ability to replace GFP in ETvmat2:GFP fish with
genes that drive optogenetic actuators to activate or silence DDNs [19], and the development of closed loop systems that drive more complex larval behaviors [20], make it likely that answers to these questions are not far off. Given the conserved genetic origins of DDNs and the similarity in their activity patterns to mammalian dopamine neurons, the zebrafish model system will surely be a reliable source for principles underlying the modulation of circuits and behavior by dopamine in years to come. References 1. Jay, M., De Faveri, F., and McDearmid, J.R. (2015). Firing dynamics and modulatory actions of supraspinal dopaminergic neurons during zebrafish locomotor behavior. Curr. Biol. 25, 435–444. 2. Jankowska, E., Jukes, M.G., Lund, S., and Lundberg, A. (1967). The effect of DOPA on the spinal cord. 5. Reciprocal organization of pathways transmitting excitatory action to alpha motoneurones of flexors and extensors. Acta Physiol. Scand. 70, 369–388. 3. Grillner, S., and Jessell, T.M. (2009). Measured motion: searching for simplicity in spinal locomotor networks. Curr. Opin. Neurobiol. 19, 572–586. 4. Miles, G.B., and Sillar, K.T. (2011). Neuromodulation of vertebrate locomotor control networks. Physiology 26, 393–411. 5. Missale, C., Nash, S.R., Robinson, S.W., Jaber, M., and Caron, M.G. (1998). Dopamine receptors: from structure to function. Physiol. Rev. 78, 189–225. 6. Clemens, S., Belin-Rauscent, A., Simmers, J., and Combes, D. (2012). Opposing modulatory effects of D1- and D2-like receptor activation on a spinal central pattern generator. J. Neurophysiol. 107, 2250–2259. 7. Lambert, A.M., Bonkowsky, J.L., and Masino, M.A. (2012). The conserved dopaminergic diencephalospinal tract mediates vertebrate locomotor development in zebrafish larvae. J. Neurosci. 32, 13488–13500. 8. Thirumalai, V., and Cline, H.T. (2008). Endogenous dopamine suppresses initiation of swimming in prefeeding zebrafish larvae. J. Neurophysiol. 100, 1635–1648. 9. Tay, T.L., Ronneberger, O., Ryu, S., Nitschke, R., and Driever, W. (2011). Comprehensive catecholaminergic projectome analysis reveals
Photoreceptor Evolution: Ancient ‘Cones’ Turn Out to Be Rods Vertebrate rod photoreceptors are thought to have evolved from cone photoreceptors only after the divergence of the jawed and jawless fishes, but this idea is questioned by new evidence that the short ‘cones’ of jawless sea lampreys are physiologically equivalent to rods. Eric J. Warrant About 540 million years ago one of the most spectacular events in the history of the evolution of animal life
began: in the space of just 20 million years — a blink of an eye in geological terms — many of our familiar modern animal lineages suddenly appeared on the Earth. This explosion of
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
single-neuron integration of zebrafish ascending and descending dopaminergic systems. Nat. Commun. 2, 171. McLean, D.L., and Fetcho, J.R. (2004). Relationship of tyrosine hydroxylase and serotonin immunoreactivity to sensorimotor circuitry in larval zebrafish. J. Comp. Neurol. 480, 57–71. Koblinger, K., Fuzesi, T., Ejdrygiewicz, J., Krajacic, A., Bains, J.S., and Whelan, P.J. (2014). Characterization of A11 neurons projecting to the spinal cord of mice. PLoS One 9, e109636. Wen, L., Wei, W., Gu, W., Huang, P., Ren, X., Zhang, Z., Zhu, Z., Lin, S., and Zhang, B. (2008). Visualization of monoaminergic neurons and neurotoxicity of MPTP in live transgenic zebrafish. Dev. Biol. 314, 84–92. Surmeier, D.J., Mercer, J.N., and Chan, C.S. (2005). Autonomous pacemakers in the basal ganglia: who needs excitatory synapses anyway? Curr. Opin. Neurobiol. 15, 312–318. Tsai, H.C., Zhang, F., Adamantidis, A., Stuber, G.D., Bonci, A., de Lecea, L., and Deisseroth, K. (2009). Phasic firing in dopaminergic neurons is sufficient for behavioral conditioning. Science 324, 1080–1084. Buss, R.R., and Drapeau, P. (2001). Synaptic drive to motoneurons during fictive swimming in the developing zebrafish. J. Neurophysiol. 86, 197–210. Veasey, S.C., Fornal, C.A., Metzler, C.W., and Jacobs, B.L. (1995). Response of serotonergic caudal raphe neurons in relation to specific motor activities in freely moving cats. J. Neurosci. 15, 5346–5359. McLean, D.L., and Fetcho, J.R. (2004). Ontogeny and innervation patterns of dopaminergic, noradrenergic, and serotonergic neurons in larval zebrafish. J. Comp. Neurol. 480, 38–56. Dreyer, J.K., Herrik, K.F., Berg, R.W., and Hounsgaard, J.D. (2010). Influence of phasic and tonic dopamine release on receptor activation. J. Neurosci. 30, 14273–14283. Auer, T.O., Duroure, K., Concordet, J.P., and Del Bene, F. (2014). CRISPR/Cas9-mediated conversion of eGFP- into Gal4-transgenic lines in zebrafish. Nat. Protoc. 9, 2823–2840. Engert, F. (2012). Fish in the matrix: motor learning in a virtual world. Front. Neural Circuits 6, 125.
