Hot off the presses! Oct 26 PLoS Biol

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Latest Articles Include:

• Open Education, Open Minds
  - PLoS Biol 8(10):e1000508 (2010)
  
• Parallel Lives
  - PLoS Biol 8(10):e1000528 (2010)
  
• Stress May Drive Plant Patterns
  - PLoS Biol 8(10):e1000517 (2010)
  
• Day and Night: Circadian Rhythms in Worms
  - PLoS Biol 8(10):e1000511 (2010)
  
• Nuclear Fission without Microtubules—a Return to the Past?
  - PLoS Biol 8(10):e1000515 (2010)
  
• Axonal Signaling Proteins Take the ER Highway
  - PLoS Biol 8(10):e1000504 (2010)
  
• In Living Color: Bacterial Pigments as an Untapped Resource in the Classroom and Beyond
  - PLoS Biol 8(10):e1000510 (2010)
  
• Trying to Avoid Your Sister
  - PLoS Biol 8(10):e1000519 (2010)
  
• Parasite Evolution and Life History Theory
  - PLoS Biol 8(10):e1000524 (2010)
  
• Making Marine Life Count: A New Baseline for Policy
  - PLoS Biol 8(10):e1000531 (2010)
  
• Ask A Biologist: Bringing Science to the Public
  - PLoS Biol 8(10):e1000458 (2010)
  
