The overall plan is to elucidate the epigenetic mechanisms of action of metals at subcellular and molecular levels. Central to this plan is to test the hypothesis that the cytoskeleton acts as a major target for metals and, as such, its perturbations may be an important part in the manifestation of cellular injury leading to systemic toxicity or cell death. Investigations will focus on both the mechanisms of metal-induced cytoskeletal perturbation, particularly microtubule (MT) disassembly, and the consequences of such perturbation.
The specific aims are: 1. To understand the molecular mechanisms of cytoskeletal perturbations induced by such metals as As (III), Cd (II), Hg (II), and Ni (II): a. To determine whether MT disassembly and inhibition of assembly induced by As (III), Cd (II) and Hg (II) in cultured cells are due to the loss of essential tubulin -SH groups, or the binding to and activation of calmodulin (CaM), or a combination of both. The hypothesis that heavy metal toxicity may result in part from inappropriate activation of CaM will be tested at the cellular level. b. To evaluate the role of microtubule associated proteins (MAPs) in modulating the lability of MT to CaM activation or binding of -SH groups mediated by these metals. c. To test the hypothesis that activation of CaM by Ca2+ and other metals stimulates a Ca2+/CaM dependent protein kinase resulting in MAP phosphorylation and MT disassembly. d. To identify metal induced alterations in the molecular species, phosphorylation state and distribution of MAPs and their isoelectric variants by two dimensional (2-D) gel electrophoresis. 2. To examine the consequences of cytoskeletal perturbation. a. To investigate the alterations in the cytoplasmic location, organization, and morphology of such cytoskeleton-associated organelles as mitochondria, the Golgi apparatus, endoplasmic reticulum (ER) and lysosomes following metal-induced cytoskeletal perturbation. b. To test the hypothesis that cytoskeletal perturbation and the resulting nucleotide pool imbalance may be common, coordinate events in the mechanism of cellular toxic responses to metals. Nucleotide pools will be determined by HPLC analysis. c. To identify by biochemical and microscopic analyses the metal-induced perturbations in the composition and organization of the nuclear matrix prepared by in situ detergent and high salt extraction followed digestion with nucleases.
