Organization of the lens core syncytium

描述(申请人提供)：纤维细胞之间的细胞-细胞通讯是晶状体细胞结构最显著的方面之一，对该组织的正常生理至关重要，该组织缺乏血管系统。这种交流的改变会导致人类和小鼠的白内障形成。在我们的初步实验中，我们描述了一种新的途径，它可以渗透到大分子，并在发育过程中形成在晶状体的核心。这一途径不同于缝隙连接和相互连接晶状体核心中的纤维细胞以形成真正的合胞体。 本研究的目的是描述包括人类在内的各种脊椎动物核心合胞体的发育特性，阐明合胞体形成的生理意义和分子机制。我们将解决四个问题：1)核心合胞体是所有脊椎动物晶状体的特征吗？我们将通过描述鸡、鼠和灵长类晶状体中的合胞体来寻找答案，测试胚胎和成体晶状体中合胞体形成与细胞器丢失和去核的时空相关性。我们将进行形态计量学和功能分析，以确定小鼠合胞体的发育起始时间。2)晶状体核心合体的功能是什么？为了确定这一功能，我们将测试天然晶状体蛋白是否能够从生物合成活跃的外周纤维细胞转移到合体内核心区域的静止细胞。我们将监测脉冲标记蛋白质的易位，并确定哪些蛋白质物种使用这种通讯途径。3)融合孔的形成是否代表了晶状体核心合胞体内蛋白质通透性细胞偶联的机制？我们将在小鼠和鸡胚胎中测试纤维之间的融合形成是否总是先于合体形成。我们将使用超微结构分析来确定融合孔的密度和正常大小的变化，连接合胞体内的纤维细胞以绘制晶状体上的孔分布。4)纤维细胞融合的分子机制是什么？我们将测试丰富的膜蛋白ADAM12、CX46、CX50和AQP0在晶状体细胞融合和合胞体形成中的作用。我们将检查连接蛋白基因敲除小鼠和转基因小鼠，表达显性阴性或融合形式的ADAM12，检查晶状体核心中是否存在合胞体和细胞融合。我们还将测试ADAM12、Cx46、CX50和AQP0是否相互共定位，以及是否共定位于融合孔、耦合晶状体纤维细胞。 这项研究项目将有助于揭示晶状体发育、老化和白内障形成的新方面。

DESCRIPTION (provided by applicant): Cell-cell communication between fiber cells is one of the most striking aspects of lens cell organization and is critical for the normal physiology of this tissue, which is devoid of vasculature. Alterations in such communication result in cataract formation both in human and mouse. In our preliminary experiments we characterized a new pathway, which is permeable to macromolecules and form in the core of the lens during development. This pathway is distinct from gap junctions and interconnects fiber cells in the lens core to form a true syncytium. The purpose of this Proposal is to characterize the developmental properties of the core syncytium, elucidate physiological significance and molecular mechanism of the syncytium formation in various vertebrate species, including humans. We will address four questions: 1) Is core syncytia a feature of all vertebrate lenses? We will approach the answer by characterization of syncytia in chicken, mouse and primate lenses, testing spatio-temporal correlation of syncytium formation with organelle loss and denucleation both in embryonic and adult lenses. We will perform morphometric and functional analysis to determine developmental onset time of the syncytium in the mouse. 2) What is the function of the lens core syncytium? To define the function we will test whether native lens proteins are able to translocate from biosynthetically active peripheral fiber cells into quiescent cells of the core region within the syncytium. We will monitor translocation of pulse-labeled proteins and determine which protein species use this communication pathway. 3) Does fusion pore formation represent a mechanism for protein-permeable cell coupling within lens core syncytium? We will test whether fusion formation between fibers always precedes syncytial formation in mouse and chicken embryos. We will use ultrastructural analysis to determine density and normal size variation of fusion pores, connecting fiber cells within syncytium to map pore distribution across the lens. 4) What is the molecular mechanism, underlying fiber cell fusion? We will test abundant membrane proteins ADAM12, Cx46, Cx50 and AQP0 for the role in cell fusion and syncytium formation in the lens. We will examine connexin knockout mice and transgenic mice, expressing dominant negative or fusogenic form of ADAM12, for the presence of syncytium and cell fusions in the lens core. We will also test whether ADAM12, Cx46, Cx50 and AQP0 co-localize to each other and to the fusion pores, coupling lens fiber cells. This research project will help reveal new aspects of lens development, aging and cataract formation.