Abstract
Purpose: :
Bridging integrator protein 3 or Bin3, is an evolutionarily conserved and ubiquitously expressed member of the BAR superfamily of curved membrane and GTPase binding proteins. Previous work has shown that homozygous inactivation of Bin3 causes cataracts marked by morphological defects in lens fiber cells. This study investigates the physiological functions of Bin3 in mammals and in the lens.
Methods: :
Mice lacking the Bin3 proximal promoter and exon 1 were created and backcrossed onto a C57Bl6/Har background. RT-PCR and PCR was used to evaluate the loss of Bin3 expression in lens and other tissues. Lens gross anatomy was documented by darkfield microscopy and lens weight was analyzed. Lens ultrastructure was assessed through scanning electron microscopy. Immunofluorescence was used to determine the localization and expression of Cdc42, smooth muscle actin, and ß1 integrin. Whole lens phalloidin staining was done to assess filamentous actin.
Results: :
Bin3 knockout mice had cataracts that developed around 1 to 2 months of age. The lenses were significantly smaller with defects in shape and structure. The ultrastructural analysis of the cortical fiber cells showed that the Bin3 knockout lens had a disorganization of the structurally repeated ball and socket pattern seen in wildtype controls. The broad faces of nuclear fiber cells showed a highly organized lattice like pattern in the Bin3 knockout lens compared to the random arrangement seen in the wildtype lens. Bin3 knockout lens lost Cdc42 protein in the fibers while its expression was elevated in the lens epithelium. Smooth muscle actin was seen to stain the epithelial cells in the Bin3 knockout lens and ß1 integrin was highly upregulated in the epithelial cells as well. Bin3 knockout lens also lacked normal F-actin organization in fibers compared to the wildtype lens.
Conclusions: :
Our data suggest that Bin3 plays a role in the normal development of the repeated ball and sockets pattern, membrane furrows, and the cytoskeletal network in lens fiber cells. Bin3 may also be essential to allow lens epithelial cells to maintain their phenotype and not transdifferentiate into myofibroblasts.
Keywords: cell membrane/membrane specializations • cataract • cytoskeleton