Publications

2012
Song E, Kunes S. Determinants of the Drosophila Odorant Receptor Pattern. Developmental Cell. 2012;22(2):363-376.
Dearborn, R.E. J, Dai Y, Reed B, Gray J, Kunes S. Reph, a Regulator of Eph Receptor Expression in the Drosophila melanogaster Optic Lobe. PloS . 2012;in press.
2010
Huang H-R, Chen ZJ, Kunes S, Chang G-D, Maniatis T. Endocytic pathway is required for Drosophila Toll innate immune signaling. Proceedings of the National Academy of Sciences of the United States of America. 2010;107(18):8322-7.Abstract
The Toll signaling pathway is required for the innate immune response against fungi and Gram-positive bacteria in Drosophila. Here we show that the endosomal proteins Myopic (Mop) and Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) are required for the activation of the Toll signaling pathway. This requirement is observed in cultured cells and in flies, and epistasis experiments show that the Mop protein functions upstream of the MyD88 adaptor and the Pelle kinase. Mop and Hrs, which are critical components of the ESCRT-0 endocytosis complex, colocalize with the Toll receptor in endosomes. We conclude that endocytosis is required for the activation of the Toll signaling pathway.
Murakami S, Dan C, Zagaeski B, Maeyama Y, Kunes S, Tabata T. Optimizing Drosophila olfactory learning with a semi-automated training device. Journal of neuroscience methods. 2010;188(2):195-204.Abstract
Drosophila olfactory aversive conditioning has served as a powerful model system with which to elucidate the molecular and neuronal mechanisms underlying memory formation. In the typical protocol, flies are exposed to a constant odor stream while receiving a pulsed electric shock in the conditioning tube of a manual apparatus. We have devised a simple, low-cost semi-automated conditioning apparatus that computationally controls the delivery of odor and shock. A semiconductor-based odor sensor is employed to monitor the change of odor concentration in the training tube. The system thus allows electric shocks to be precisely matched with odor concentration in the training tube. We found that short-term memory performance was improved with a pulsed odor flow protocol, in which odor is presented in short pulses, each paired with electric shock, rather than as a constant flow. The effect of pulsed odor flow might be ascribed to the phenomenon of 'conditioned approach', where approach toward an odor is induced when the electric shock is presented before odor pulse ends. Our data shows that the system is applicable to the study of olfactory memory formation and to the examination of conditioning parameters at a level of detail not practical with a manual apparatus.
Tokhunts R, Singh S, Chu T, D'Angelo G, Baubet V, Goetz JA, Huang Z, Yuan Z, Ascano M, Zavros Y, et al. The full-length unprocessed hedgehog protein is an active signaling molecule. The Journal of biological chemistry. 2010;285(4):2562-8.Abstract
The hedgehog (HH) family of ligands plays an important instructional role in metazoan development. HH proteins are initially produced as approximately 45-kDa full-length proteins, which undergo an intramolecular cleavage to generate an amino-terminal product that subsequently becomes cholesterol-modified (HH-Np). It is well accepted that this cholesterol-modified amino-terminal cleavage product is responsible for all HH-dependent signaling events. Contrary to this model we show here that full-length forms of HH proteins are able to traffic to the plasma membrane and participate directly in cell-cell signaling, both in vitro and in vivo. We were also able to rescue a Drosophila eye-specific hh loss of function phenotype by expressing a full-length form of hh that cannot be processed into HH-Np. These results suggest that in some physiological contexts full-length HH proteins may participate directly in HH signaling and that this novel activity of full-length HH may be evolutionarily conserved.
2009
de Bivort BL, Perlstein EO, Kunes S, Schreiber SL. Amino acid metabolic origin as an evolutionary influence on protein sequence in yeast. Journal of molecular evolution. 2009;68(5):490-7.Abstract
The metabolic cycle of Saccharomyces cerevisiae consists of alternating oxidative (respiration) and reductive (glycolysis) energy-yielding reactions. The intracellular concentrations of amino acid precursors generated by these reactions oscillate accordingly, attaining maximal concentration during the middle of their respective yeast metabolic cycle phases. Typically, the amino acids themselves are most abundant at the end of their precursor's phase. We show that this metabolic cycling has likely biased the amino acid composition of proteins across the S. cerevisiae genome. In particular, we observed that the metabolic source of amino acids is the single most important source of variation in the amino acid compositions of functionally related proteins and that this signal appears only in (facultative) organisms using both oxidative and reductive metabolism. Periodically expressed proteins are enriched for amino acids generated in the preceding phase of the metabolic cycle. Proteins expressed during the oxidative phase contain more glycolysis-derived amino acids, whereas proteins expressed during the reductive phase contain more respiration-derived amino acids. Rare amino acids (e.g., tryptophan) are greatly overrepresented or underrepresented, relative to the proteomic average, in periodically expressed proteins, whereas common amino acids vary by a few percent. Genome-wide, we infer that 20,000 to 60,000 residues have been modified by this previously unappreciated pressure. This trend is strongest in ancient proteins, suggesting that oscillating endogenous amino acid availability exerted genome-wide selective pressure on protein sequences across evolutionary time.
