The need for new model systems to elucidate protein subcellular localization and function: Making the case for osteoclast V-ATPases

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Background: Vacuolar H+ ATPases (V-ATPases) are proton pumps responsible for the regulation of intracellular and extracellular pH in all cells. Mammalian V-ATPases are composed of 16 different subunits, most of which have paralogues and splice isoforms, resulting in a bewildering array of holoenzymes. Subcellular localization of V-ATPases is primarily determined by the `a' subunit; however, the spatial relationship between different `a' paralogues (a1-a4 in mammals) and V-ATPase holoenzyme composition is not known. Furthermore, V-ATPases act as scaffolds to assemble signalling complexes which not only influence V-ATPase functions, but also control the orderly maturation of intracellular vesicles (from early endosomes to lysosomes). Our understanding of signalosome formation and localization is currently in its infancy. Problem: We believe that the contradictory results reported in the literature are largely due to the use of terminally differentiated cell lines containing underlying/uncharacterised defects in signal transduction, leading to non-physiological systems. Solution: To decipher V-ATPase structure/function relationships we require a self-contained cell system devoid of intrinsic mutations, sensitive to V-ATPase function, easily manipulatable, capable of a multistep differentiation program, easily quantifiable, and sharing common, as well as unique processes limited to specialized cells. Induced myeloid restricted precursor (iMRP) cells represent a self-contained system characterized by cellular processes common to all cells, as well as the processes unique to highly specialized cells, such as bone-resorbing osteoclasts (OCs). Hypothesis: We hypothesize that studying V-ATPases using OCs derived from iMRP cells will elucidate V-ATPase structure-function relationships that mirror in vivo processes. To test this hypothesis, we propose to: (1) Generate iMRP cells capable of OCgenesis. We will generate iMRP cell lines capable of differentiating down the OC, macrophage, or neutrophil lineage, using published protocols and established growth factor cocktails. (2) Determine the function and real-time position/movement of `a' V-ATPase isocomplexes during OCgenesis. Here we will answer the basic question of whether different `a' isocomplexes exist in the same cellular compartment, and will establish whether domains restricted to `a' are the limiting factor. (3) Determine subunit composition of `a' V-ATPase isocomplexes and their associated signalosome components. We will tag each of the a1-4 paralogues and isolate each `a' isocomplex. This will provide information whether V-ATPase subunit composition changes as it moves through subcellular structures and identify associated signalosome components. Significance: This proposal will provide insight into V-ATPase function and signalling mechanisms in a physiologically relevant cell system devoid of the intrinsic mutations common to currently used culture systems.