Association of clathrin, AP-2 adaptor and actin cytoskeleton with developing interlocking membrane domains of lens fibre cells.

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.