Biology subjects

Benton, R.

Publications and source records attributed to Benton, R..

6 recordsLinked to original sources

bioRxiv2018

In vivo assembly and trafficking of olfactory Ionotropic Receptors

lonotropic Receptors (IRs) are a large, divergent subfamily of ionotropic glutamate receptors(iGluRs), with roles in chemosensation, thermosensation and hygrosensation. Analogous to the synaptic targeting mechanisms of their iGluR ancestors, IRs are thought to form complexes of broadly-expressed co-receptors and selectively-expressed tuning receptors to localise to sensory cilia. While tuning receptors extracellular ligand-binding domain (LBD) defines sensory specificity, the role of this domain in co-receptors is unclear. We identify a coreceptor-specific sequence in the LBD, which contains a single N-glycosylation site. Combining molecular genetic and cell biological analyses, we show that this site is dispensable for assembly of IR complexes in olfactory sensory neurons, but essential for endoplasmic reticulum exit of some,but not all, IR complexes. Our data reveal an important role for the IR co-receptor LBD in control of intracellular transport, provide novel insights into the stoichiometry and assembly of IR complexes, and uncover an unexpected heterogeneity in the trafficking regulation of this sensory receptor family.

neuroscience↗

bioRxiv2018

Molecular evolution of juvenile hormone esterase-like proteins in a socially exchanged fluid

Socially exchanged fluids are a direct means for organisms to influence conspecifics. When orally feeding larval offspring via trophallaxis, Camponotus floridanus ant workers were shown to transfer Juvenile Hormone (JH), a key developmental regulator, as well as paralogs of JH esterase (JHE), an enzyme that hydrolyzes JH. We combine proteomic, phylogenetic and selection analyses to investigate the evolution of this esterase subfamily. We show that Camponotus JHE-like proteins have sustained multiple duplications, positive selection, and changed localization to become abundantly and selectively present in trophallactic fluid. To assess their potential role in larval development, we fed workers a JHE-specific inhibitor to introduce it into the trophallactic network. This increased the proportion larvae reared to metamorphosis by these workers, similar to supplementation with JH. Together these findings suggest that JHE-like proteins have evolved new roles in inter-individual regulation of larval development in Camponotus.

evolutionary biology↗

bioRxiv2018

Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system

Nervous systems exhibit myriad cell types, but understanding how this diversity arises is hampered by the difficulty to visualize and genetically-interrogate specific lineages, especially at early developmental stages prior to expression of unique molecular markers. Here, we use a genetic immortalization method to analyze the development of sensory neuron lineages in the Drosophila olfactory system, from their origin to terminal differentiation. We apply this approach to first define a fate map of all olfactory lineages and refine the model of temporal patterns of lineage divisions. Taking advantage of a selective marker for the lineage that gives rise to Or67d pheromone-sensing neurons and a genome-wide transcription factor RNAi screen, we identify the spatial and temporal requirements for Pointed, an ETS family member, in this developmental pathway. Transcriptomic analysis of wild-type and Pointed-depleted olfactory tissue reveals a universal requirement for this factor as a switch-like determinant of fates in these sensory lineages.

neuroscience↗

bioRxiv2018

An expression atlas of chemosensory ionotropic glutamate receptors identifies a molecular basis of carbonation detection

Taste perception is thought to involve the encoding of appetitive and aversive chemical cues in food through a limited number of sensory pathways. Through expression analysis of the complete repertoire of Drosophila Ionotropic Receptors (IRs), a sensory subfamily of ionotropic glutamate receptors, we reveal that the majority of IRs is expressed in diverse peripheral neuron populations across gustatory organs in both larvae and adults, implying numerous roles in taste-evoked behaviours. We characterise Ir56d, which labels two anatomically-distinct classes of neurons in the proboscis: one represents a subset of sugar- and fatty acid-sensing neurons, while the other responds to carbonated solutions and fatty acids. Mutational analysis shows that IR56d, together with the broadly-expressed co-receptors IR25a and IR76b, is essential for physiological activation by carbonation and fatty acids, but not sucrose. We further demonstrate that carbonation is behaviourally attractive to flies (in an IR56d-dependent manner), but in a distinct way to other appetitive stimuli. Our work provides a valuable toolkit for investigating the taste functions of IRs, defines a molecular basis of carbonation sensing, and illustrates how the gustatory system uses combinatorial expression of sensory receptors in distinct neuron types to coordinate behaviour.

