Accurate measurement of the amount of specific protein a cell produces is important for investigating basic molecular processes. We have developed a technique that allows for quantitation of protein levels in single cells in vivo. This protein quantitation ratioing (PQR) technique uses a genetic tag that produces a stoichiometric ratio of a fluorescent protein reporter and the protein of interest during protein translation. The fluorescence intensity is proportional to the number of molecules produced of the protein of interest and is used to determine the relative amount of protein within the cell. We use PQR to quantify protein expression of different genes using quantitative imaging, electrophysiology, and phenotype. We use genome editing to insert Protein Quantitation Reporters into endogenous genomic loci in three different genomes for quantitation of endogenous protein levels. The PQR technique will allow for a wide range of quantitative experiments examining gene-to-phenotype relationships with greater accuracy.

In the originally published version of this article, the DNA sequence reported in the Experimental Procedures for the PQR in Drosophila cells was the incorrect variant. The correct DNA sequence for the PQR in Drosophila cells is 5'-GGAAGCGGAGAAGGTCGTGGTAGT CTACTAACGTGTGGTGATGTAGAAGAAAATCCTGGACCT-3'.

We describe a protocol to image the complex axonal branching structure of identified mechanosensory neurons in Drosophila, combined with a behavioral assay to evaluate the functional output of the neuron. The stimulation of identified mechanosensory neurons in live animals produces a stereotyped grooming reflex. The mechanosensory axonal arbor within the CNS is subsequently labeled with a lipophilic fluorescent dye and imaged by fluorescence microscopy. The behavioral output can therefore be correlated to the axonal morphology of the stimulated neuron in the same animal. Combining this protocol with genetic analysis provides a powerful tool for identifying the roles of molecules involved in different aspects of hard-wired neural circuit formation underlying an innate behavior. From behavioral analysis to axonal imaging, the protocol takes 4 d.

Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel gated by noxious heat, vanilloids and extracellular protons. TRPV1 is acting as an important signal integrator in sensory nociceptors under physiological and pathological conditions including inflammation and neuropathy. Because of its integrative signaling properties in response to inflammatory stimuli, TRPV1 agonists and antagonists are predicted to inhibit the sensation of ongoing or burning pain that is reported by patients suffering from chronic pain, therefore offering an unprecedented advantage in selectively inhibiting painful signaling from where it is initiated. In this article, we firstly summarize recent advances in the understanding of the role of TRPV1 in pain signaling, including a overview of clinical pharmacological trials using TRPV1 agonists and antagonists. Finally, we also present an update on the mechanistic understanding and controlling of hyperthermia caused by TRPU1 antagonists, and provide perspective for future study.

Single-cell analysis overcomes the problems of cellular heterogeneity by revealing the individual differences between cells in tissue. The current tools used to profile gene expression at the single-cell level are arduous and often require specialized equipment. We have previously developed a technique to quantify protein expression levels in single living cells. Here, we combine quantification of protein expression with absolute measurement of mRNA amounts of the same gene in the same cell, to profile the expression of genes at the transcriptional and translational levels. We show that high heterogeneity exists at both the mRNA and protein levels for multiple genes, even among monoclonal cells. We demonstrate a rapid, straightforward approach to single-cell profiling of RNA and protein production.