Department of Neurobiology, Northwestern University, Evanston, IL 60208, USA. *E-mail: [email protected]
http://dx.doi.org/10.1016/j.cub.2015.01.001
new animal forms ushered in the Cambrian epoch, and at its end, a little more than 500 million years ago, the earliest true vertebrates appeared. These were the so-called jawless fishes, or Agnathans, of which only two lineages survive until the present day, the hagfishes and the lampreys. From the Agnatha evolved the jawed fishes, or Gnathostomes, and from these arose all the vertebrate lineages we are familiar with today, including our own. The eyes of these early jawed fishes were probably very much like
Dispatch R149
our eyes – well developed camera eyes with a duplex retina containing rod and cone photoreceptors. In contrast, the eyes of their jawless ancestors — if the eyes of present day hagfishes and lampreys are any indication — were probably quite different. Hagfishes have rudimentary lensless eyes with a reduced retina and a single photoreceptor class of unknown type. Lampreys, in contrast, have well-developed camera eyes, with a sophisticated retina containing at least two classes of photoreceptors — all of these, however, morphologically resemble cones. In the sea lamprey Petromyzon marinus (Figure 1) there are two classes of these cone-like photoreceptors, one ‘long’ and one ‘short’. Because rods are known to have evolved from cones, the apparently rod-free retinas of hagfishes and lampreys have led to the conjecture that the modern vertebrate duplex retina evolved only after the jawed fishes diverged from the jawless fishes [1,2]. Now, in a landmark study reported in this issue of Current Biology, Morshedian and Fain [3] have discovered that the sea lamprey short photoreceptor — despite having a cone-like morphology — is physiologically much more like a rod than a cone, notably (and critically) having the necessary sensitivity to respond to single photons of light, a hallmark property of rods. This discovery not only implies that the vertebrate duplex retina evolved prior to the divergence of the jawed and jawless fishes, it also suggests that the classic rod outer segment morphology, with cytosolic disks surrounded by a continuous plasma membrane, is not necessary for the high-gain transduction of single photon responses. Rods and cones, the defining elements of the vertebrate duplex retina, have evolved to give us highly sensitive but coarse monochromatic scotopic vision at night (rods) and highly resolved photopic colour vision during the day (cones). Not surprisingly, they differ significantly from each other in several key respects (Figure 2), both in terms of morphology and physiology (reviewed in [2]). Morphologically, the most obvious
Figure 1. The sea lamprey Petromyzon marinus. (A) The gills slits and well developed eyes. (B) The mouth with its concentric rows of horny teeth. Images used with kind permission of the photographers: A. Muir (A) and T. Lawrence (B) of the Great Lakes Fishery Commission, USA.
distinguishing feature is the topology of the membrane of the outer segment, the region of the photoreceptor housing the visual pigment and responsible for the absorption of light and its transduction into an electrical signal. In cones, the entire outer segment membrane is continuous with the plasma membrane of the inner segment, creating a stack of plate-like lamellae. The outer segment so created is cylindrical in mammals, but somewhat conical in other vertebrates. In rods, these lamellae become internalised during the synthesis of the outer segment, creating a stack of membrane-bound disks, whose membranes are separated from the main plasma membrane (and thus from the extracellular matrix). Other morphological differences — such as the presence of an ellipsoid in cones (filled with mitochondria or a spectrally filtering oil droplet), deep longitudinal incisures in the outer
segment of rods (thought to increase the diffusion of intracellular messengers) and considerable differences in the morphologies of their synaptic terminals — further distinguish rods from cones. Physiologically, the defining feature of a rod is its ability to respond to single photons of light in the dark-adapted state [4], an ability largely due to the fact that transduction has a much higher gain in rods than in cones. This high rod sensitivity — which is at least 25 times greater than the sensitivity of cones [5] — is further enhanced by very slow response kinetics (five times slower than in cones), slow transduction machinery (reaction product lifetimes 10–20 times longer than in cones [2]) and a slow recovery of visual sensitivity following bleaching (10 times longer than in cones [6]). The price paid for this high sensitivity is that rods saturate at very dim light levels [5], although the
Current Biology Vol 25 No 4 R150
D
Outer segment
L
Inner segment
Synaptic terminal Rod
Cone Current Biology
Figure 2. The morphology of rods (left) and cones (right) in modern jawed vertebrates. Note the outer segment lamellae (L) and internalised cytosolic disks (D) characteristic of cones and rods, respectively. After [12].
