K. VijayRaghavan

A Protein Complex Network of Drosophila melanogaster

Determining the composition of protein complexes is an essential step toward understanding the cell as an integrated system. Using coaffinity purification coupled to mass spectrometry analysis, we examined protein associations involving nearly 5,000 individual, FLAG-HA epitope-tagged Drosophila proteins. Stringent analysis of these data, based on a statistical framework designed to define individual protein-protein interactions, led to the generation of a Drosophila protein interaction map (DPiM) encompassing 556 protein complexes. The high quality of the DPiM and its usefulness as a paradigm for metazoan proteomes are apparent from the recovery of many known complexes, significant enrichment for shared functional attributes, and validation in human cells. The DPiM defines potential novel members for several important protein complexes and assigns functional links to 586 protein-coding genes lacking previous experimental annotation. The DPiM represents, to our knowledge, the largest metazoan protein complex map and provides a valuable resource for analysis of protein complex evolution.

Nanoscale Organization of Hedgehog Is Essential for Long-Range Signaling

Hedgehog (Hh) plays crucial roles in tissue-patterning and activates signaling in Patched (Ptc)-expressing cells. Paracrine signaling requires release and transport over many cell diameters away by a process that requires interaction with heparan sulfate proteoglycans (HSPGs). Here, we examine the organization of functional, fluorescently tagged variants in living cells by using optical imaging, FRET microscopy, and mutational studies guided by bioinformatics prediction. We find that cell-surface Hh forms suboptical oligomers, further concentrated in visible clusters colocalized with HSPGs. Mutation of a conserved Lys in a predicted Hh-protomer interaction interface results in an autocrine signaling-competent Hh isoform--incapable of forming dense nanoscale oligomers, interacting with HSPGs, or paracrine signaling. Thus, Hh exhibits a hierarchical organization from the nanoscale to visible clusters with distinct functions.

even-skipped Determines the Dorsal Growth of Motor Axons in Drosophila

Axon pathfinding and target choice are governed by cell type-specific responses to external cues. Here, we show that in the Drosophila embryo, motorneurons with targets in the dorsal muscle field express the homeobox gene even-skipped and that this expression is necessary and sufficient to direct motor axons into the dorsal muscle field. Previously, it was shown that motorneurons projecting to ventral targets express the LIM homeobox gene islet, which is sufficient to direct axons to the ventral muscle field. Thus, even-skipped complements the function of islet, and together these two genes constitute a bimodal switch regulating axonal growth and directing motor axons to ventral or to dorsal regions of the muscle field.

A genome-wide resource for the analysis of protein localisation in Drosophila

The Drosophila genome contains >13000 protein-coding genes, the majority of which remain poorly investigated. Important reasons include the lack of antibodies or reporter constructs to visualise these proteins. Here, we present a genome-wide fosmid library of 10000 GFP-tagged clones, comprising tagged genes and most of their regulatory information. For 880 tagged proteins, we created transgenic lines, and for a total of 207 lines, we assessed protein expression and localisation in ovaries, embryos, pupae or adults by stainings and live imaging approaches. Importantly, we visualised many proteins at endogenous expression levels and found a large fraction of them localising to subcellular compartments. By applying genetic complementation tests, we estimate that about two-thirds of the tagged proteins are functional. Moreover, these tagged proteins enable interaction proteomics from developing pupae and adult flies. Taken together, this resource will boost systematic analysis of protein expression and localisation in various cellular and developmental contexts. DOI: http://dx.doi.org/10.7554/eLife.12068.001

Muscle pattern diversification in Drosophila: the story of imaginal myogenesis.

There are two phases of somatic muscle formation in Drosophila. During embryonic development, one phase of myogenesis generates larval muscle elements that mediate the relatively simple behavioural repertoire of the larva. During pupal metamorphosis, a diverse pattern of muscle fibres are assembled, and these facilitate the more elaborate behavioural patterns of the adult fly. In this review, we discuss the current status of understanding of the cellular, genetic, and molecular mechanisms of pattern formation during the second phase, imaginal muscle development. We briefly compare aspects of embryonic and adult myogenesis in Drosophila and muscle development in vertebrates and highlight conserved themes and disparities between these diverse myogenic programmes. BioEssays 21:486–498, 1999.

Founder myoblasts and fibre number during adult myogenesis in Drosophila.

We have examined the mechanisms underlying the setting of myotubes and choice of myotube number in adult Drosophila. We find that the pattern of adult myotubes is prefigured by a pattern of duf-lacZ-expressing myoblasts at appropriate locations. Selective expression of duf-lacZ in single myoblasts emerges from generalized, low-level expression in all adult myoblasts during the third larval instar. The number of founders, thus chosen, corresponds to the number of fibres in a muscle. In contrast to the embryo, the selection of individual adult founder cells during myogenesis does not depend on Notch-mediated lateral inhibition. Our results suggest a general mechanism by which multi-fibre muscles can be patterned.

