Publications

2014
Baqri RM, Pietron AV, Gokhale RH, Turner BA, Kaguni LS, Shingleton AW, Kunes S, Miller KE. Mitochondrial chaperone TRAP1 activates the mitochondrial UPR and extends healthspan in Drosophila. Mech Ageing Dev. 2014;141-142 :35-45.Abstract
The molecular mechanisms influencing healthspan are unclear but mitochondrial function, resistance to oxidative stress and proteostasis are recurring themes. Tumor necrosis factor Receptor Associated Protein 1 (TRAP1), the mitochondrial analog of Hsp75, regulates levels of reactive oxygen species in vitro and is found expressed at higher levels in tumor cells where it is thought to play a pro-survival role. While TRAP1-directed compartmentalized protein folding is a promising target for cancer therapy, its role at the organismal level is unclear. Here we report that overexpression of TRAP1 in Drosophila extends healthspan by enhancing stress resistance, locomotor activity and fertility while depletion of TRAP1 has the opposite effect, with little effect on lifespan under both conditions. In addition, modulating TRAP1 expression promotes the nuclear translocation of homeobox protein Dve and increases expression of genes associated with the mitochondrial unfolded protein response (UPR(mt)), indicating an activation of this proteostasis pathway. Notably, independent genetic knockdown of components of the UPR(mt) pathway dampen the enhanced stress resistance observed in TRAP1 overexpression flies. Together these studies suggest that TRAP1 regulates healthspan, potentially through activation of the UPR(mt).
2012
Song E, de Bivort B, Dan C, Kunes S. Determinants of the Drosophila odorant receptor pattern. Dev Cell. 2012;22 (2) :363-76.Abstract
In most olfactory systems studied to date, neurons that express the same odorant receptor (Or) gene are scattered across sensory epithelia, intermingled with neurons that express different Or genes. In Drosophila, olfactory sensilla that express the same Or gene are dispersed on the antenna and the maxillary palp. Here we show that Or identity is specified in a spatially stereotyped pattern by the cell-autonomous activity of the transcriptional regulators Engrailed and Dachshund. Olfactory sensilla then become highly motile and disperse beneath the epidermis. Thus, positional information and cell motility underlie the dispersed patterns of Drosophila Or gene expression.
Dearborn RE, Dai Y, Reed B, Karian T, Gray J, Kunes S. Reph, a regulator of Eph receptor expression in the Drosophila melanogaster optic lobe. PLoS One. 2012;7 (5) :e37303.Abstract
Receptors of the Eph family of tyrosine kinases and their Ephrin ligands are involved in developmental processes as diverse as angiogenesis, axon guidance and cell migration. However, our understanding of the Eph signaling pathway is incomplete, and could benefit from an analysis by genetic methods. To this end, we performed a genetic modifier screen for mutations that affect Eph signaling in Drosophila melanogaster. Several dozen loci were identified on the basis of their suppression or enhancement of an eye defect induced by the ectopic expression of Ephrin during development; many of these mutant loci were found to disrupt visual system development. One modifier locus, reph (regulator of eph expression), was characterized in molecular detail and found to encode a putative nuclear protein that interacts genetically with Eph signaling pathway mutations. Reph is an autonomous regulator of Eph receptor expression, required for the graded expression of Eph protein and the establishment of an optic lobe axonal topographic map. These results reveal a novel component of the regulatory pathway controlling expression of eph and identify reph as a novel factor in the developing visual system.
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. Proc Natl Acad Sci U S A. 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.
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. J Neurosci 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.
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. J Biol Chem. 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.
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. J Mol Evol. 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.
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. Curr Opin Neurobiol. 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?
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. Dev 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.
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.
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.