2007
Perlstein EO, de Bivort BL, Kunes S, Schreiber SL. Evolutionarily conserved optimization of amino acid biosynthesis. Journal of molecular evolution. 2007;65(2):186-96.Abstract
The "cognate bias hypothesis" states that early in evolutionary history the biosynthetic enzymes for amino acid x gradually lost residues of x, thereby reducing the threshold for deleterious effects of x scarcity. The resulting reduction in cognate amino acid composition of the enzymes comprising a particular amino acid biosynthetic pathway is predicted to confer a selective growth advantage on cells. Bioinformatic evidence from protein-sequence data of two bacterial species previously demonstrated reduced cognate bias in amino acid biosynthetic pathways. Here we show that cognate bias in amino acid biosynthesis is present in the other domains of life-Archaebacteria and Eukaryota. We also observe evolutionarily conserved underrepresentations (e.g., glycine in methionine biosynthesis) and overrepresentations (e.g., tryptophan in asparagine biosynthesis) of amino acids in noncognate biosynthetic pathways, which can be explained by secondary amino acid metabolism. Additionally, we experimentally validate the cognate bias hypothesis using the yeast Saccharomyces cerevisiae. Specifically, we show that the degree to which growth declines following amino acid deprivation is negatively correlated with the degree to which an amino acid is underrepresented in the enzymes that comprise its cognate biosynthetic pathway. Moreover, we demonstrate that cognate fold representation is more predictive of growth advantage than a host of other potential growth-limiting factors, including an amino acid's metabolic cost or its intracellular concentration and compartmental distribution.
2006
Ashraf SI, Kunes S. A trace of silence: memory and microRNA at the synapse. Current opinion in neurobiology. 2006;16(5):535-9.Abstract
Identifying the neural circuits that mediate particular behaviors and uncovering their plasticity is an endeavor at the heart of neuroscience. This effort is allied with the elucidation of plasticity mechanisms, because the molecular determinants of plasticity can be markers for the neurons and synapses that are modified by experience. Of particular interest is protein synthesis localized to the synapse, which might establish and maintain the stable modification of neuronal properties, including the pattern and strength of synaptic connections. Recent studies reveal that microRNAs and the RISC pathway regulate synaptic protein synthesis. Is synaptic activity of the RISC pathway a molecular signature of memory?
Chu T, Chiu M, Zhang E, Kunes S. A C-terminal motif targets Hedgehog to axons, coordinating assembly of the Drosophila eye and brain. Developmental cell. 2006;10(5):635-46.Abstract
The developmental signal Hedgehog is distributed to two receptive fields by the photoreceptor neurons of the developing Drosophila retina. Delivery to the retina propagates ommatidial development across a precursor field. Transport along photoreceptor axons induces the development of postsynaptic neurons in the brain. Hedgehog is composed of N-terminal and C-terminal domains that dissociate in an autoproteolytic reaction that attaches cholesterol to the N-terminal cleavage product. Here, we show that the N-terminal domain is targeted to the retina when synthesized in the absence of the C-terminal domain. In contrast to studies that have focused on cholesterol as a determinant of subcellular localization, we find that the C-terminal domain harbors a conserved motif that overrides retinal localization, sending most of the autocleavage products into vesicles bound for growth cones or synapses. Competition between targeting signals at the opposite ends of Hedgehog apparently controls the match between eye and brain development.
Ashraf SI, McLoon AL, Sclarsic SM, Kunes S. Synaptic protein synthesis associated with memory is regulated by the RISC pathway in Drosophila. Cell. 2006;124(1):191-205.Abstract
Long-lasting forms of memory require protein synthesis, but how the pattern of synthesis is related to the storage of a memory has not been determined. Here we show that neural activity directs the mRNA of the Drosophila Ca(2+), Calcium/Calmodulin-dependent Kinase II (CaMKII), to postsynaptic sites, where it is rapidly translated. These features of CaMKII synthesis are recapitulated during the induction of a long-term memory and produce patterns of local protein synthesis specific to the memory. We show that mRNA transport and synaptic protein synthesis are regulated by components of the RISC pathway, including the SDE3 helicase Armitage, which is specifically required for long-lasting memory. Armitage is localized to synapses and lost in a memory-specific pattern that is inversely related to the pattern of synaptic protein synthesis. Therefore, we propose that degradative control of the RISC pathway underlies the pattern of synaptic protein synthesis associated with a stable memory.