Myoblast Diversification and Ectodermal Signaling in Drosophila

The flight muscles of Drosophila derive from myoblasts found on the third instar disc. We demonstrate that these myoblasts already show distinctive properties and examine how this diversity is generated. In the late larva, Vestigial and low levels of Cut are expressed in myoblasts that will contribute to the indirect flight muscles. Other myoblasts, which express high levels of Cut but no Vestigial, are required for the formation of the direct flight muscles. Vestigial and Cut expression are stabilized by a mutually repressive feedback loop. Vestigial expression begins in the embryo in a subset of adult myoblasts, and Wingless signaling is required later to maintain this expression. Thus, myoblasts are divided into identifiable populations, consistent with their allocation to different muscles, and ectodermal signals act to maintain these differences.

Dendritic Targeting in the Leg Neuropil of Drosophila: The Role of Midline Signalling Molecules in Generating a Myotopic Map

During development of the Drosophila motor system, global guidance cues control and coordinate the targeting of both input and output elements of the neural system.

FMRP and Ataxin-2 function together in long-term olfactory habituation and neuronal translational control

Significance This work explores the endogenous functions of two disease- and RNA-associated proteins, FMRP (fragile X protein) and Ataxin-2, in memory, synaptic plasticity, RNA regulation, and messenger ribo-nucleoprotein particle assembly. By documenting an array of common phenotypic consequences from loss of Atx2 or dFMR1, the results argue that both proteins have similar in vivo functions and that differences in spinocerebellar ataxia 2 and fragile X disease pathologies may arise from the distinct features of the respective disease causative mutations. This work provides insight into the in vivo mechanisms of long-term memory (LTM)-associated synaptic plasticity and into the roles of FMRP and Atx2 in neuronal translational control. Fragile X mental retardation protein (FMRP) and Ataxin-2 (Atx2) are triplet expansion disease- and stress granule-associated proteins implicated in neuronal translational control and microRNA function. We show that Drosophila FMRP (dFMR1) is required for long-term olfactory habituation (LTH), a phenomenon dependent on Atx2-dependent potentiation of inhibitory transmission from local interneurons (LNs) to projection neurons (PNs) in the antennal lobe. dFMR1 is also required for LTH-associated depression of odor-evoked calcium transients in PNs. Strong transdominant genetic interactions among dFMR1, atx2, the deadbox helicase me31B, and argonaute1 (ago1) mutants, as well as coimmunoprecitation of dFMR1 with Atx2, indicate that dFMR1 and Atx2 function together in a microRNA-dependent process necessary for LTH. Consistently, PN or LN knockdown of dFMR1, Atx2, Me31B, or the miRNA-pathway protein GW182 increases expression of a Ca2+/calmodulin-dependent protein kinase II (CaMKII) translational reporter. Moreover, brain immunoprecipitates of dFMR1 and Atx2 proteins include CaMKII mRNA, indicating respective physical interactions with this mRNA. Because CaMKII is necessary for LTH, these data indicate that fragile X mental retardation protein and Atx2 act via at least one common target RNA for memory-associated long-term synaptic plasticity. The observed requirement in LNs and PNs supports an emerging view that both presynaptic and postsynaptic translation are necessary for long-term synaptic plasticity. However, whereas Atx2 is necessary for the integrity of dendritic and somatic Me31B-containing particles, dFmr1 is not. Together, these data indicate that dFmr1 and Atx2 function in long-term but not short-term memory, regulating translation of at least some common presynaptic and postsynaptic target mRNAs in the same cells.

Metamorphosis of an identified serotonergic neuron in the Drosophila olfactory system

BackgroundOdors are detected by sensory neurons that carry information to the olfactory lobe where they connect to projection neurons and local interneurons in glomeruli: anatomically well-characterized structures that collect, integrate and relay information to higher centers. Recent studies have revealed that the sensitivity of such networks can be modulated by wide-field feedback neurons. The connectivity and function of such feedback neurons are themselves subject to alteration by external cues, such as hormones, stress, or experience. Very little is known about how this class of central neurons changes its anatomical properties to perform functions in altered developmental contexts. A mechanistic understanding of how central neurons change their anatomy to meet new functional requirements will benefit greatly from the establishment of a model preparation where cellular and molecular changes can be examined in an identified central neuron.ResultsIn this study, we examine a wide-field serotonergic neuron in the Drosophila olfactory pathway and map the dramatic changes that it undergoes from larva to adult. We show that expression of a dominant-negative form of the ecdysterone receptor prevents remodeling. We further use different transgenic constructs to silence neuronal activity and report defects in the morphology of the adult-specific dendritic trees. The branching of the presynaptic axonal arbors is regulated by mechanisms that affect axon growth and retrograde transport. The neuron develops its normal morphology in the absence of sensory input to the antennal lobe, or of the mushroom bodies. However, ablation of its presumptive postsynaptic partners, the projection neurons and/or local interneurons, affects the growth and branching of terminal arbors.ConclusionOur studies establish a cellular system for studying remodeling of a central neuromodulatory feedback neuron and also identify key elements in this process. Understanding the morphogenesis of such neurons, which have been shown in other systems to modulate the sensitivity and directionality of response to odors, links anatomy to the development of olfactory behavior.