Using awake and freely behaving mice, this study employs a high-throughput in vivo RNA-seq approach to identify odorant receptor repertoires. The authors find sets of odorant receptors for two odorants encompassing 69 odorant receptor-odor pairs and develop models to predict receptor activation. The mammalian olfactory system uses a large family of odorant receptors (ORs) to detect and discriminate amongst a myriad of volatile odor molecules. Understanding odor coding requires comprehensive mapping between ORs and corresponding odors. We developed a means of high-throughput in vivo identification of OR repertoires responding to odorants using phosphorylated ribosome immunoprecipitation of mRNA from olfactory epithelium of odor-stimulated mice followed by RNA-Seq. This approach screened the endogenously expressed ORs against an odor in one set of experiments using awake and freely behaving mice. In combination with validations in a heterologous system, we identified sets of ORs for two odorants, acetophenone and 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), encompassing 69 OR-odorant pairs. We also identified shared amino acid residues specific to the acetophenone or TMT receptors and developed models to predict receptor activation by acetophenone. Our results provide a method for understanding the combinatorial coding of odors in vivo.

The mouse olfactory sensory neuron (OSN) repertoire is composed of 10 million cells and each expresses one olfactory receptor (OR) gene from a pool of over 1000. Thus, the nose is sub-stratified into more than a thousand OSN subtypes. Here, we employ and validate an RNA-sequencing-based method to quantify the abundance of all OSN subtypes in parallel, and investigate the genetic and environmental factors that contribute to neuronal diversity. We find that the OSN subtype distribution is stereotyped in genetically identical mice, but varies extensively between different strains. Further, we identify cis-acting genetic variation as the greatest component influencing OSN composition and demonstrate independence from OR function. However, we show that olfactory stimulation with particular odorants results in modulation of dozens of OSN subtypes in a subtle but reproducible, specific and time-dependent manner. Together, these mechanisms generate a highly individualized olfactory sensory system by promoting neuronal diversity.

Endocytic sorting is achieved through the formation of morphologically and functionally distinct sub-domains within early endosomes. Cargoes destined for recycling are sorted to and transported through newly-formed tubular membranes, but the processes that regulate membrane tubulation are poorly understood. Here, we identified a novel Caenorhabditis elegans Cdc50 family protein, CHAT-1, which acts as the chaperone of the TAT-1 P4-ATPase to regulate membrane phosphatidylserine (PS) asymmetry and endocytic transport. In chat-1 and tat-1 mutants, the endocytic sorting process is disrupted, leading to defects in both cargo recycling and degradation. TAT-1 and CHAT-1 colocalize to the tubular domain of the early endosome, the tubular endocytic recycling compartment (ERC), and the recycling endosome where PS is enriched on the cytosolic surface. Loss of tat-1 and chat-1 function disrupts membrane PS asymmetry and abrogates the tubular membrane structure. Our data suggest that CHAT-1 and TAT-1 maintain membrane phosphatidylserine asymmetry, thus promoting membrane tubulation and regulating endocytic sorting and recycling. The process by which cells take up nutrients and other large molecules from the extracellular environment is known as endocytosis. At the cell surface, external molecules become enclosed in membrane spheres called endosomes. Early endosomes serve as a sorting station, directing the contents (cargo molecules) to the correct compartment within the cell. This is thought to be achieved by the formation of membrane structures with distinct shape and function. For example, cargoes destined for recycling and degradation are processed through tubular membrane structures and big vesicular compartments, respectively. However, it is poorly understood how early endosome membranes are shaped into different structures. Here we show that two proteins, CHAT-1 and TAT-1, regulate membrane structure and are important for normal endocytic transport in the nematode worm C. elegans. TAT-1 and CHAT-1 are found in tubular membrane structures along the sorting and recycling pathway, where they enrich the outer membrane layer with a lipid called phosphatidylserine (PS) and probably change the membrane curvature. Loss of tat-1 and chat-1 function disrupts the asymmetric distribution of PS, abolishes tubular membrane structures, and abrogates endocytic sorting/recycling. Our data support a role of TAT-1/CHAT-1–regulated membrane PS asymmetry in promoting membrane tubulation for endocytic cargo sorting and recycling.