Kunes S, Chu T, Chiu M, Zhang E. A C-terminal motif targets hedgehog to axons, coordinating assembly of the Drosophila eye and brain. Developmental Cell [Internet]. 2006;10(5):635-46. WebsiteAbstract
The developmental signal Hedgehog is distributed to two receptive fields by the photoreceptor neurons of the developing Drosophila retina. Delivery to the retina propagates ommatidial development across a precursor field. Transport along photoreceptor axons induces the development of postsynaptic neurons in the brain. Hedgehog is composed of N-terminal and C-terminal domains that dissociate in an autoproteolytic reaction that attaches cholesterol to the N-terminal cleavage product. Here, we show that the N-terminal domain is targeted to the retina when synthesized in the absence of the C-terminal domain. In contrast to studies that have focused on cholesterol as a determinant of subcellular localization, we find that the C-terminal domain harbors a conserved motif that overrides retinal localization, sending most of the autocleavage products into vesicles bound for growth cones or synapses. Competition between targeting signals at the opposite ends of Hedgehog apparently controls the match between eye and brain development.
2004
Yang H, Kunes S. Nonvesicular release of acetylcholine is required for axon targeting in the Drosophila visual system. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(42):15213-8.Abstract
We report evidence for a developmental role of acetylcholine in axon pathfinding in the Drosophila visual system. Acetylcholine was detected on photoreceptor axons during their navigation to target sites in the brain, a time well before the formation of functional synapses. The pattern of photoreceptor axon projections was severely disrupted when acetylcholine synthesis or metabolism was altered or eliminated, or when transgenic alpha-bungarotoxin, a nicotinic acetylcholine receptor antagonist, was expressed in the developing eye or brain. The requirement for acetylcholine signaling exists before photoreceptor neurons form synaptic connections and does not require the function of vesicular acetylcholine transporter protein. That this early effect of acetylcholine is mediated through nonvesicular release is further supported by the observation that transgenic expression of tetanus toxin, a blocker of neurotransmitter release via synaptic vesicles, did not cause similar photoreceptor axon projection defects. These observations support the notion that a form of acetylcholine secretion mediates the behavior of growth cones during axon pathfinding.
Dearborn R, Kunes S. An axon scaffold induced by retinal axons directs glia to destinations in the Drosophila optic lobe. Development (Cambridge, England). 2004;131(10):2291-303.Abstract
In the developing Drosophila visual system, glia migrate into stereotyped positions within the photoreceptor axon target fields and provide positional information for photoreceptor axon guidance. Glial migration conversely depends on photoreceptor axons, as glia precursors stall in their progenitor zones when retinal innervation is eliminated. Our results support the view that this requirement for retinal innervation reflects a role of photoreceptor axons in the establishment of an axonal scaffold that guides glial cell migration. Optic lobe cortical axons extend from dorsal and ventral positions towards incoming photoreceptor axons and establish at least four separate pathways that direct glia to proper destinations in the optic lobe neuropiles. Photoreceptor axons induce the outgrowth of these scaffold axons. Most glia do not migrate when the scaffold axons are missing. Moreover, glia follow the aberrant pathways of scaffold axons that project aberrantly, as occurs in the mutant dachsous. The local absence of glia is accompanied by extensive apoptosis of optic lobe cortical neurons. These observations reveal a mechanism for coordinating photoreceptor axon arrival in the brain with the distribution of glia to multiple target destinations, where they are required for axon guidance and neuronal survival.
2002
Dearborn R, He Q, Kunes S, Dai Y. Eph receptor tyrosine kinase-mediated formation of a topographic map in the Drosophila visual system. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2002;22(4):1338-49.Abstract
Roles for Eph receptor tyrosine kinase signaling in the formation of topographic patterns of axonal connectivity have been well established in vertebrate visual systems. Here we describe a role for a Drosophila Eph receptor tyrosine kinase (EPH) in the control of photoreceptor axon and cortical axon topography in the developing visual system. Although uniform across the developing eye, EPH is expressed in a concentration gradient appropriate for conveying positional information during cortical axon guidance in the second-order optic ganglion, the medulla. Disruption of this graded pattern of EPH activity by double-stranded RNA interference or by ectopic expression of wild-type or dominant-negative transgenes perturbed the establishment of medulla cortical axon topography. In addition, abnormal midline fasciculation of photoreceptor axons resulted from the eye-specific expression of the dominant-negative EPH transgene. These observations reveal a conserved role for Eph kinases as determinants of topographic map formation in vertebrates and invertebrates.