upside of this saturation is a substantial reduction in the metabolic cost of the retina in bright light [7]. Cones, in contrast, effectively never saturate, no matter how bright the source of light they are viewing [8]. Now with these features of rods and cones in mind, let us return to lampreys, those living descendants of the jawless fishes from which all vertebrates evolved. These ancient aquatic vertebrates, of which there are 38 extant species, are characterised by their round, funnel-like sucking mouths with concentric rows of horny teeth which, in parasitic species, are used to attach and bore into the flesh of a larger host (Figure 1B). Their eyes, as we mentioned above, are well developed. In the southern hemisphere lamprey Geotria australis, five distinct classes of cone-like photoreceptors have evolved [9], four of which possess cone opsins and one of which is likely to contain rhodopsin (Rh1) [10], the visual pigment found in modern rods. The two photoreceptor types found in the northern
hemisphere sea lamprey — the species studied by Morshedian and Fain [3] — are also cone-like [11]. The outer segments of both types lack deep longitudinal incisures and possess lamellae whose collective membrane is continuous with the plasma membrane of the inner segment. Moreover, their synaptic terminals resemble the typical pedicles of cones rather than the spherules of rods. It is thus little wonder that the jawless ancestors of vertebrates are generally thought to have lacked rods, and as a direct consequence, a duplex retina and scotopic vision [1,2]. Following this reasoning, such visual advances could then only have arisen after the divergence of the jawed and jawless fishes. The clinching piece of evidence needed to prove the contrary — that rods and scotopic vision evolved before this divergence — would be a clear demonstration that one or more of the cone-like photoreceptor classes of an extant jawless fish, like a lamprey or hagfish, is in fact a rod. Physiologically, this would involve proving that the photoreceptor is capable of detecting single photons, the defining characteristic of a rod. This, it turns out, is precisely what Morshedian and Fain have done [3]. By placing small slices of sea lamprey retina in a dish beneath a microscope, Morshedian and Fain [3] were able to suck an individual photoreceptor outer segment into the end of a glass micropipette and record its responses to light. When they did this, they noticed that the responses of the ‘short’ and ‘long’ photoreceptor types differed considerably, particularly in their response dynamics and sensitivity. The short photoreceptors responded to flashes of light very slowly, taking around half a second to reach peak response and then several seconds to decay. The long photoreceptors, in contrast, responded rapidly, peaking and decaying in less than a second. They also discovered that the short photoreceptors were at least 40 times more sensitive to light than the long photoreceptors. These differences are very reminiscent of the differences between rods and cones we described above, suggesting that even though the long photoreceptors are physiologically like cones, the short photoreceptors are much more like
rods. But the ‘clinching piece of evidence’, which sealed the identities of these photoreceptors once and for all, was that the short photoreceptors had responses to single photons of light of about the same magnitude as rods in other vertebrates, making them by every definition rods. In this ground-breaking study, Morshedian and Fain [3] have shown that the two photoreceptor classes of the sea lamprey are actually rods and cones. The immediate implication of this finding is that the evolution of the vertebrate duplex retina predated the divergence of the jawed and jawless fishes and that the last common ancestor of Agnathans and Gnathostomes had scotopic visual capacities. Despite this major advance in our understanding of visual evolution, a vexing question nonetheless arises from their study. Because the closed internalised cytosolic disks of rods are clearly not necessary to generate enhanced sensitivity, why then did the characteristic rod morphology of all modern jawed vertebrates evolve? Morshedian and Fain [3] suggest that the answer may lie in benefits for the transport of visual pigments and the renewal of the outer segment. Whatever the reason, there is no doubt that this question provides a promising avenue for future research. References 1. Lamb, T.D., Collin, S.P., and Pugh, E.N., Jr. (2007). Evolution of the vertebrate eye: opsins, photoreceptors, retina and eye cup. Nat. Rev. Neurosci. 8, 960–976. 2. Lamb, T.D. (2013). Evolution of phototransduction, vertebrate photoreceptors and retina. Prog. Retin. Eye Res. 36, 52–119. 3. Morshedian, A., and Fain, G.L. (2015). Singlephoton sensitivity of lamprey rods with cone-like outer segments. Curr. Biol. 25, 484–487. 4. Baylor, D.A., Lamb, T.D., and Yau, K.-W. (1979). Responses of retinal rods to single photons. J. Physiol. 288, 613–634. 5. Fain, G.L., and Dowling, J.E. (1973). Intracellular recordings from single rods and cones in the mudpuppy retina. Science 180, 1178–1181. 6. Kenkre, J.S., Moran, N.A., Lamb, T.D., and Mahroo, O.A.R. (2005). Extremely rapid recovery of human cone circulating current at the extinction of bleaching exposures. J. Physiol. 567, 95–112. 7. Okawa, H., Sampath, A.P., Laughlin, S.B., and Fain, G.L. (2008). ATP consumption by mammalian rod photoreceptors in darkness and in light. Curr. Biol. 18, 1917–1921. 8. Normann, R.S., and Werblin, F.S. (1974). Control of retinal sensitivity. I. Light and dark adaptation of vertebrate rods and cones. J. Gen. Physiol. 63, 37–61. 9. Collin, S.P., Hart, N.S., Shand, J., and Potter, I.C. (2003). Morphology and spectral absorption characteristics of retinal photoreceptors in the southern hemisphere
Dispatch R151
lamprey (Geotria australis). Vis. Neurosci. 20, 119–130. 10. Pisani, D., Mohun, S.M., Harris, S.R., McInerney, J.O., and Wilkinson, M. (2006). Molecular evidence for dim-light vision in the last common ancestor of the vertebrates. Curr. Biol. 16, R318–R319.
11. Dickson, D.H., and Graves, D.A. (1979). Fine structure of the lamprey photoreceptors and retinal pigment epithelium (Petromyzon marinus L.). Exp. Eye Res. 29, 45–60. 12. Fain, G.L. (2014). Molecular and Cellular Physiology of Neurons, 2nd edn. (Cambridge USA: Harvard University Press).