• Non-Canonical NF-κB Activation and Abnormal B Cell Accumulation in Mice Expressing Ubiquitin Protein Ligase-Inactive c-IAP2
  - PLoS Biol 8(10):e1000518 (2010)
  Chromosomal translocations between loci encoding MALT1 and c-IAP2 are common in MALT lymphomas. The resulting fusion proteins lack the c-IAP2 RING domain, the region responsible for its ubiquitin protein ligase (E3) activity. Ectopic expression of the fusion protein activates the canonical NF-κB signaling cascade, but how it does so is controversial and how it promotes MALT lymphoma is unknown. Considering recent reports implicating c-IAP1 and c-IAP2 E3 activity in repression of non-canonical NF-κB signaling, we asked if the c-IAP2/MALT fusion protein can initiate non-canonical NF-κB activation. Here we show that in addition to canonical activation, the fusion protein stabilizes NIK and activates non-canonical NF-κB. Canonical but not non-canonical activation depended on MALT1 paracaspase activity, and expression of E3-inactive c-IAP2 activated non-canonical NF-κB. Mice in which endogenous c-IAP2 was replaced with an E3-inactive mutant accumulated abnormal B cells! with elevated non-canonical NF-κB and had increased numbers of B cells with a marginal zone phenotype, gut-associated lymphoid hyperplasia, and other features of MALT lymphoma. Thus, the c-IAP2/MALT1 fusion protein activates NF-κB by two distinct mechanisms, and loss of c-IAP2 E3 activity in vivo is sufficient to induce abnormalities common to MALT lymphoma.
• A Protein Inventory of Human Ribosome Biogenesis Reveals an Essential Function of Exportin 5 in 60S Subunit Export
  - PLoS Biol 8(10):e1000522 (2010)
  The assembly of ribosomal subunits in eukaryotes is a complex, multistep process so far mostly studied in yeast. In S. cerevisiae, more than 200 factors including ribosomal proteins and trans-acting factors are required for the ordered assembly of 40S and 60S ribosomal subunits. To date, only few human homologs of these yeast ribosome synthesis factors have been characterized. Here, we used a systematic RNA interference (RNAi) approach to analyze the contribution of 464 candidate factors to ribosomal subunit biogenesis in human cells. The screen was based on visual readouts, using inducible, fluorescent ribosomal proteins as reporters. By performing computer-based image analysis utilizing supervised machine-learning techniques, we obtained evidence for a functional link of 153 human proteins to ribosome synthesis. Our data show that core features of ribosome assembly are conserved from yeast to human, but differences exist for instance with respect to 60S subunit expor! t. Unexpectedly, our RNAi screen uncovered a requirement for the export receptor Exportin 5 (Exp5) in nuclear export of 60S subunits in human cells. We show that Exp5, like the known 60S exportin Crm1, binds to pre-60S particles in a RanGTP-dependent manner. Interference with either Exp5 or Crm1 function blocks 60S export in both human cells and frog oocytes, whereas 40S export is compromised only upon inhibition of Crm1. Thus, 60S subunit export is dependent on at least two RanGTP-binding exportins in vertebrate cells.
• Field Studies Reveal Strong Postmating Isolation between Ecologically Divergent Butterfly Populations
  - PLoS Biol 8(10):e1000529 (2010)
  Gene flow between populations that are adapting to distinct environments may be restricted if hybrids inherit maladaptive, intermediate phenotypes. This phenomenon, called extrinsic postzygotic isolation (EPI), is thought to play a critical role in the early stages of speciation. However, despite its intuitive appeal, we know surprisingly little about the strength and prevalence of EPI in nature, and even less about the specific phenotypes that tend to cause problems for hybrids. In this study, we searched for EPI among allopatric populations of the butterfly Euphydryas editha that have specialized on alternative host plants. These populations recall a situation thought typical of the very early stages of speciation. They lack consistent host-associated genetic differentiation at random nuclear loci and show no signs of reproductive incompatibility in the laboratory. However, they do differ consistently in diverse host-related traits. For each of these traits, we first! asked whether hybrids between populations that use different hosts (different-host hybrids) were intermediate to parental populations and to hybrids between populations that use the same host (same-host hybrids). We then conducted field experiments to estimate the effects of intermediacy on fitness in nature. Our results revealed strong EPI under field conditions. Different-host hybrids exhibited an array of intermediate traits that were significantly maladaptive, including four behaviors. Intermediate foraging height slowed the growth of larvae, while intermediate oviposition preference, oviposition site height, and clutch size severely reduced the growth and survival of the offspring of adult females. We used our empirical data to construct a fitness surface on which different-host hybrids can be seen to fall in an adaptive valley between two peaks occupied by same-host hybrids. These findings demonstrate how ecological selection against hybrids can create a strong barri! er to gene flow at the early stages of adaptive divergence.
• iCLIP Predicts the Dual Splicing Effects of TIA-RNA Interactions
  - PLoS Biol 8(10):e1000530 (2010)
