The hypotheses of this project are: a) The ATP depletion in epithelial cells results in the depolymerization of actin microfilaments that form the microvillar core, and, thus, in either the release/depolarization of apical cytoskeletally anchored plasma membrane proteins, the loss of vectorial delivery of membrane proteins, or both. b) The intermediate filaments of the terminal web carry organizers to reassemble the rootlets of the microvillar cores during the recovery after an ischemia-like injury. To test these hypotheses, I propose the following specific aims; 1- Determine the extent, morphology and kinetics of actin depolymerization and repolymerization due to ischemia/ recovery situations in epithelial cells. The actin depolymerization/repolymerization will be analyzed in epithelial cells in tissue culture depleted in ATP by biochemical methods, immunofluorescence and immunoelectronmicroscopy. 2- Determine the role of terminal web and an apical 53 kD cytokeratin as potential organizers of actin repolymerization after an ischemia-like injury. Specific antibodies against an apically localized 53 kD cytokeratin and fimbrin introduced in the cytoplasm will be used to block of microvillar reassembly. Anti-sense oligonucleotides will be used to hinder the synthesis of the 53 kD protein. Organization of microfilaments on IF will be studied after ATP depletion. 3- Analyze the relationship between actin depolymerization following an ischemia-like injury and loss in epithelial plasma membrane polarity. Polarization of apical and basolateral plasma membrane markers will be analyzed as a function of cytoskeletal disruption after ischemia.
| Ameen, N A; Salas, P J (2000) Microvillus inclusion disease: a genetic defect affecting apical membrane protein traffic in intestinal epithelium. Traffic 1:76-83 |
| Salas, P J (1999) Insoluble gamma-tubulin-containing structures are anchored to the apical network of intermediate filaments in polarized CACO-2 epithelial cells. J Cell Biol 146:645-58 |
| Salas, P J; Rodriguez, M L; Viciana, A L et al. (1997) The apical submembrane cytoskeleton participates in the organization of the apical pole in epithelial cells. J Cell Biol 137:359-75 |