Membrane Trafficking: ER Export Encounters Dualism Cytoplasmic coat protein complexes perform central roles in sorting protein constituents within the endomembrane system. A new study reveals that the COPII coat operates through dual recognition of signals in a sorting receptor and its bound cargo to promote efficient export from the endoplasmic reticulum. Charles Barlowe Nascent secretory proteins span a range of sizes, subunit arrangements and membrane topologies, yet each is folded in the endoplasmic reticulum (ER) and then packaged into vesicles bearing coat protein complex II (COPII) that transport these cargo proteins forward in the secretory pathway. A framework for coat-protein-dependent trafficking has emerged wherein sorting signals displayed by protein cargo are recognized and bound by cytoplasmic coat adaptor proteins for selective incorporation into specific classes of carrier vesicles [1]. Transmembrane sorting receptors expand the connections by which soluble and membrane cargo can be linked to specific coat adaptors. Segregation of cargo during rounds of membrane trafficking dynamically localizes proteins to their proper intracellular sites. Investigators had set out to define specific sorting signals and modes of recognition in order to understand cellular organization. However, the rules just got a bit more complex and quite probably more discerning in how folded transmembrane cargo are selected for ER export. Instead of single recognition signals in cargo to specify packaging, in some instances dual signals are recognized by the Sec24 adaptor to impart efficient export from the ER in COPII vesicles. In this issue of Current Biology, Pagant et al. [2] show that the nascent form of the membrane protein Yor1 assembles with the Erv14 cargo receptor: this
complex is then packaged through simultaneous recognition of sorting signals on Yor1 and on Erv14 by distinct binding sites within the Sec24 adaptor subunit. Sequential assembly of the COPII subunits Sar1, Sec23–Sec24 and the outer layer Sec13–Sec31 on the surface of ER membranes segregates cargo for incorporation into ER-derived transport intermediates [3]. The Sec24 subunit and its homologs contain multiple cargo recognition sites, each capable of binding distinct ER export motifs. But precisely how this coat complex reversibly binds diverse cargos and how the ER export machinery distinguishes folded from unfolded cargo remain open questions. To address these issues the Miller laboratory has a longstanding interest in folding and ER sorting of Yor1, a polytopic membrane protein in yeast that traffics to the plasma membrane. Yor1 is a member of the ATP-binding cassette (ABC) family of membrane transporters and shares homology with mammalian cystic fibrosis transmembrane conductance regulator (CFTR). Moreover, deletion of a highly conserved phenylalanine residue in Yor1 produces a misfolded variant that is retained in the ER and mimics some properties of the disease-causing CFTR-DF508 mutation [4]. Investigation of Yor1 in Saccharomyces cerevisiae has facilitated genetic analyses of ABC transporter folding and trafficking. Initial studies of wild-type Yor1 identified a cytoplasmically exposed diacidic DXE motif at residues 71–73
Department Biology, University of Lund, So¨lvegatan 35, S-22362 Lund, Sweden. E-mail: [email protected]
http://dx.doi.org/10.1016/j.cub.2015.01.005
that was required for efficient ER export and delivery of Yor1 to the cell surface [5,6]. Structural studies of the Sec23–Sec24 complex had revealed at least three distinct cargo-binding sites in Sec24 [7,8] and it was demonstrated that the diacidic motif in Yor1 depended on the Sec24 B-site (residues R230, R235) for packaging into COPII vesicles [6]. All this made good sense until genetic interaction mapping revealed that wild-type Yor1 also displayed partial dependence on the Erv14 cargo receptor for efficient export from the ER [9]. Erv14 is a small hydrophobic protein with three transmembrane segments and belongs to a highly conserved family that was founded by the Drosophila Cornichon protein [10,11]. Erv14/Cornichon directs specific integral membrane secretory cargo into COPII vesicles through interactions with the Sec23–Sec24 complex and actively cycles between ER and Golgi compartments [12]. Several lines of study have now identified multiple transmembrane cargo proteins in yeast and animal cells that depend on the Erv14/Cornichon family for ER export [13–16]. Most Erv14-dependent cargos are polytopic membrane proteins that reside at the plasma membrane or in late endomembrane compartments and contain longer transmembrane domains that are common for proteins in these locations. Indeed, the transmembrane domain length of one such cargo was shown to control Erv14 dependence in export from the ER [14]. Therefore, Erv14 is thought to link specific cargo to the Sec24 adaptor complex for COPII packaging and to help usher proteins with lengthy transmembrane domains out of the ER. In the new study, Pagant et al. [2] were intrigued by why a plasma membrane cargo protein such as Yor1 would need an ER export receptor, since it already has a perfectly good diacidic ER export motif. To explore this mechanism they first showed that deletion of both ERV14 and its close