neuroscience↗

bioRxiv2016

Second Generation Drosophila Chemical Tags: Sensitivity, Versatility and Speed

Labeling and visualizing cells and sub-cellular structures within thick tissues, whole organs and even intact animals is key to studying biological processes. This is particularly true for studies of neural circuits where neurons form sub-micron synapses but have arbors that may span millimeters in length. Traditionally labeling is achieved by immunofluorescence; however diffusion of antibody molecules (>100 kDa) is slow and often results in uneven labeling with very poor penetration into the centre of thick specimens; these limitations can be partially addressed by extending staining protocols to over a week (Drosophila brain) and months (mice). Recently we developed an alternative approach using genetically encoded chemical tags CLIP, SNAP, Halo and TMP for tissue labeling; this resulted in >100 fold increase in labeling speed in both mice and Drosophila, at the expense of a considerable drop in absolute sensitivity when compared to optimized immunofluorescence staining. We now present a second generation of UAS and LexA responsive CLIP, SNAPf and Halo chemical labeling reagents for flies. These multimerized tags with translational enhancers display up to 64 fold increase in sensitivity over first generation reagents. In addition we developed a suite of conditional reporters (4xSNAPf tag and CLIP-SNAP-Halo) that are activated by the DNA recombinase Bxb1. Our new reporters can be used with weak and strong GAL4 and LexA drivers and enable stochastic, intersectional and multicolor Brainbow labeling. These improvements in sensitivity and experimental versatility, while still retaining the substantial speed advantage that is a signature of chemical labeling, should significantly increase the scope of this technology.

neuroscience↗

bioRxiv2016

FlyLimbTracker: an active contour based approach for leg segment tracking in unmarked, freely behaving Drosophila

Understanding the biological underpinnings of movement and action requires the development of tools for precise, quantitative, and high-throughput measurements of animal behavior. Drosophila melanogaster provides an ideal model for developing such tools: the fly has unparalleled genetic accessibility and depends on a relatively compact nervous system to generate sophisticated limbed behaviors including walking, reaching, grooming, courtship, and boxing. Here we describe a method that uses active contours to semi-automatically track body and leg segments from video image sequences of unmarked, freely behaving Drosophila. We show that this approach is robust to wide variations in video spatial and temporal resolution and that it can be used to measure leg segment motions during a variety of locomotor and grooming behaviors. FlyLimbTracker, the software implementation of this method, is open-source and our approach is generalizable. This opens up the possibility of tracking leg movements in other species by modifications of underlying active contour models.\n\nAuthor SummaryIn terrestrial animals, including humans, fundamental actions like locomotion and grooming emerge from the displacement of multiple limbs through space. Therefore, precise measurements of limb movements are critical for investigating and, ultimately, understanding the neural basis for behavior. The vinegar fly, Drosophila melanogaster, is an attractive animal model for uncovering general principles about limb control since its genome and nervous system are easy to manipulate. However, existing methods for measuring leg movements in freely behaving Drosophila have significant drawbacks: they require complicated experimental setups and provide limited information about each leg. Here we report a new method - and provide its open-source software implementation, FlyLimbTracker - for tracking the body and leg segments of freely behaving flies using only computational image processing approaches. We illustrate the power of this method by tracking fly limbs during five distinct walking and grooming behaviors and from videos across a wide range of spatial and temporal resolutions. Our approach is generalizable, allowing researchers to use and customize our software for limb tracking in Drosophila and in other species.

bioinformatics↗