Lens alpha-crystallin is an aggregate of two polypeptides alphaA- and alphaB, which are small heat shock proteins and act as molecular chaperones. Mutations in alphaA- and alphaB-crystallin are associated with early onset human cataract. Targeted disruption of alphaA gene induces cataract at an early age implying a critical role for this protein in maintaining fiber cell transparency. AlphaA knockout lenses are 40% smaller than wild type lenses. We found that lens epithelial cells derived from alphaA knockout mice have 50% slower growth; a fraction of alphaA knockout lens epithelial cells die during mitotic (cell division) phase of the cell cycle in vivo with a phenotype that correlates with abnormal microtubule assembly. AlphaA-crystallin is concentrated in centrosomes and intercellular bridge microtubules of dividing wild type cells, and its expression is cell cycle phase-dependent in synchronized primary mouse lens epithelial cultures. We now propose a series of biochemical and cell biological experiments to further examine whether alphaA-crystallin and proliferation and apoptosis are linked directly in lens epithelium in vivo.
Aim 1 tests the hypothesis that one of the functions of alpha-crystallin is to stabilize the tubulin cytoskeleton in lens epithelial cells. We will determine the assembly of microtubules from wild type and alphaA knockout lens epithelial extracts and examine the interaction of alphaA and alphaB-crystallin with microtubules.
Aim 2 tests the hypothesis that mutations in alphaA- and alphaB-crystallin are cytotoxic to lens epithelial cells in vivo. We are generating knock-in mouse models to analyze the in vivo effect of two mutations (R120G in alphaB-crystallin and R49C in alphaA-crystallin) associated with human cataract. We will determine proliferation and apoptosis in lens epithelium of these mouse models to gain insight into the mechanism by which these mutations lead to cataract formation.
Aim 3 tests the hypothesis that the function of alphaA-crystallin in regulating apoptosis and proliferation is dependent on its interaction with signaling proteins and survival factors. We will resolve the cell survival-related proteins that alpha-crystallin interacts with in the lens epithelium. These mechanistic studies may provide insight into how the loss of normal alpha-crystallin function can lead to cataract formation.
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