  The regulation of alternative splicing involves interactions between RNA-binding proteins and pre-mRNA positions close to the splice sites. T-cell intracellular antigen 1 (TIA1) and TIA1-like 1 (TIAL1) locally enhance exon inclusion by recruiting U1 snRNP to 5′ splice sites. However, effects of TIA proteins on splicing of distal exons have not yet been explored. We used UV-crosslinking and immunoprecipitation (iCLIP) to find that TIA1 and TIAL1 bind at the same positions on human RNAs. Binding downstream of 5′ splice sites was used to predict the effects of TIA proteins in enhancing inclusion of proximal exons and silencing inclusion of distal exons. The predictions were validated in an unbiased manner using splice-junction microarrays, RT-PCR, and minigene constructs, which showed that TIA proteins maintain splicing fidelity and regulate alternative splicing by binding exclusively downstream of 5′ splice sites. Surprisingly, TIA binding at 5′ splice sites sile! nced distal cassette and variable-length exons without binding in proximity to the regulated alternative 3′ splice sites. Using transcriptome-wide high-resolution mapping of TIA-RNA interactions we evaluated the distal splicing effects of TIA proteins. These data are consistent with a model where TIA proteins shorten the time available for definition of an alternative exon by enhancing recognition of the preceding 5′ splice site. Thus, our findings indicate that changes in splicing kinetics could mediate the distal regulation of alternative splicing.
• Quiescent Fibroblasts Exhibit High Metabolic Activity
  - PLoS Biol 8(10):e1000514 (2010)
  Many cells in mammals exist in the state of quiescence, which is characterized by reversible exit from the cell cycle. Quiescent cells are widely reported to exhibit reduced size, nucleotide synthesis, and metabolic activity. Much lower glycolytic rates have been reported in quiescent compared with proliferating lymphocytes. In contrast, we show here that primary human fibroblasts continue to exhibit high metabolic rates when induced into quiescence via contact inhibition. By monitoring isotope labeling through metabolic pathways and quantitatively identifying fluxes from the data, we show that contact-inhibited fibroblasts utilize glucose in all branches of central carbon metabolism at rates similar to those of proliferating cells, with greater overflow flux from the pentose phosphate pathway back to glycolysis. Inhibition of the pentose phosphate pathway resulted in apoptosis preferentially in quiescent fibroblasts. By feeding the cells labeled glutamine, we also det! ected a "backwards" flux in the tricarboxylic acid cycle from α-ketoglutarate to citrate that was enhanced in contact-inhibited fibroblasts; this flux likely contributes to shuttling of NADPH from the mitochondrion to cytosol for redox defense or fatty acid synthesis. The high metabolic activity of the fibroblasts was directed in part toward breakdown and resynthesis of protein and lipid, and in part toward excretion of extracellular matrix proteins. Thus, reduced metabolic activity is not a hallmark of the quiescent state. Quiescent fibroblasts, relieved of the biosynthetic requirements associated with generating progeny, direct their metabolic activity to preservation of self integrity and alternative functions beneficial to the organism as a whole.
• Alignment between PIN1 Polarity and Microtubule Orientation in the Shoot Apical Meristem Reveals a Tight Coupling between Morphogenesis and Auxin Transport
  - PLoS Biol 8(10):e1000516 (2010)
  Morphogenesis during multicellular development is regulated by intercellular signaling molecules as well as by the mechanical properties of individual cells. In particular, normal patterns of organogenesis in plants require coordination between growth direction and growth magnitude. How this is achieved remains unclear. Here we show that in Arabidopsis thaliana, auxin patterning and cellular growth are linked through a correlated pattern of auxin efflux carrier localization and cortical microtubule orientation. Our experiments reveal that both PIN1 localization and microtubule array orientation are likely to respond to a shared upstream regulator that appears to be biomechanical in nature. Lastly, through mathematical modeling we show that such a biophysical coupling could mediate the feedback loop between auxin and its transport that underlies plant phyllotaxis.
• Frequent and Efficient Use of the Sister Chromatid for DNA Double-Strand Break Repair during Budding Yeast Meiosis
  - PLoS Biol 8(10):e1000520 (2010)
  Recombination between homologous chromosomes of different parental origin (homologs) is necessary for their accurate segregation during meiosis. It has been suggested that meiotic inter-homolog recombination is promoted by a barrier to inter-sister-chromatid recombination, imposed by meiosis-specific components of the chromosome axis. Consistent with this, measures of Holliday junction–containing recombination intermediates (joint molecules [JMs]) show a strong bias towards inter-homolog and against inter-sister JMs. However, recombination between sister chromatids also has an important role in meiosis. The genomes of diploid organisms in natural populations are highly polymorphic for insertions and deletions, and meiotic double-strand breaks (DSBs) that form within such polymorphic regions must be repaired by inter-sister recombination. Efforts to study inter-sister recombination during meiosis, in particular to determine recombination frequencies and mechanisms, ha! ve been constrained by the inability to monitor the products of inter-sister recombination. We present here molecular-level studies of