dopamine neurons suggest that tonic versus burst firing would result in differences in the relative occupancy of receptors with different affinities [18]. In support, pharmacological experiments in zebrafish have demonstrated that dopamine can shorten the duration of drug-evoked episodic swimming bouts in spinalized larvae, something that is attributed to the progressive innervation of the spinal cord by DDNs [7]. However, this observation is difficult to reconcile with the fact that early ablation of DDNs produced no effects on the patterning of spontaneous, real swimming. While the authors suggest possible explanations for this discrepancy, including potential off-target effects of the pharmacological manipulations, it remains to be seen exactly what DDN bursting contributes to zebrafish locomotion. In this sense, the work by Jay et al. [1] achieves the goal of all high quality studies, in that it generates more questions than it answers. The description of different modes of firing not only helps put pharmacological observations in a proper context, but also provides a framework for investigating how dynamic changes in dopamine levels in the spinal cord and elsewhere may exert differential effects on locomotor behavior. Are different dopamine receptor subtypes located on the same or different spinal circuit elements? How about targets in the brainstem or the periphery? Does the transition from tonic to burst firing orchestrate a common behavioral goal via these distributed targets? If so, what is this behavior? The ability to replace GFP in ETvmat2:GFP fish with
genes that drive optogenetic actuators to activate or silence DDNs [19], and the development of closed loop systems that drive more complex larval behaviors [20], make it likely that answers to these questions are not far off. Given the conserved genetic origins of DDNs and the similarity in their activity patterns to mammalian dopamine neurons, the zebrafish model system will surely be a reliable source for principles underlying the modulation of circuits and behavior by dopamine in years to come. References 1. Jay, M., De Faveri, F., and McDearmid, J.R. (2015). Firing dynamics and modulatory actions of supraspinal dopaminergic neurons during zebrafish locomotor behavior. Curr. Biol. 25, 435–444. 2. Jankowska, E., Jukes, M.G., Lund, S., and Lundberg, A. (1967). The effect of DOPA on the spinal cord. 5. Reciprocal organization of pathways transmitting excitatory action to alpha motoneurones of flexors and extensors. Acta Physiol. Scand. 70, 369–388. 3. Grillner, S., and Jessell, T.M. (2009). Measured motion: searching for simplicity in spinal locomotor networks. Curr. Opin. Neurobiol. 19, 572–586. 4. Miles, G.B., and Sillar, K.T. (2011). Neuromodulation of vertebrate locomotor control networks. Physiology 26, 393–411. 5. Missale, C., Nash, S.R., Robinson, S.W., Jaber, M., and Caron, M.G. (1998). Dopamine receptors: from structure to function. Physiol. Rev. 78, 189–225. 6. Clemens, S., Belin-Rauscent, A., Simmers, J., and Combes, D. (2012). Opposing modulatory effects of D1- and D2-like receptor activation on a spinal central pattern generator. J. Neurophysiol. 107, 2250–2259. 7. Lambert, A.M., Bonkowsky, J.L., and Masino, M.A. (2012). The conserved dopaminergic diencephalospinal tract mediates vertebrate locomotor development in zebrafish larvae. J. Neurosci. 32, 13488–13500. 8. Thirumalai, V., and Cline, H.T. (2008). Endogenous dopamine suppresses initiation of swimming in prefeeding zebrafish larvae. J. Neurophysiol. 100, 1635–1648. 9. Tay, T.L., Ronneberger, O., Ryu, S., Nitschke, R., and Driever, W. (2011). Comprehensive catecholaminergic projectome analysis reveals
Photoreceptor Evolution: Ancient ‘Cones’ Turn Out to Be Rods Vertebrate rod photoreceptors are thought to have evolved from cone photoreceptors only after the divergence of the jawed and jawless fishes, but this idea is questioned by new evidence that the short ‘cones’ of jawless sea lampreys are physiologically equivalent to rods. Eric J. Warrant About 540 million years ago one of the most spectacular events in the history of the evolution of animal life
began: in the space of just 20 million years — a blink of an eye in geological terms — many of our familiar modern animal lineages suddenly appeared on the Earth. This explosion of
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
single-neuron integration of zebrafish ascending and descending dopaminergic systems. Nat. Commun. 2, 171. McLean, D.L., and Fetcho, J.R. (2004). Relationship of tyrosine hydroxylase and serotonin immunoreactivity to sensorimotor circuitry in larval zebrafish. J. Comp. Neurol. 480, 57–71. Koblinger, K., Fuzesi, T., Ejdrygiewicz, J., Krajacic, A., Bains, J.S., and Whelan, P.J. (2014). Characterization of A11 neurons projecting to the spinal cord of mice. PLoS One 9, e109636. Wen, L., Wei, W., Gu, W., Huang, P., Ren, X., Zhang, Z., Zhu, Z., Lin, S., and Zhang, B. (2008). Visualization of monoaminergic neurons and neurotoxicity of MPTP in live transgenic zebrafish. Dev. Biol. 314, 84–92. Surmeier, D.J., Mercer, J.N., and Chan, C.S. (2005). Autonomous pacemakers in the basal ganglia: who needs excitatory synapses anyway? Curr. Opin. Neurobiol. 15, 312–318. Tsai, H.C., Zhang, F., Adamantidis, A., Stuber, G.D., Bonci, A., de Lecea, L., and Deisseroth, K. (2009). Phasic firing in dopaminergic neurons is sufficient for behavioral conditioning. Science 324, 1080–1084. Buss, R.R., and Drapeau, P. (2001). Synaptic drive to motoneurons during fictive swimming in the developing zebrafish. J. Neurophysiol. 86, 197–210. Veasey, S.C., Fornal, C.A., Metzler, C.W., and Jacobs, B.L. (1995). Response of serotonergic caudal raphe neurons in relation to specific motor activities in freely moving cats. J. Neurosci. 15, 5346–5359. McLean, D.L., and Fetcho, J.R. (2004). Ontogeny and innervation patterns of dopaminergic, noradrenergic, and serotonergic neurons in larval zebrafish. J. Comp. Neurol. 480, 38–56. Dreyer, J.K., Herrik, K.F., Berg, R.W., and Hounsgaard, J.D. (2010). Influence of phasic and tonic dopamine release on receptor activation. J. Neurosci. 30, 14273–14283. Auer, T.O., Duroure, K., Concordet, J.P., and Del Bene, F. (2014). CRISPR/Cas9-mediated conversion of eGFP- into Gal4-transgenic lines in zebrafish. Nat. Protoc. 9, 2823–2840. Engert, F. (2012). Fish in the matrix: motor learning in a virtual world. Front. Neural Circuits 6, 125.
