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

背景：液泡H+-ATPase(V-ATPase)是一种质子泵，负责调节所有细胞的胞内和胞外pH。哺乳动物的V-ATPase由16个不同的亚基组成，大多数亚基都有类似物和剪接异构体，导致了一系列令人困惑的全酶。V-ATPase的亚细胞定位主要由‘a’亚基决定；然而，不同的‘a’对映体(哺乳动物中的A1-A4)与V-ATPase全酶组成之间的空间关系尚不清楚。此外，V-ATPase作为支架组装信号复合体，不仅影响V-ATPase的功能，而且控制细胞内小泡(从早期的内体到溶酶体)的有序成熟。我们对信号体的形成和定位的理解目前还处于初级阶段。问题：我们认为，文献中报道的相互矛盾的结果在很大程度上是由于使用了终末分化的细胞系，这些细胞系在信号转导中包含潜在的/不典型的缺陷，导致了非生理系统。解决方案：为了破译V-ATPase结构/功能关系，我们需要一个没有内在突变、对V-ATPase功能敏感、易于操作、能够进行多步分化程序、易于量化、共享共同的、仅限于特定细胞的独特过程的独立细胞系统。诱导的髓系限制性前体细胞(IMRP)是一种自给自足的细胞系统，具有所有细胞共有的细胞过程，以及高度专业化的细胞所特有的过程，如骨吸收破骨细胞(OCS)。假设：我们假设，使用来自IMRP细胞的OCs研究V-ATPase将阐明反映体内过程的V-ATPase结构-功能关系。为了验证这一假设，我们建议：(1)生成具有肿瘤形成能力的IMRP细胞。我们将使用已公布的方案和已确定的生长因子鸡尾酒，建立能够向OC、巨噬细胞或中性粒细胞系分化的IMRP细胞系。(2)确定‘a’V-ATPase异构体在肿瘤发生过程中的功能和实时位置/运动。在这里，我们将回答一个基本问题，即不同的‘a’异构体是否存在于同一细胞隔室中，并将确定限制在‘a’的结构域是否为限制因素。(3)测定‘a’V-ATPase异构体及其相关信号体成分的亚基组成。我们将标记每个A1-4并列，并分离每个‘a’异构体。这将提供V-ATPase亚单位组成在穿过亚细胞结构时是否发生变化的信息，并识别相关的信号体成分。意义：这项提议将提供对V-ATPase功能和信号机制的洞察，在一个生理相关的细胞系统中，没有目前使用的培养系统常见的内在突变。

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.