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. 2014 Jul;13(7):1828-43.
doi: 10.1074/mcp.M113.035378. Epub 2014 Apr 18.

The membrane proteome of sensory cilia to the depth of olfactory receptors

Katja Kuhlmann  1 , Astrid Tschapek  1 , Heike Wiese  2 , Martin Eisenacher  1 , Helmut E Meyer  3 , Hanns H Hatt  4 , Silke Oeljeklaus  2 , Bettina Warscheid  5
Affiliations

The membrane proteome of sensory cilia to the depth of olfactory receptors

Katja Kuhlmann et al. Mol Cell Proteomics. 2014 Jul.

Abstract

In the nasal cavity, the nonmotile cilium of olfactory sensory neurons (OSNs) constitutes the chemosensory interface between the ambient environment and the brain. The unique sensory organelle facilitates odor detection for which it includes all necessary components of initial and downstream olfactory signal transduction. In addition to its function in olfaction, a more universal role in modulating different signaling pathways is implicated, for example, in neurogenesis, apoptosis, and neural regeneration. To further extend our knowledge about this multifunctional signaling organelle, it is of high importance to establish a most detailed proteome map of the ciliary membrane compartment down to the level of transmembrane receptors. We detached cilia from mouse olfactory epithelia via Ca(2+)/K(+) shock followed by the enrichment of ciliary membrane proteins at alkaline pH, and we identified a total of 4,403 proteins by gel-based and gel-free methods in conjunction with high resolution LC/MS. This study is the first to report the detection of 62 native olfactory receptor proteins and to provide evidence for their heterogeneous expression at the protein level. Quantitative data evaluation revealed four ciliary membrane-associated candidate proteins (the annexins ANXA1, ANXA2, ANXA5, and S100A5) with a suggested function in the regulation of olfactory signal transduction, and their presence in ciliary structures was confirmed by immunohistochemistry. Moreover, we corroborated the ciliary localization of the potassium-dependent Na(+)/Ca(2+) exchanger (NCKX) 4 and the plasma membrane Ca(2+)-ATPase 1 (PMCA1) involved in olfactory signal termination, and we detected for the first time NCKX2 in olfactory cilia. Through comparison with transcriptome data specific for mature, ciliated OSNs, we finally delineated the membrane ciliome of OSNs. The membrane proteome of olfactory cilia established here is the most complete today, thus allowing us to pave new avenues for the study of diverse molecular functions and signaling pathways in and out of olfactory cilia and thus to advance our understanding of the biology of sensory organelles in general.

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Figures

Fig. 1.
Fig. 1.
Experimental strategy. A, cilia were detached from OE of mice by Ca2+/K+ shock and further subjected to Na2CO3 washes at alkaline pH. Aliquots of ciliary membrane-enriched fractions were separated by SDS-PAGE or SCX chromatography followed by high resolution LC/MS and bioinformatics data analysis. In total, 162 samples from three independent replicates were analyzed. B, immunostaining of cryosections of OE from transgenic OMP-GFP mice. Mature olfactory sensory neurons as well as dendritic knobs at the ciliary layer are labeled green by GFP. Olfactory cilia were stained specifically using an antibody against adenylyl cyclase III. Confocal micrographs show OE sections before (left) and after (right) Ca2+/K+ shock. Scale bars indicate 20 μm. For further details, refer to main text.
Fig. 2.
Fig. 2.
Venn diagrams showing the intersections of protein identifications from mouse olfactory cilia membranes. A, overlap of protein identifications (i.e. MaxQuant protein groups) obtained from three independent replicates by gel-based (left) and gel-free (right) proteomics analysis. B, comparison of protein identifications obtained by gel-based and gel-free analyses.
Fig. 3.
Fig. 3.
Distribution of proteins identified in olfactory cilia membrane fractions. A, subcellular localization of membrane proteins (blue) as well as soluble proteins (gray) identified by gel-based and gel-free LC/MS analyses. Information about the subcellular localization of identified proteins was derived from UniProt and the Gene Ontology database. Unchar., uncharacterized. B, distribution of membrane proteins according to the number of transmembrane domains predicted by Phobius (31) and according to the detection approach.
Fig. 4.
Fig. 4.
Identification and abundance of olfactory receptors. A, overlap of soluble proteins (top), integral membrane proteins (middle), and olfactory receptors (bottom) identified in olfactory cilia membrane fractions following the gel-based and gel-free approach. B, IBAQ values of all ORs identified in individual replicates of gel-based and gel-free LC/MS analyses. ORs are listed according to the mean of their iBAQ values derived from gel-free experiments. C, relative abundance of ORs. ORs (open diamonds) and all other proteins identified (filled squares) exhibiting a molecular mass of ≤50 kDa are plotted versus their mean iBAQ value determined across all gel-free replicates.
Fig. 5.
Fig. 5.
Relative abundance of proteins with known and putative function in olfactory signaling. Olfactory signaling proteins (A), anoctamins (B), chloride and calcium transporters (C and D), annexins (E), and S100A proteins (F) detected in olfactory cilia membrane fractions are plotted against their mean iBAQ value determined across all replicates. ▿, new components of olfactory cilia validated in this work. Mean iBAQ values of proteins in square brackets were in the range of 2.82E + 03 to 9.03E + 04.
Fig. 6.
Fig. 6.
Localization of the candidate proteins ANXA1, ANXA2, ANXA5, and S100A5 to the olfactory cilia membrane. ANXA1, ANXA2, ANXA5, and S100A5 as well as the known ciliary proteins NCKX4 and PMCA1 (all depicted in red) show co-localization with the olfactory cilia marker protein AC III (blue) in cryosections of olfactory epithelia from transgenic OMP-GFP mice. In the overlay, mature olfactory sensory neurons and dendritic knobs are labeled green. Scale bars indicate 10 μm.
Fig. 7.
Fig. 7.
Extended olfactory membrane ciliome. A, depicted are all 178 proteins defined as olfactory cilium-specific membrane and membrane-associated proteins as well as 35 further membrane or regulatory proteins with ciliary localization or associated with ciliary functions identified in this work but not defined mature OSN-specific by transcriptome analysis (24). For more details about the proteins, please refer to the text and supplemental Table S1A. Bold, cilium-specific membrane proteins; bold and italic, cilium-specific soluble proteins; regular type, non-cilium-specific proteins. B, components of the olfactory membrane ciliome (dark gray circles) and all other proteins identified in olfactory cilia membrane-enriched fractions (light gray circles) are plotted versus their mean iBAQ value. Relative abundance levels of known proteins of the canonical pathway of olfactory signal transduction (blue circles) including 62 ORs (white circles) and components of the Ca2+ signaling network (red circles) are highlighted.
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