Department of Neurobiology, Northwestern University, Evanston, IL 60208, USA. *E-mail: [email protected]
http://dx.doi.org/10.1016/j.cub.2015.01.001
new animal forms ushered in the Cambrian epoch, and at its end, a little more than 500 million years ago, the earliest true vertebrates appeared. These were the so-called jawless fishes, or Agnathans, of which only two lineages survive until the present day, the hagfishes and the lampreys. From the Agnatha evolved the jawed fishes, or Gnathostomes, and from these arose all the vertebrate lineages we are familiar with today, including our own. The eyes of these early jawed fishes were probably very much like
Dispatch R149
our eyes – well developed camera eyes with a duplex retina containing rod and cone photoreceptors. In contrast, the eyes of their jawless ancestors — if the eyes of present day hagfishes and lampreys are any indication — were probably quite different. Hagfishes have rudimentary lensless eyes with a reduced retina and a single photoreceptor class of unknown type. Lampreys, in contrast, have well-developed camera eyes, with a sophisticated retina containing at least two classes of photoreceptors — all of these, however, morphologically resemble cones. In the sea lamprey Petromyzon marinus (Figure 1) there are two classes of these cone-like photoreceptors, one ‘long’ and one ‘short’. Because rods are known to have evolved from cones, the apparently rod-free retinas of hagfishes and lampreys have led to the conjecture that the modern vertebrate duplex retina evolved only after the jawed fishes diverged from the jawless fishes [1,2]. Now, in a landmark study reported in this issue of Current Biology, Morshedian and Fain [3] have discovered that the sea lamprey short photoreceptor — despite having a cone-like morphology — is physiologically much more like a rod than a cone, notably (and critically) having the necessary sensitivity to respond to single photons of light, a hallmark property of rods. This discovery not only implies that the vertebrate duplex retina evolved prior to the divergence of the jawed and jawless fishes, it also suggests that the classic rod outer segment morphology, with cytosolic disks surrounded by a continuous plasma membrane, is not necessary for the high-gain transduction of single photon responses. Rods and cones, the defining elements of the vertebrate duplex retina, have evolved to give us highly sensitive but coarse monochromatic scotopic vision at night (rods) and highly resolved photopic colour vision during the day (cones). Not surprisingly, they differ significantly from each other in several key respects (Figure 2), both in terms of morphology and physiology (reviewed in [2]). Morphologically, the most obvious
Figure 1. The sea lamprey Petromyzon marinus. (A) The gills slits and well developed eyes. (B) The mouth with its concentric rows of horny teeth. Images used with kind permission of the photographers: A. Muir (A) and T. Lawrence (B) of the Great Lakes Fishery Commission, USA.
distinguishing feature is the topology of the membrane of the outer segment, the region of the photoreceptor housing the visual pigment and responsible for the absorption of light and its transduction into an electrical signal. In cones, the entire outer segment membrane is continuous with the plasma membrane of the inner segment, creating a stack of plate-like lamellae. The outer segment so created is cylindrical in mammals, but somewhat conical in other vertebrates. In rods, these lamellae become internalised during the synthesis of the outer segment, creating a stack of membrane-bound disks, whose membranes are separated from the main plasma membrane (and thus from the extracellular matrix). Other morphological differences — such as the presence of an ellipsoid in cones (filled with mitochondria or a spectrally filtering oil droplet), deep longitudinal incisures in the outer
segment of rods (thought to increase the diffusion of intracellular messengers) and considerable differences in the morphologies of their synaptic terminals — further distinguish rods from cones. Physiologically, the defining feature of a rod is its ability to respond to single photons of light in the dark-adapted state [4], an ability largely due to the fact that transduction has a much higher gain in rods than in cones. This high rod sensitivity — which is at least 25 times greater than the sensitivity of cones [5] — is further enhanced by very slow response kinetics (five times slower than in cones), slow transduction machinery (reaction product lifetimes 10–20 times longer than in cones [2]) and a slow recovery of visual sensitivity following bleaching (10 times longer than in cones [6]). The price paid for this high sensitivity is that rods saturate at very dim light levels [5], although the
Current Biology Vol 25 No 4 R150
D
Outer segment
L
Inner segment
Synaptic terminal Rod
Cone Current Biology
Figure 2. The morphology of rods (left) and cones (right) in modern jawed vertebrates. Note the outer segment lamellae (L) and internalised cytosolic disks (D) characteristic of cones and rods, respectively. After [12].
