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

需要新的模型系统来阐明蛋白质亚细胞定位和功能：为破骨细胞 V-ATP 酶提供依据

• 批准号: RGPIN-2022-05169
• 负责人: Manolson, Morris
• 金额: $ 2.33万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=750711
• 关键词: need; new; model; systems; elucidate; need; new; model; systems; elucidate

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.

背景：液泡H+ ATPases（V-ATPases）是负责调节所有细胞内细胞内和细胞外pH的质子泵。哺乳动物V-ATPase由16个不同的亚基组成，其中大多数具有旁产词和剪接同工型，导致了令人困惑的全酶。 V-ATPases的亚细胞定位主要由“ A”亚基确定。然而，尚不清楚不同的“ A”旁生（哺乳动物中的A1-A4）和V-ATPase Holoenzyme组成之间的空间关系。此外，V-ATP酶充当组装信号复合物的支架，不仅影响V-ATPase功能，还可以控制细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本地化的理解目前仍处于起步阶段。 问题：我们认为，文献中报告的矛盾结果在很大程度上是由于使用终端分化的细胞系在信号转导中包含潜在/未表征的缺陷，从而导致非生理系统。 解决方案：为了解密V-ATPase结构/功能关系，我们需要一个独立的细胞系统，这些细胞系统没有固有的突变，对V-ATPase函数敏感，易于操纵，能够具有多步分化程序，易于量化，共享共享，并且共享常见，以及限于专用细胞的独特过程。诱导的髓样限制前体（IMRP）细胞代表一个以所有细胞共有的细胞过程以及高度专业的细胞（例如骨响应骨骼反应破骨细胞（OCS））为特征的独立系统。 假设：我们假设使用源自IMRP细胞的OC研究V-ATP酶将阐明反映在体内过程中的V-ATPase结构 - 函数函数函数关系。为了检验这一假设，我们建议：（1）生成能够抗原发生的IMRP细胞。我们将使用已发表的方案和已建立的生长因子鸡尾酒来生成能够区分OC，巨噬细胞或中性粒细胞谱系的IMRP细胞系。 （2）确定oc基因期间“ A” V-ATPase同源物的功能和实时位置/运动。在这里，我们将回答一个基本问题，即在同一蜂窝室内是否存在不同的“ A”同源物，并确定限制为“ A”的域是否是限制因素。 （3）确定“ A” V-ATPase同源物及其相关信号组成分的亚基组成。我们将标记每个A1-4旁系同源物，并隔离每个``A'iSocomplex。这将提供信息，即V-ATPase亚基组成在通过亚细胞结构移动并识别相关的信号体成分时是否会发生变化。意义：该提案将提供有关V-ATPase功能和信号传导机制的洞察力，这些生理相关的细胞系统中没有目前使用的培养系统常见的固有突变。