细胞间粘附(CTCA),这是建立镜头透明度的关键,是水通道蛋白0(AQP0)的关键功能。这项研究的目的是找出AQP0在成纤维细胞之间发挥CTCA的可能机制,由于目前有两个建议,AQP0-AQP0相互作用或AQP0-脂质相互作用。我们研究了完整AQP0和C端裂解的AQP0(CTC-AQP0)中AQP0诱导的CTCA的机制。测定显示用完整AQP0或CTC-AQP0转染的L-细胞与亲本L-细胞之间的CTCA,表明AQP0-膜相互作用。AQP0的两种形式均显着(P<0.001)促进了与带负电荷的1-α-磷脂酰丝氨酸脂质囊泡的粘附,这表明AQP0-脂质相互作用。表达AQP0的L细胞还显着促进WT和AQP0-KO小鼠晶状体纤维细胞膜囊泡(FCMV)的粘附(P<0.001)。然而,当WT或AQP0-KO的FCMV铺在亲本L细胞上时,只有WT囊泡明显粘附,证实AQP0-膜相互作用。与细胞外结构域特异性AQP0抗体孵育后,表达完整AQP0或CTC-AQP0的L细胞在AQP0-KOFCMV的粘附中显示出显着降低(P<0.001),表明CTCA中的胞外环参与。来自外皮层和内皮层的WTFCMV促进了对亲本L细胞的粘附,粘连效率无统计学差异(P>0.05)。WT的超微结构研究,AQP0-KO和转基因晶状体显示AQP0对纤维CTCA和压实至关重要。收集的数据清楚地表明,AQP0胞外环结构域中的带正电荷的氨基酸与质膜中的带负电荷的脂质相互作用以促进CTCA用于成纤维细胞的压实。
Cell-to-cell adhesion (CTCA), which is key for establishing lens transparency, is a critical function of Aquaporin 0 (AQP0). The aim of this investigation was to find out the possible mechanism by which AQP0 exerts CTCA between fiber cells, since there are two proposals currently, either an
AQP0-
AQP0 interaction or an
AQP0-lipid interaction. We studied the mechanism of
AQP0-induced CTCA in intact
AQP0 and C-terminally cleaved
AQP0 (CTC-
AQP0). Assays showed CTCA between L-cells transfected with intact AQP0 or CTC-AQP0 and parental L-cells indicating
AQP0-membrane interaction. Both forms of AQP0 significantly (P < 0.001) promoted adhesion to negatively charged l-α-phosphatidylserine lipid vesicles signifying AQP0-lipid interaction.
AQP0-expressing L-cells also promoted adhesion of WT and
AQP0-KO mouse lens fiber cell membrane vesicles (FCMVs) significantly (P < 0.001). However, when FCMVs of WT or AQP0-KO were plated over parental L-cells, only WT vesicles adhered significantly, corroborating
AQP0-membrane interaction. After incubating with extracellular domain-specific
AQP0 antibody, L-cells expressing intact
AQP0 or CTC-AQP0 showed a significant reduction (P < 0.001) in the adhesion of AQP0-KO FCMVs indicating extracellular loop involvement in CTCA. WT FCMVs from outer cortex and inner cortex promoted adhesion to parental L-cells, without any statistically significant difference in adhesion efficiency (P > 0.05). Ultrastructure studies of WT, AQP0-KO and transgenic lenses showed AQP0 is critical for fiber CTCA and compaction. The data collected clearly demonstrate that the positively charged amino acids in the AQP0 extracellular loop domains interact with the negatively charged lipids in the plasma membrane to promote CTCA for compaction of fiber cells.