2000
Song Y, Chung S, Kunes S. Combgap relays wingless signal reception to the determination of cortical cell fate in the Drosophila visual system. Molecular cell. 2000;6(5):1143-54.Abstract
The dorsoventral axis of the Drosophila visual cortex is patterned by nonautonomous signals expressed at its dorsal and ventral margins. wingless (wg) expression at the margins induces decapentaplegic (dpp), optomotor blind (omb), and aristaless in adjacent domains. We show that Combgap, a zinc finger protein, represses Wg target gene expression in the visual cortex. Wg signal reception downregulates combgap expression and derepresses target gene transcription. Combgap participates in a Hedgehog-controlled circuit in the developing wing and leg by regulating the expression of Cubitus interruptus. Combgap is thus a tissue-specific relay between Wingless and its target genes for the determination of cell fate in the visual cortex.
Kunes S. Axonal signals in the assembly of neural circuitry. Current opinion in neurobiology. 2000;10(1):58-62.Abstract
Recent work in Drosophila and rodents has revealed that proteins transported along axons and delivered to pathway and target cell populations play important roles in the construction of neural circuitry. Interestingly, the parallels between these systems may extend to the identities of some of the molecules involved.
1999
Kunes S. Stop or go in the target zone. Neuron. 1999;22(4):639-40.Abstract
n/a
1998
Huang Z, Kunes S. Signals transmitted along retinal axons in Drosophila: Hedgehog signal reception and the cell circuitry of lamina cartridge assembly. Development (Cambridge, England). 1998;125(19):3753-64.Abstract
The arrival of retinal axons in the brain of Drosophila triggers the assembly of glial and neuronal precursors into a 'neurocrystalline' array of lamina synaptic 'cartridges'. Hedgehog, a secreted protein, is an inductive signal delivered by retinal axons for the initial steps of lamina differentiation. In the development of many tissues, Hedgehog acts in a signal relay cascade via the induction of secondary secreted factors. Here we show that lamina neuronal precursors respond directly to Hedgehog signal reception by entering S-phase, a step that is controlled by the Hedgehog-dependent transcriptional regulator Cubitus interruptus. The terminal differentiation of neuronal precursors and the migration and differentiation of glia appear to be controlled by other retinal axon-mediated signals. Thus retinal axons impose a program of developmental events on their postsynaptic field utilizing distinct signals for different precursor populations.
Huang Z, Shilo BZ, Kunes S. A retinal axon fascicle uses spitz, an EGF receptor ligand, to construct a synaptic cartridge in the brain of Drosophila. Cell. 1998;95(5):693-703.Abstract
Photoreceptor axons arriving in the Drosophila brain organize their postsynaptic target field into a precise array of five neuron "cartridge" ensembles. Here we show that Hedgehog, an initial inductive signal transported along retinal axons from the developing eye, induces postsynaptic precursor cells to express the Drosophila homolog of the epidermal growth factor receptor (EGFR). The EGFR ligand Spitz, a signal for ommatidial assembly in the compound eye, is transported to retinal axon termini in the brain where it acts as a local cue for the recruitment of five cells into a cartridge ensemble. Hedgehog and Spitz thus bring about the concerted assembly of ommatidial and synaptic cartridge units, imposing the "neurocrystalline" order of the compound eye on the postsynaptic target field.
1996
Huang Z, Kunes S. Hedgehog, transmitted along retinal axons, triggers neurogenesis in the developing visual centers of the Drosophila brain. Cell. 1996;86(3):411-22.Abstract
The development of the visual centers of the Drosophila brain is tightly regulated by the ingrowth of retinal axons from the developing eye. In the first optic ganglion, the lamina, arriving retinal axons trigger the precursors of their synaptic partners to complete a final cell division and commence neural differentiation. The secreted product of the hedgehog gene regulates the temporal assembly of photoreceptor precursor cells into ommatidial clusters in the compound eye. Here, we show that Hedgehog is transmitted along the retinal axons to serve as the inductive signal in the brain. Hedgehog acts in the first of two retinal axon-mediated steps in the assembly of lamina synaptic cartridges. These observations provide a novel insight into the molecular interactions that orchestrate the assembly of neural precursor cells into precise synaptic circuits.