olfaction; signal tranduction; scaffolding protein; Anoctamin; STED
570 Biowissenschaften; Biologie
Over 1.000 olfactory receptors and more than 100 vomeronasal receptors translate a tremendous amount of information into electrical signals in sensory neurons. These receptors are part of complex signaling cascades localized in specialized cell compartments. The elements of these cascades are quite well understood, but their spatial arrangement inside the signaling compartments remains elusive.
The existence of signaling microdomains in the olfactory systems, so called olfactosomes, has been already proposed in the early 2000‘s. It has been recently demonstrated that MUPP1 recruits members of the signaling cascade from olfactory sensory neurons into a functional complex. In this thesis, we investigated organization of signaling proteins and provide first optical evidence for the formation of signaling microdomains in cilia of olfactory sensory neurons and microvilli of vomeronasal sensory neurons.
The investigations comprised three aspects. (1) We identified potential PDZ domain containing sca_olding proteins which could organize the signal transduction cascade in the vomeronasal organ, and identified NHERF1 and DLG1 as potential organizers of vomeronasal signal transduction. NHERF1 and DLG1 were found to be expressed in the vomeronasal organ, localized in microvilli of vomeronasal sensory neurons, and demonstrated to interact with vomeronasal receptors in an in vitro interaction assay. (2) We analyzed the spatial organization of signaling proteins in the main olfactory epithelium and the vomeronasal organ using super-resolution microscopy. We demonstrated that signaling proteins in both neuron types are organized in spatially segregated microdomains. This observation includes Anoctamins, NHERF1 and the G protein G_i2. (3) We investigated direct interaction between Anoctamin proteins to elucidate their function as calcium-gated chloride channels in olfactory signal transduction.
Anoctamin 2 was recently identified as the major component of calciumgated chloride conductance in olfactory sensory neurons. Vomeronasal sensory neurons also rely on calcium-gated chloride channels for signal transduction, their identity, however, remains elusive. We identified Anoctamin 1 and Anoctamin 2 expression microvilli of vomeronasal sensory neurons, and demonstrate co-localization with the signaling protein G_i2. We also identified Anoctamin 6 in cilia of olfactory sensory neurons. Anoctamin 6 is also organized in segregated microdomains, co-localizing with Anoctamin 2. Furthermore, we demonstrated direct interaction of Anoctamin 2 with Anoctamin 6 and Anoctamin 1, and this interaction has direct e_ects on the chloride currents mediated by recombinantly expressed Anoctamin proteins. Co-expression of di_erent Anoctamins could therefore have physiological relevance for olfactory signaling.
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