inter-sister recombination during budding yeast meiosis. We examined events initiated by DSBs in regions that lack corresponding sequences on the homolog, and show that these DSBs are efficiently repaired by inter-sister recombination. This occurs with the same timing as inter-homolog recombination, but with reduced (2- to 3-fold) yields of JMs. Loss of the meiotic-chromosome-axis-associated kinase Mek1 accelerates inter-sister DSB repair and markedly increases inter-sister JM frequencies. Furthermore, inter-sister JMs formed in mek1Δ mutants are preferentially lost, while inter-homolog JMs are maintained. These findings indicate that inter-sister recombination occurs frequently during budding yeast meiosis, with the possibility that up to one-third of all recombination events occur between sister chromatids. We suggest that a Mek1-dependen! t reduction in the rate of inter-sister repair, combined with ! the destabilization of inter-sister JMs, promotes inter-homolog recombination while retaining the capacity for inter-sister recombination when inter-homolog recombination is not possible.
• Filarial Parasites Develop Faster and Reproduce Earlier in Response to Host Immune Effectors That Determine Filarial Life Expectancy
  - PLoS Biol 8(10):e1000525 (2010)
  Humans and other mammals mount vigorous immune assaults against helminth parasites, yet there are intriguing reports that the immune response can enhance rather than impair parasite development. It has been hypothesized that helminths, like many free-living organisms, should optimize their development and reproduction in response to cues predicting future life expectancy. However, immune-dependant development by helminth parasites has so far eluded such evolutionary explanation. By manipulating various arms of the immune response of experimental hosts, we show that filarial nematodes, the parasites responsible for debilitating diseases in humans like river blindness and elephantiasis, accelerate their development in response to the IL-5 driven eosinophilia they encounter when infecting a host. Consequently they produce microfilariae, their transmission stages, earlier and in greater numbers. Eosinophilia is a primary host determinant of filarial life expectancy, operat! ing both at larval and at late adult stages in anatomically and temporally separate locations, and is implicated in vaccine-mediated protection. Filarial nematodes are therefore able to adjust their reproductive schedules in response to an environmental predictor of their probability of survival, as proposed by evolutionary theory, thereby mitigating the effects of the immune attack to which helminths are most susceptible. Enhancing protective immunity against filarial nematodes, for example through vaccination, may be less effective at reducing transmission than would be expected and may, at worst, lead to increased transmission and, hence, pathology.
• Genome-Wide Analysis of Light- and Temperature-Entrained Circadian Transcripts in Caenorhabditis elegans
  - PLoS Biol 8(10):e1000503 (2010)
  Most organisms have an endogenous circadian clock that is synchronized to environmental signals such as light and temperature. Although circadian rhythms have been described in the nematode Caenorhabditis elegans at the behavioral level, these rhythms appear to be relatively non-robust. Moreover, in contrast to other animal models, no circadian transcriptional rhythms have been identified. Thus, whether this organism contains a bona fide circadian clock remains an open question. Here we use genome-wide expression profiling experiments to identify light- and temperature-entrained oscillating transcripts in C. elegans. These transcripts exhibit rhythmic expression with temperature-compensated 24-h periods. In addition, their expression is sustained under constant conditions, suggesting that they are under circadian regulation. Light and temperature cycles strongly drive gene expression and appear to entrain largely nonoverlapping gene sets. We show that mutations in a cy! clic nucleotide-gated channel required for sensory transduction abolish both light- and temperature-entrained gene expression, implying that environmental cues act cell nonautonomously to entrain circadian rhythms. Together, these findings demonstrate circadian-regulated transcriptional rhythms in C. elegans and suggest that further analyses in this organism will provide new information about the evolution and function of this biological clock.
• Subtle Alterations in PCNA-Partner Interactions Severely Impair DNA Replication and Repair
  - PLoS Biol 8(10):e1000507 (2010)
  The robustness of complex biological processes in the face of environmental and genetic perturbations is a key biological trait. However, while robustness has been extensively studied, little is known regarding the fragility of biological processes. Here, we have examined the susceptibility of DNA replication and repair processes mediated by the proliferating cell nuclear antigen (PCNA). Using protein directed evolution, biochemical, and genetic approaches, we have generated and characterized PCNA mutants with increased affinity for several key partners of the PCNA-partner network. We found that increases in PCNA-partner interaction affinities led to severe in vivo phenotypic defects. Surprisingly, such defects are much more severe than those induced by complete abolishment of the respective interactions. Thus, the subtle and tunable nature of these affinity perturbations produced different phenotypic effects than realized with traditional "on-off" analysis using g! ene knockouts. Our findings indicate that biological systems can be robust to one set of perturbations yet fragile to others.
