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

背景：胞内质子H+ ATP酶（V-ATP酶）是一种质子泵，在所有细胞中负责调节细胞内外pH。哺乳动物V-ATP酶由16个不同的亚基组成，其中大多数具有旁系同源物和剪接异构体，从而产生令人困惑的全酶阵列。V-ATP酶的亚细胞定位主要由“a”亚基决定;然而，不同的“a”旁系同源物（哺乳动物中的a1-a4）与V-ATP酶全酶组成之间的空间关系尚不清楚。此外，V-ATP酶作为支架组装信号复合物，其不仅影响V-ATP酶功能，而且控制胞内囊泡的有序成熟（从早期内体到溶酶体）。我们对信号体形成和定位的理解目前还处于起步阶段。 问题：我们认为，文献中报道的矛盾结果主要是由于使用了含有潜在/未表征的信号转导缺陷的终末分化细胞系，导致非生理系统。 解决方法：为了破译V-ATP酶的结构/功能关系，我们需要一个独立的细胞系统，没有内在的突变，敏感的V-ATP酶功能，易于操作，能够进行多步分化程序，易于量化，并共享共同的，以及独特的过程限于专门的细胞。诱导的骨髓限制性前体（iMRP）细胞代表了一个独立的系统，其特征在于所有细胞共有的细胞过程，以及高度特化细胞（如骨吸收破骨细胞（OC））特有的过程。 假设：我们假设，使用来自iMRP细胞的OC研究V-ATP酶将阐明反映体内过程的V-ATP酶结构-功能关系。为了验证这一假设，我们提出：（1）产生能够OC发生的iMRP细胞。我们将使用已发表的方案和已建立的生长因子混合物产生能够向下分化OC、巨噬细胞或中性粒细胞谱系的iMRP细胞系。(2)确定OC发生过程中“a”V-ATP酶同工复合物的功能和实时位置/运动。在这里，我们将回答是否不同的'a' isocomplex存在于同一个细胞室的基本问题，并将建立是否域限制到'a'是限制因素。(3)确定'a' V-ATP酶同工复合物及其相关信号体组分的亚基组成。我们将标记每个a1-4旁系同源物并分离每个“a”同功复合物。这将提供V-ATP酶亚基组成在其通过亚细胞结构时是否发生变化的信息，并确定相关的信号体组分。重要性：该提案将提供洞察V-ATP酶功能和信号传导机制的生理相关的细胞系统缺乏的内在突变常见的目前使用的文化系统。

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.