upside of this saturation is a substantial reduction in the metabolic cost of the retina in bright light [7]. Cones, in contrast, effectively never saturate, no matter how bright the source of light they are viewing [8]. Now with these features of rods and cones in mind, let us return to lampreys, those living descendants of the jawless fishes from which all vertebrates evolved. These ancient aquatic vertebrates, of which there are 38 extant species, are characterised by their round, funnel-like sucking mouths with concentric rows of horny teeth which, in parasitic species, are used to attach and bore into the flesh of a larger host (Figure 1B). Their eyes, as we mentioned above, are well developed. In the southern hemisphere lamprey Geotria australis, five distinct classes of cone-like photoreceptors have evolved [9], four of which possess cone opsins and one of which is likely to contain rhodopsin (Rh1) [10], the visual pigment found in modern rods. The two photoreceptor types found in the northern
hemisphere sea lamprey — the species studied by Morshedian and Fain [3] — are also cone-like [11]. The outer segments of both types lack deep longitudinal incisures and possess lamellae whose collective membrane is continuous with the plasma membrane of the inner segment. Moreover, their synaptic terminals resemble the typical pedicles of cones rather than the spherules of rods. It is thus little wonder that the jawless ancestors of vertebrates are generally thought to have lacked rods, and as a direct consequence, a duplex retina and scotopic vision [1,2]. Following this reasoning, such visual advances could then only have arisen after the divergence of the jawed and jawless fishes. The clinching piece of evidence needed to prove the contrary — that rods and scotopic vision evolved before this divergence — would be a clear demonstration that one or more of the cone-like photoreceptor classes of an extant jawless fish, like a lamprey or hagfish, is in fact a rod. Physiologically, this would involve proving that the photoreceptor is capable of detecting single photons, the defining characteristic of a rod. This, it turns out, is precisely what Morshedian and Fain have done [3]. By placing small slices of sea lamprey retina in a dish beneath a microscope, Morshedian and Fain [3] were able to suck an individual photoreceptor outer segment into the end of a glass micropipette and record its responses to light. When they did this, they noticed that the responses of the ‘short’ and ‘long’ photoreceptor types differed considerably, particularly in their response dynamics and sensitivity. The short photoreceptors responded to flashes of light very slowly, taking around half a second to reach peak response and then several seconds to decay. The long photoreceptors, in contrast, responded rapidly, peaking and decaying in less than a second. They also discovered that the short photoreceptors were at least 40 times more sensitive to light than the long photoreceptors. These differences are very reminiscent of the differences between rods and cones we described above, suggesting that even though the long photoreceptors are physiologically like cones, the short photoreceptors are much more like
rods. But the ‘clinching piece of evidence’, which sealed the identities of these photoreceptors once and for all, was that the short photoreceptors had responses to single photons of light of about the same magnitude as rods in other vertebrates, making them by every definition rods. In this ground-breaking study, Morshedian and Fain [3] have shown that the two photoreceptor classes of the sea lamprey are actually rods and cones. The immediate implication of this finding is that the evolution of the vertebrate duplex retina predated the divergence of the jawed and jawless fishes and that the last common ancestor of Agnathans and Gnathostomes had scotopic visual capacities. Despite this major advance in our understanding of visual evolution, a vexing question nonetheless arises from their study. Because the closed internalised cytosolic disks of rods are clearly not necessary to generate enhanced sensitivity, why then did the characteristic rod morphology of all modern jawed vertebrates evolve? Morshedian and Fain [3] suggest that the answer may lie in benefits for the transport of visual pigments and the renewal of the outer segment. Whatever the reason, there is no doubt that this question provides a promising avenue for future research. References 1. Lamb, T.D., Collin, S.P., and Pugh, E.N., Jr. (2007). Evolution of the vertebrate eye: opsins, photoreceptors, retina and eye cup. Nat. Rev. Neurosci. 8, 960–976. 2. Lamb, T.D. (2013). Evolution of phototransduction, vertebrate photoreceptors and retina. Prog. Retin. Eye Res. 36, 52–119. 3. Morshedian, A., and Fain, G.L. (2015). Singlephoton sensitivity of lamprey rods with cone-like outer segments. Curr. Biol. 25, 484–487. 4. Baylor, D.A., Lamb, T.D., and Yau, K.-W. (1979). Responses of retinal rods to single photons. J. Physiol. 288, 613–634. 5. Fain, G.L., and Dowling, J.E. (1973). Intracellular recordings from single rods and cones in the mudpuppy retina. Science 180, 1178–1181. 6. Kenkre, J.S., Moran, N.A., Lamb, T.D., and Mahroo, O.A.R. (2005). Extremely rapid recovery of human cone circulating current at the extinction of bleaching exposures. J. Physiol. 567, 95–112. 7. Okawa, H., Sampath, A.P., Laughlin, S.B., and Fain, G.L. (2008). ATP consumption by mammalian rod photoreceptors in darkness and in light. Curr. Biol. 18, 1917–1921. 8. Normann, R.S., and Werblin, F.S. (1974). Control of retinal sensitivity. I. Light and dark adaptation of vertebrate rods and cones. J. Gen. Physiol. 63, 37–61. 9. Collin, S.P., Hart, N.S., Shand, J., and Potter, I.C. (2003). Morphology and spectral absorption characteristics of retinal photoreceptors in the southern hemisphere
Dispatch R151
lamprey (Geotria australis). Vis. Neurosci. 20, 119–130. 10. Pisani, D., Mohun, S.M., Harris, S.R., McInerney, J.O., and Wilkinson, M. (2006). Molecular evidence for dim-light vision in the last common ancestor of the vertebrates. Curr. Biol. 16, R318–R319.