• Genome-Wide Analyses Reveal a Role for Peptide Hormones in Planarian Germline Development
  - PLoS Biol 8(10):e1000509 (2010)
  Bioactive peptides (i.e., neuropeptides or peptide hormones) represent the largest class of cell-cell signaling molecules in metazoans and are potent regulators of neural and physiological function. In vertebrates, peptide hormones play an integral role in endocrine signaling between the brain and the gonads that controls reproductive development, yet few of these molecules have been shown to influence reproductive development in invertebrates. Here, we define a role for peptide hormones in controlling reproductive physiology of the model flatworm, the planarian Schmidtea mediterranea. Based on our observation that defective neuropeptide processing results in defects in reproductive system development, we employed peptidomic and functional genomic approaches to characterize the planarian peptide hormone complement, identifying 51 prohormone genes and validating 142 peptides biochemically. Comprehensive in situ hybridization analyses of prohormone gene expression reveal! ed the unanticipated complexity of the flatworm nervous system and identified a prohormone specifically expressed in the nervous system of sexually reproducing planarians. We show that this member of the neuropeptide Y superfamily is required for the maintenance of mature reproductive organs and differentiated germ cells in the testes. Additionally, comparative analyses of our biochemically validated prohormones with the genomes of the parasitic flatworms Schistosoma mansoni and Schistosoma japonicum identified new schistosome prohormones and validated half of all predicted peptide-encoding genes in these parasites. These studies describe the peptide hormone complement of a flatworm on a genome-wide scale and reveal a previously uncharacterized role for peptide hormones in flatworm reproduction. Furthermore, they suggest new opportunities for using planarians as free-living models for understanding the reproductive biology of flatworm parasites.
• Fission Yeast Cells Undergo Nuclear Division in the Absence of Spindle Microtubules
  - PLoS Biol 8(10):e1000512 (2010)
  Mitosis in eukaryotic cells employs spindle microtubules to drive accurate chromosome segregation at cell division. Cells lacking spindle microtubules arrest in mitosis due to a spindle checkpoint that delays mitotic progression until all chromosomes have achieved stable bipolar attachment to spindle microtubules. In fission yeast, mitosis occurs within an intact nuclear membrane with the mitotic spindle elongating between the spindle pole bodies. We show here that in fission yeast interference with mitotic spindle formation delays mitosis only briefly and cells proceed to an unusual nuclear division process we term nuclear fission, during which cells perform some chromosome segregation and efficiently enter S-phase of the next cell cycle. Nuclear fission is blocked if spindle pole body maturation or sister chromatid separation cannot take place or if actin polymerization is inhibited. We suggest that this process exhibits vestiges of a primitive nuclear division proce! ss independent of spindle microtubules, possibly reflecting an evolutionary intermediate state between bacterial and Archeal chromosome segregation where the nucleoid divides without a spindle and a microtubule spindle-based eukaryotic mitosis.
• Emergence of Noise-Induced Oscillations in the Central Circadian Pacemaker
  - PLoS Biol 8(10):e1000513 (2010)
  Bmal1 is an essential transcriptional activator within the mammalian circadian clock. We report here that the suprachiasmatic nucleus (SCN) of Bmal1-null mutant mice, unexpectedly, generates stochastic oscillations with periods that overlap the circadian range. Dissociated SCN neurons expressed fluctuating levels of PER2 detected by bioluminescence imaging but could not generate circadian oscillations intrinsically. Inhibition of intercellular communication or cyclic-AMP signaling in SCN slices, which provide a positive feed-forward signal to drive the intracellular negative feedback loop, abolished the stochastic oscillations. Propagation of this feed-forward signal between SCN neurons then promotes quasi-circadian oscillations that arise as an emergent property of the SCN network. Experimental analysis and mathematical modeling argue that both intercellular coupling and molecular noise are required for the stochastic rhythms, providing a novel biological example of n! oise-induced oscillations. The emergence of stochastic circadian oscillations from the SCN network in the absence of cell-autonomous circadian oscillatory function highlights a previously unrecognized level of circadian organization.
• Chromosomal Redistribution of Male-Biased Genes in Mammalian Evolution with Two Bursts of Gene Gain on the X Chromosome
  - PLoS Biol 8(10):e1000494 (2010)
