The Membrane Proteome of Sensory Cilia to the Depth of Olfactory Receptors

Kim

Proc. Natl. Acad. Sci. U.S.A.

et al. 2016

In mammals, initial detection of olfactory stimuli is mediated by sensory neurons in the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). The heterotrimeric GTP-binding protein G o is widely expressed in the MOE and VNO of mice. Early studies indicated that G o expression in VNO sensory neurons is critical for directing social and sexual behaviors in female mice [Oboti L, et al. (2014) BMC Biol 12:31]. However, the physiological functions of G o in the MOE have remained poorly defined. Here, we examined the role of G o in the MOE using mice lacking the α subunit of G o . Development of the olfactory bulb (OB) was perturbed in mutant mice as a result of reduced neurogenesis and increased cell death. The balance between cell types of OB interneurons was altered in mutant mice, with an increase in the number of tyrosine hydroxylase-positive interneurons at the expense of calbindin-positive interneurons. Sexual behavior toward female mice and preference for female urine odors by olfactory sensory neurons in the MOE were abolished in mutant male mice. Our data suggest that G o signaling is essential for the structural and functional integrity of the MOE and for specification of OB interneurons, which in turn are required for the transmission of pheromone signals and the initiation of mating behavior with the opposite sex.heterotrimeric G o protein | olfactory mucosa | olfactory bulb interneuron | tyrosine monooxygenase | sexual behavior P heromones evoke diverse social and sexual behaviors in animals of the same species. In mammals, initial detection of olfactory stimuli is mediated by sensory neurons in the main olfactory epithelium (MOE) and vomeronasal organ (VNO). Early studies indicated that the VNO is largely responsible for the detection of pheromones and the consequent direction of social and sexual behaviors (1). VNO sensory neurons (VSNs) express distinct types of vomeronasal receptors: type 1 (V1Rs) in the apical region and type 2 (V2Rs) in the basal region. These receptors couple, respectively, to G i and G o members of the heterotrimeric GTPbinding protein family. It has been suggested that, following ligand binding to V1Rs and V2Rs, the Gβγ subunits released from G i and G o activate phospholipase C and increase the intracellular concentration of Ca 2+ . Ca 2+ ions induce opening of small Ca 2+ -activated K + (SK3) channels, subsequently activating Ca 2+ -activated Cl − channels (CACCs). In the VNO, Gβγ can also directly induce the opening of G protein-activated inward rectifying K + (GIRK) channels independently of transient receptor potential cation channel C2 (TRPC2) (2). Because of the VNO's high luminal K + concentration (2), opening of SK3 and GIRK channels leads to depolarization of VSNs and transmission of pheromone signals. Targeted deletion of the TRPC2 gene abrogates gender identification and intermale aggression without affecting male-to-female mating behavior in male mice (3, 4). Targeted deletion of the GIRK1 gene partially attenuates mating behavior of the male toward fem...

Section: Discussionmentioning

confidence: 99%

Development of the main olfactory system and main olfactory epithelium-dependent male mating behavior are altered in G _o -deficient mice

Kim

Proc. Natl. Acad. Sci. U.S.A.

et al. 2016

“…After a successful verification of the enrichment of olfactory cilia membranes by immunoblot assays, a gelbased and a gel-free (including strong cation exchange (SCX)-chromatography) LC-MS/MS was performed. The usage of two different approaches (gel-free and gel-based) was carried out to ensure a comprehensive inventory of olfactory ciliary membranes to the depth of low abundant ORs (Kuhlmann et al, 2014). In total, 4403 unique protein groups (false discovery rate <0.01) were identified, from which 3750 protein groups (85.2%) were detected after the gel-based and 4257 protein groups (96.7%) after the gel-free approach.…”

Section: Relative and Absolute Quantification Strategiesmentioning

confidence: 99%

“…The signal transduction pathway is well established, but the molecular processes in OSN cilia are not fully understood. The study of Kuhlmann et al focused on the analysis of the mouse olfactory membrane ciliome to discover new resident proteins and low-abundant central components (mainly focused on ORs) in the OSNs (Kuhlmann et al, 2014). The used workflow includes a selective enrichment of olfactory ciliary membrane proteins, with an established Ca 2+ /K + -shock method from transgenic mice expressing the gene coding for the green fluorescent protein under control of the olfactory marker protein (OMP)-promoter (Mayer et al, 2009).…”

Section: Relative and Absolute Quantification Strategiesmentioning

confidence: 99%

“…Recently, labelfree quantitative proteomics is increasingly used for global interaction studies. One example for protein-protein interaction mapping is the label-free based quantitative analysis of the membrane proteome of sensory cilia to the depth of olfactory receptors by (Kuhlmann et al, 2014).…”

Section: Label-free Quantificationmentioning

confidence: 99%

“…SCX, strong cation exchange; LC-MS/MS, liquid chromatography-tandem mass spectrometry. Revised from Kuhlmann et al (2014). 1982). After a successful verification of the enrichment of olfactory cilia membranes by immunoblot assays, a gelbased and a gel-free (including strong cation exchange (SCX)-chromatography) LC-MS/MS was performed.…”

Section: Relative and Absolute Quantification Strategiesmentioning

confidence: 99%

See 2 more Smart Citations

Strategies in relative and absolute quantitative mass spectrometry based proteomics

Lindemann

Thomanek

Hundt

et al. 2017

Biological Chemistry

Self Cite

Quantitative mass spectrometry approaches are used for absolute and relative quantification in global proteome studies. To date, relative and absolute quantification techniques are available that differ in quantification accuracy, proteome coverage, complexity and robustness. This review focuses on most common relative or absolute quantification strategies exemplified by three experimental studies. A label-free relative quantification approach was performed for the investigation of the membrane proteome of sensory cilia to the depth of olfactory receptors in Mus musculus. A SILAC-based relative quantification approach was successfully applied for the identification of core components and transient interactors of the peroxisomal importomer in Saccharomyces cerevisiae. Furthermore, AQUA using stable isotopes was exemplified to unraveling the prenylome influenced by novel prenyltransferase inhibitors. Characteristic enrichment and fragmentation strategies for a robust quantification of the prenylome are also summarized.

Possible ATP trafficking by ATP‐shuttles in the olfactory cilia and glucose transfer across the olfactory mucosa

et al. 2019

Odor transduction in the cilia of olfactory sensory neurons involves several ATP‐requiring enzymes. ATP is generated by glycolysis in the ciliary lumen, using glucose incorporated from surrounding mucus, and by oxidative phosphorylation in the dendrite. During prolonged stimulation, the cilia maintain ATP levels along their length, by unknown means. We used immunochemistry, RT‐PCR, and immunoblotting to explore possible underlying mechanisms. We found the ATP‐shuttles, adenylate and creatine kinases, capable of equilibrating ATP. We also investigated how glucose delivered by blood vessels in the olfactory mucosa reaches the mucus. We detected, in sustentacular and Bowman's gland cells, the crucial enzyme in glucose secretion glucose‐6‐phosphatase, implicating both cell types as putative glucose pathways. We propose a model accounting for both processes.