B–Crystallin Strongly Localizes to the Leading Edges of the Cell Membrane in Migrating Lens Primary Epithelial Cells

Abstract

Abstract: : Purpose: To determine the sub–cellular distribution of αB–crystallin in semi–confluent and contact–inhibited confluent lens epithelial cell cultures. Methods:Primary lens epithelial cells isolated from porcine lenses were grown to confluence or semi–confluence on gelatin coated glass coverslips or plastic Petri dishes. Cells grown on glass coverslips were immunolabelled for αB–crystallin using polyclonal antibodies against recombinant αB crystallin or ser59 phosphospecific αB–crystallin, along with appropriate controls, and images were recorded using confocal microscopy. Cells were also double–labeled for αB–crystallin and either beta–catenin, vinculin, actin or phosphotyrosine. Levels of αB–crystallin (both total and phosphorylated) were determined in porcine lens epithelial cell cytosolic, membrane and nuclear fractions by Western blot analysis. Subcellular distribution of αB–crystallin was evaluated in cells pretreated with inhibitors of P38 MAP kinase, ERK kinase, Src kinase, integrins or tyrosine kinases. Results:αB–crystallin staining was confined predominantly to cytosolic and nuclear regions in the confluent cells, where as in semi–confluent cells, intense pattern of staining was noted at the leading edges of the cell membrane, using either αB–crystallin or Ser59 phosphopecific αB–crystallin polycolonal antibody. Subcellular fractionation and Western blotting revealed enrichment of αB–crystallin (both total and phosphospecific) in membrane fractions from semi–confluent lens cells. Interestingly, treatment of semi–confluent cells with inhibitors of P38 MAP kinae (SB 202190, 5µM), integrins (RGD peptide, 5 µM), or Src kinase (PP2, 20 µM) caused dramatic reduction in αB–crystallin staining at the leading edges of the cell membrane. Furthermore, the strong αB–crystallin staining at the leading edges of cell membrane appears to co–localize with beta–catenin, phosphotyrosine, and, to some extent, with cortical actin fibers. Conclusions: These preliminary data reveal a predominant localization of αB–crystallin at the leading edges of the cell membrane in a phosphorylation–dependent fashion, in semi–confluent, migrating and proliferating lens epithelial cells. Interestingly, αB–crystallin also appears to co–localize with other signaling molecules known to participate in cell migration and proliferation.

Keywords: crystallins • cell adhesions/cell junctions • cytoskeleton