Zhou Cheng-Jing, Lo Woo-Kuen
Department of Anatomy and Neurobiology, Morehouse School of Medicine, Atlanta, GA 30310, USA.
Exp Eye Res. 2003 Oct;77(4):423-32. doi: 10.1016/s0014-4835(03)00171-4.
Interlocking membrane domains are specialized membrane interdigitations in the form of ball-and-sockets and protrusions between lens fibre cells of all species. They are believed to play a key role in maintaining fibre-fibre stability and are therefore, important for normal lens function. Here we report the specific association of the clathrin/AP-2 adaptor complex and the branching F-actin network with the development of interlocking domains in rats and several other species. By thin-section electron microscopy we consistently observed a layer of distinct coating (approximately 25-nm thick) on the concave membrane surface of small and intermediate-sized developing interlocking domains. These membrane coats remarkably resembled the clathrin-coat of endocytic vesicles in which clathrin and the AP-2 adaptor are involved in the induction of coated pit formation during receptor-mediated endocytosis. We hypothesize that the clathrin/AP-2 complex is directly involved in the induction of interlocking domains in fibre cells. By immunoconfocal microscopy, co-labelling of a dotted-pattern of clathrin and AP-2 adaptor antibodies was seen along the cortical fibre cells. Immunoblot analysis further confirmed that clathrin and AP-2 adaptor antibodies specifically stained a polypeptide band of 180 and 106kD, respectively, in the membrane fractions prepared separately from the outer and inner cortical fibres where interlocking domains are abundant but endocytic vesicles are absent. Immunoelectron microscopy showed that the clathrin antibody was localized along the interlocking membrane. In addition, branching actin filament networks were frequently observed within the cytoplasmic compartment of developing interlocking domains by TEM, in consistent with the findings by fluorescence and immunogold labelling of the F-actin antibody in the domains. These results demonstrate for the first time that the clathrin/AP-2 complex plays a new role for the formation of interlocking domains in lens fibre cells. Branching actin networks and possibly other cytoskeletal components are also associated with the development and maintenance of these interlocking domains. The coordinated 'pulling and pushing' actions generated by the clathrin/AP-2 complex and branching actin networks during interlocking domain formation are discussed.
连锁膜结构域是所有物种晶状体纤维细胞之间以球窝和突起形式存在的特殊膜交错结构。它们被认为在维持纤维 - 纤维稳定性方面起关键作用，因此对晶状体的正常功能很重要。在此我们报道了网格蛋白/AP - 2衔接蛋白复合物和分支状F - 肌动蛋白网络与大鼠及其他几种物种连锁结构域发育的特异性关联。通过超薄切片电子显微镜，我们持续观察到在小型和中型发育中的连锁结构域的凹面膜表面有一层明显的包被（约25纳米厚）。这些膜包被与内吞小泡的网格蛋白包被非常相似，在受体介导的内吞过程中，网格蛋白和AP - 2衔接蛋白参与包被小窝的形成诱导。我们推测网格蛋白/AP - 2复合物直接参与纤维细胞中连锁结构域的诱导形成。通过免疫共聚焦显微镜观察，沿皮质纤维细胞可见网格蛋白和AP - 2衔接蛋白抗体的点状共标记。免疫印迹分析进一步证实，在分别从外皮质纤维和内皮质纤维制备的膜组分中，网格蛋白和AP - 2衔接蛋白抗体分别特异性地染出了一条180kD和106kD的多肽带，这些区域连锁结构域丰富但内吞小泡不存在。免疫电子显微镜显示网格蛋白抗体定位于连锁膜上。此外，通过透射电子显微镜在发育中的连锁结构域的细胞质区域内经常观察到分支状肌动蛋白丝网络，这与该结构域中F - 肌动蛋白抗体的荧光和免疫金标记结果一致。这些结果首次证明网格蛋白/AP - 2复合物在晶状体纤维细胞连锁结构域的形成中发挥新作用。分支状肌动蛋白网络以及可能的其他细胞骨架成分也与这些连锁结构域的发育和维持相关。文中还讨论了在连锁结构域形成过程中由网格蛋白/AP - 2复合物和分支状肌动蛋白网络产生的协同“推拉”作用。
