Project Details
Projekt
Molecular composition and structure of the SLC26A5 microenvironment in sensory outer hair cells
Applicant
Professor Dr. Dominik Oliver
Subject Area
Anatomy and Physiology
Term
since 2020
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 426950122
SLC26 anion transporters function in cellular context, e.g. by colocalizing and physically interacting with functionally linked ion transporters and by precisely targeting to the appropriate membrane domain. There is evidence that localization and functional interactions are remodeled dynamically to adapt SLC26 activity to changing physiological needs. These notions imply that the SLC26 transporters are scaffolded into supramolecular protein assemblies. Little is known, however, about the nature and composition of these assemblies. Here we propose to address the microenvironment of SLC26A5 (prestin), which, although physiologically unique among SLC26 transporters, functions through conserved molecular mechanisms. SLC26A5 is selectively expressed in auditory outer hair cells and drives electro-mechanical cellular length changes, that are essential for cochlear amplification and sensory acuity. SLC26A5 is associated with a submembrane supramolecular scaffold, which is presumed to be pivotal for cell mechanics. Its molecular components and supramolecular architecture are unknown. Besides the immediate relevance for sensory physiology and pathophysiology, this cellular structure may serve as a model system for the supramolecular organization of epithelial SLC26 transporters. To identify the protein components of the submembrane scaffold, we will use proteomic strategies that take advantage of the spatial proximity of the submembrane structure to membrane-localized SLC26A5. We will use cross-linking mass spectrometry to selectively capture proteins immediately adjacent to SLC26A5, and proximity-dependent biotinylation to label proteins that populate the subcellular environment. Once molecular components have been identified, we will determine their supramolecular organization by super-resolution fluorescence microscopy and by cryo electron tomography. To study the function of identified scaffold components and their interaction with SLC26A5, we will reconstitute the presumptive protein assemblies by heterologous expression in cell culture. Their function for outer hair cell structure and physiology will be addressed by murine knock-out models, by pharmacological manipulation, and in organotypic culture models. In summary, the project will reveal the first integrative picture of a SLC26 protein in its nanoscale cellular environment.
DFG Programme
Research Units