11. Dickson, D.H., and Graves, D.A. (1979). Fine structure of the lamprey photoreceptors and retinal pigment epithelium (Petromyzon marinus L.). Exp. Eye Res. 29, 45–60. 12. Fain, G.L. (2014). Molecular and Cellular Physiology of Neurons, 2nd edn. (Cambridge USA: Harvard University Press).
Membrane Trafficking: ER Export Encounters Dualism Cytoplasmic coat protein complexes perform central roles in sorting protein constituents within the endomembrane system. A new study reveals that the COPII coat operates through dual recognition of signals in a sorting receptor and its bound cargo to promote efficient export from the endoplasmic reticulum. Charles Barlowe Nascent secretory proteins span a range of sizes, subunit arrangements and membrane topologies, yet each is folded in the endoplasmic reticulum (ER) and then packaged into vesicles bearing coat protein complex II (COPII) that transport these cargo proteins forward in the secretory pathway. A framework for coat-protein-dependent trafficking has emerged wherein sorting signals displayed by protein cargo are recognized and bound by cytoplasmic coat adaptor proteins for selective incorporation into specific classes of carrier vesicles [1]. Transmembrane sorting receptors expand the connections by which soluble and membrane cargo can be linked to specific coat adaptors. Segregation of cargo during rounds of membrane trafficking dynamically localizes proteins to their proper intracellular sites. Investigators had set out to define specific sorting signals and modes of recognition in order to understand cellular organization. However, the rules just got a bit more complex and quite probably more discerning in how folded transmembrane cargo are selected for ER export. Instead of single recognition signals in cargo to specify packaging, in some instances dual signals are recognized by the Sec24 adaptor to impart efficient export from the ER in COPII vesicles. In this issue of Current Biology, Pagant et al. [2] show that the nascent form of the membrane protein Yor1 assembles with the Erv14 cargo receptor: this
complex is then packaged through simultaneous recognition of sorting signals on Yor1 and on Erv14 by distinct binding sites within the Sec24 adaptor subunit. Sequential assembly of the COPII subunits Sar1, Sec23–Sec24 and the outer layer Sec13–Sec31 on the surface of ER membranes segregates cargo for incorporation into ER-derived transport intermediates [3]. The Sec24 subunit and its homologs contain multiple cargo recognition sites, each capable of binding distinct ER export motifs. But precisely how this coat complex reversibly binds diverse cargos and how the ER export machinery distinguishes folded from unfolded cargo remain open questions. To address these issues the Miller laboratory has a longstanding interest in folding and ER sorting of Yor1, a polytopic membrane protein in yeast that traffics to the plasma membrane. Yor1 is a member of the ATP-binding cassette (ABC) family of membrane transporters and shares homology with mammalian cystic fibrosis transmembrane conductance regulator (CFTR). Moreover, deletion of a highly conserved phenylalanine residue in Yor1 produces a misfolded variant that is retained in the ER and mimics some properties of the disease-causing CFTR-DF508 mutation [4]. Investigation of Yor1 in Saccharomyces cerevisiae has facilitated genetic analyses of ABC transporter folding and trafficking. Initial studies of wild-type Yor1 identified a cytoplasmically exposed diacidic DXE motif at residues 71–73
Department Biology, University of Lund, So¨lvegatan 35, S-22362 Lund, Sweden. E-mail: [email protected]
http://dx.doi.org/10.1016/j.cub.2015.01.005
that was required for efficient ER export and delivery of Yor1 to the cell surface [5,6]. Structural studies of the Sec23–Sec24 complex had revealed at least three distinct cargo-binding sites in Sec24 [7,8] and it was demonstrated that the diacidic motif in Yor1 depended on the Sec24 B-site (residues R230, R235) for packaging into COPII vesicles [6]. All this made good sense until genetic interaction mapping revealed that wild-type Yor1 also displayed partial dependence on the Erv14 cargo receptor for efficient export from the ER [9]. Erv14 is a small hydrophobic protein with three transmembrane segments and belongs to a highly conserved family that was founded by the Drosophila Cornichon protein [10,11]. Erv14/Cornichon directs specific integral membrane secretory cargo into COPII vesicles through interactions with the Sec23–Sec24 complex and actively cycles between ER and Golgi compartments [12]. Several lines of study have now identified multiple transmembrane cargo proteins in yeast and animal cells that depend on the Erv14/Cornichon family for ER export [13–16]. Most Erv14-dependent cargos are polytopic membrane proteins that reside at the plasma membrane or in late endomembrane compartments and contain longer transmembrane domains that are common for proteins in these locations. Indeed, the transmembrane domain length of one such cargo was shown to control Erv14 dependence in export from the ER [14]. Therefore, Erv14 is thought to link specific cargo to the Sec24 adaptor complex for COPII packaging and to help usher proteins with lengthy transmembrane domains out of the ER. In the new study, Pagant et al. [2] were intrigued by why a plasma membrane cargo protein such as Yor1 would need an ER export receptor, since it already has a perfectly good diacidic ER export motif. To explore this mechanism they first showed that deletion of both ERV14 and its close