  Mammalian X chromosomes evolved under various mechanisms including sexual antagonism, the faster-X process, and meiotic sex chromosome inactivation (MSCI). These forces may contribute to nonrandom chromosomal distribution of sex-biased genes. In order to understand the evolution of gene content on the X chromosome and autosome under these forces, we dated human and mouse protein-coding genes and miRNA genes on the vertebrate phylogenetic tree. We found that the X chromosome recently acquired a burst of young male-biased genes, which is consistent with fixation of recessive male-beneficial alleles by sexual antagonism. For genes originating earlier, however, this pattern diminishes and finally reverses with an overrepresentation of the oldest male-biased genes on autosomes. MSCI contributes to this dynamic since it silences X-linked old genes but not X-linked young genes. This demasculinization process seems to be associated with feminization of the X chromosome with mo! re X-linked old genes expressed in ovaries. Moreover, we detected another burst of gene originations after the split of eutherian mammals and opossum, and these genes were quickly incorporated into transcriptional networks of multiple tissues. Preexisting X-linked genes also show significantly higher protein-level evolution during this period compared to autosomal genes, suggesting positive selection accompanied the early evolution of mammalian X chromosomes. These two findings cast new light on the evolutionary history of the mammalian X chromosome in terms of gene gain, sequence, and expressional evolution.
• The DNA/RNA-Dependent RNA Polymerase QDE-1 Generates Aberrant RNA and dsRNA for RNAi in a Process Requiring Replication Protein A and a DNA Helicase
  - PLoS Biol 8(10):e1000496 (2010)
  The production of aberrant RNA (aRNA) is the initial step in several RNAi pathways. How aRNA is produced and specifically recognized by RNA-dependent RNA polymerases (RdRPs) to generate double-stranded RNA (dsRNA) is not clear. We previously showed that in the filamentous fungus Neurospora, the RdRP QDE-1 is required for rDNA-specific aRNA production, suggesting that QDE-1 may be important in aRNA synthesis. Here we show that a recombinant QDE-1 is both an RdRP and a DNA-dependent RNA polymerase (DdRP). Its DdRP activity is much more robust than the RdRP activity and occurs on ssDNA but not dsDNA templates. We further show that Replication Protein A (RPA), a single-stranded DNA-binding complex that interacts with QDE-1, is essential for aRNA production and gene silencing. In vitro reconstitution assays demonstrate that QDE-1 can produce dsRNA from ssDNA, a process that is strongly promoted by RPA. Furthermore, the interaction between QDE-1 and RPA requires the RecQ DNA! helicase QDE-3, a homolog of the human Werner/Bloom Syndrome proteins. Together, these results suggest a novel small RNA biogenesis pathway in Neurospora and a new mechanism for the production of aRNA and dsRNA in RNAi pathways.
• Protein Evolution by Molecular Tinkering: Diversification of the Nuclear Receptor Superfamily from a Ligand-Dependent Ancestor
  - PLoS Biol 8(10):e1000497 (2010)
  Understanding how protein structures and functions have diversified is a central goal in molecular evolution. Surveys of very divergent proteins from model organisms, however, are often insufficient to determine the features of ancestral proteins and to reveal the evolutionary events that yielded extant diversity. Here we combine genomic, biochemical, functional, structural, and phylogenetic analyses to reconstruct the early evolution of nuclear receptors (NRs), a diverse superfamily of transcriptional regulators that play key roles in animal development, physiology, and reproduction. By inferring the structure and functions of the ancestral NR, we show—contrary to current belief—that NRs evolved from a ligand-activated ancestral receptor that existed near the base of the Metazoa, with fatty acids as possible ancestral ligands. Evolutionary tinkering with this ancestral structure generated the extraordinary diversity of modern receptors: sensitivity to different li! gands evolved because of subtle modifications of the internal cavity, and ligand-independent activation evolved repeatedly because of various mutations that stabilized the active conformation in the absence of ligand. Our findings illustrate how a mechanistic dissection of protein evolution in a phylogenetic context can reveal the deep homology that links apparently "novel" molecular functions to a common ancestral form.
• An Integrated Micro- and Macroarchitectural Analysis of the Drosophila Brain by Computer-Assisted Serial Section Electron Microscopy
  - PLoS Biol 8(10):e1000502 (2010)
  The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval ! brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect neuropile.
• Polarized Secretion of Drosophila EGFR Ligand from Photoreceptor Neurons Is Controlled by ER Localization of the Ligand-Processing Machinery
  - PLoS Biol 8(10):e1000505 (2010)
  The release of signaling molecules from neurons must be regulated, to accommodate their highly polarized structure. In the developing Drosophila visual system, photoreceptor neurons secrete the epidermal growth factor receptor ligand Spitz (Spi) from their cell bodies, as well as from their axonal termini. Here we show that subcellular localization of Rhomboid proteases, which process Spi, determines the site of Spi release from neurons. Endoplasmic reticulum (ER) localization of Rhomboid 3 is essential for its ability to promote Spi secretion from axons, but not from cell bodies. We demonstrate that the ER extends throughout photoreceptor axons, and show that this feature facilitates the trafficking of the Spi precursor, the ligand chaperone Star, and Rhomboid 3 to axonal termini. Following this trafficking step, secretion from the axons is regulated in a manner similar to secretion from cell bodies. These findings uncover a role for the ER in trafficking proteins fro! m the neuronal cell body to axon terminus.