Biology subjects

Miller, R. M.

Publications and source records attributed to Miller, R. M..

4 recordsLinked to original sources

bioRxiv2023

An accessible and generalizable in vitro luminescence assay for detecting GPCR activation

G-protein coupled receptors (GPCRs) serve critical physiological roles as the most abundant family of receptors. Here we describe the design of a generalizable and accessible In vitro GPCR split NanoLuc ligand Triggered Reporter (IGNiTR), having broad and diverse applications. IGNiTR leverages the interaction between a conformationspecific binder and agonist-activated GPCR to reconstitute a split nanoluciferase. We have demonstrated IGNiTR with three Gs-coupled GPCRs and a Gi-coupled GPCR with three classes of conformation-specific binders: nanobodies, miniG proteins, and G-protein peptidomimetics. IGNiTR demonstrated binding efficacy and potency values of various Dopamine Receptor D1 (DRD1) ligands that agree well with reported values. IGNiTR also allows the use of a synthetic G protein peptidomimetic, providing easily standardized reagents for characterizing GPCRs and ligands. We demonstrated three applications of IGNiTR: 1) characterizing GPCR functionality during Nanodisc-based reconstitution process; 2) highthroughput screening of ligands against DRD1; 3) detection of opioids for in the field applications. Due to its convenience, accessibility and consistency, IGNiTR will find extensive applications in GPCR ligand detection, screening and GPCR characterization.

biochemistry↗

bioRxiv2021

Sex-specific topology of the nociceptive circuit shapes dimorphic behavior in C. elegans

How sexually dimorphic behavior is encoded in the nervous system is poorly understood. Here, we characterize the dimorphic nociceptive behavior in C. elegans and study the underlying circuits, which are composed of the same neurons but are wired differently. We show that while sensory transduction is similar in the two sexes, the downstream network topology markedly shapes behavior. We fit a network model that replicates the observed dimorphic behavior in response to external stimuli, and use it to predict simple network rewirings that would switch the behavior between the sexes. We then show experimentally that these subtle synaptic rewirings indeed flip behavior. Strikingly, when presented with aversive cues, rewired males were compromised in finding mating partners, suggesting that network topologies that enable efficient avoidance of noxious cues have a reproductive "cost". Our results present a deconstruction of the design of a neural circuit that controls sexual behavior, and how to reprogram it.

neuroscience↗

bioRxiv2020

Spritz: A Proteogenomic Database Engine

Proteoforms are the workhorses of the cell, and subtle differences between their amino acid sequence or post-translational modifications (PTMs) can change their biological function. To most effectively identify and quantify proteoforms in genetically diverse samples by mass spectrometry (MS), it is advantageous to search the MS data against a sample-specific protein database that is tailored to the sample being analyzed, in that it contains the correct amino acid sequences and relevant PTMs for that sample. To this end, we have developed Spritz (https://smith-chem-wisc.github.io/Spritz/), an open-source software tool for generating protein databases annotated with sequence variations and PTMs. We provide a simple graphical user interface (GUI) for Windows and scripts that can be run on any operating system. Spritz automatically sets up and executes approximately 20 tools, which enable construction of a proteogenomic database from only raw RNA sequencing data. Sequence variations that are discovered in RNA sequencing data upon comparison to the Ensembl reference genome are annotated on proteins in these databases, and PTM annotations are transferred from UniProt. Modifications can also be discovered and added to the database using bottom-up mass spectrometry data and global PTM discovery in MetaMorpheus. We demonstrate that such sample-specific databases allow the identification of variant peptides, modified variant peptides, and variant proteoforms by searching bottom-up and top-down proteomic data from the Jurkat human T lymphocyte cell line and demonstrate the identification of phosphorylated variant sites with phosphoproteomic data from the U2OS human osteosarcoma cell line.

bioinformatics↗

bioRxiv2019

A neuroendocrine feedback loop in C. elegans males integrates food detection and biological sex to modulate chemoreceptor expression and behavioral flexibility

Dynamic integration of internal and external cues is essential for flexible, adaptive animal behavior. In C. elegans, biological sex and feeding state regulate expression of the food-associated chemoreceptor odr-10, contributing to plasticity in food detection and the decision between feeding and exploration. In adult hermaphrodites, odr-10 expression is high; in well-fed adult males, odr-10 expression is low, promoting exploratory mate-searching behavior. Food-deprivation transiently activates male odr-10 expression, heightening food sensitivity and reducing food-leaving. Here, we identify a neuroendocrine feedback loop that sex-specifically regulates odr-10 in response to food deprivation. In well-fed males, insulin-like (IIS) and TGF{beta} signaling repress odr-10 expression. Upon food deprivation, odr-10 is directly activated by DAF-16/FoxO, the canonical C. elegans IIS effector. The TGF{beta} ligand DAF-7 acts upstream of IIS, and, likely because of its sexually dimorphic expression in the nervous system, links feeding to odr-10 only in males. Surprisingly, these responses to food deprivation are not triggered by internal metabolic cues, but rather by the loss of sensory signals from food. In the presence of inedible food, males become metabolically starved but express levels of odr-10 and daf-7 comparable to those of well-fed males. Further, exposing food-deprived males to inedible food is sufficient to restore low odr-10 expression. Food signals are detected by a small number of sensory neurons whose activity non-autonomously regulates daf-7 expression, IIS, and odr-10. Thus, adult C. elegans males employ a neuroendocrine feedback loop that integrates food detection and genetic sex to dynamically modulate chemoreceptor expression and sensory behavior.

neuroscience↗