Department of Biochemistry and Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee, United States.
Invest Ophthalmol Vis Sci. 2023 Aug 1;64(11):28. doi: 10.1167/iovs.64.11.28.
Lens transparency relies on the precise organization of lens fiber cells. The formation of the highly ordered lens architecture results from not only cell-cell adhesion along the lateral interfaces, but also from proper organization of fiber cells tips at lens sutures. Little is known about the cell adhesion between fiber tips at the sutures. The purpose of this study is to map suture-specific protein distributions.
Tissue sections were obtained from fresh frozen bovine lenses and washes were performed to remove soluble proteins and to retain membrane and membrane associated proteins. Imaging mass spectrometry (IMS) combined with on-tissue trypsin digestion was used to visualize protein spatial distributions. Sutures and adjacent regions were captured by laser capture microdissection and samples were digested by trypsin. Proteins were analyzed by liquid chromatography tandem MS and quantified by label-free quantification. Protein spatial distributions were confirmed by immunofluorescence.
IMS results showed enrichment of adherens junction proteins cadherin-2 and armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF) in both anterior and posterior sutures of bovine lenses. Liquid chromatography tandem MS confirmed higher expression of cadherin-2 and ARVCF and other adherens junction proteins including catenin α2 (CTNNA2) and catenin β1 (CTNNB1) in sutures. In contrast, IMS indicated low expression of gap junction protein connexin 50 and connexin 46 in the suture regions. The localization of cadherin-2 and connexin 50 was confirmed by immunofluorescence.
The complementary expression of adherens junction proteins and gap junction proteins in lens suture regions implicates adherens junctions in fiber cell tip adhesion and in maintaining the integrity of the lens.
晶状体的透明度依赖于晶状体纤维细胞的精确排列。高度有序的晶状体结构的形成不仅依赖于沿侧向界面的细胞-细胞黏附，还依赖于晶状体缝线处纤维细胞尖端的适当组织。关于缝线处纤维尖端之间的细胞黏附知之甚少。本研究旨在绘制特定于缝线的蛋白质分布。
从新鲜冷冻牛晶状体中获取组织切片，并进行洗涤以去除可溶性蛋白质，并保留膜和膜相关蛋白质。将成像质谱 (IMS) 与组织内胰蛋白酶消化相结合，用于可视化蛋白质空间分布。通过激光捕获显微切割捕获缝线和相邻区域，并用胰蛋白酶消化样品。通过液相色谱串联质谱分析蛋白质，并通过无标记定量进行定量。通过免疫荧光确认蛋白质空间分布。
IMS 结果显示，黏附连接蛋白钙黏蛋白-2 和唐氏综合征相关嵴蛋白缺失基因（ARVCF）在前、后缝线中均有富集。液相色谱串联质谱证实，钙黏蛋白-2 和 ARVCF 以及其他黏附连接蛋白（包括连环蛋白α2 (CTNNA2) 和连环蛋白β1 (CTNNB1)）在缝线中的表达更高。相比之下，IMS 表明，缝隙连接蛋白连接蛋白 50 和连接蛋白 46 在缝线区域的表达较低。钙黏蛋白-2 和连接蛋白 50 的定位通过免疫荧光证实。
晶状体缝线区域黏附连接蛋白和缝隙连接蛋白的互补表达表明黏附连接在纤维细胞尖端黏附和维持晶状体完整性中发挥作用。
