Partial reconstruction of the microvillus core bundle: Characterization of villin as a Ca++-dependent, actin-bundling/depolymerizing protein

Abstract

The brush border, isolated from chicken intestine epithelial cells, contains the 95,000 relative molecular mass (Mr) polypeptide, villin. This report describes the purification and characterization of villin as a Ca++-dependent, actin bundling/depolymerizing protein. The 100,000 g supernatant from a Ca++ extract of isolated brush borders is composed of three polypeptides of 95,000 (villin), 68,000 (fimbrin), and 42,000 Mr (actin). Villin, following purification from this extract by differential ammonium sulfate precipitation and ion-exchange chromatography, was mixed with skeletal muscle F-actin. Electron microscopy of negatively stained preparations of these villin-actin mixtures showed that filament bundles were present. The viscosity, sedimentability, and ultrastructural morphology of filament bundles are dependent on the villin:actin molar ratio, the pH, and the free Ca++ concentration in solution. At low free Ca++ (<10-6M), the amount of protein in bundles, when measured by sedimentation, increased as the villin:actin molar ratio increased and reached a plateau at approximately a 4:10 ratio. This behavior correlates with the conversion of single actin filaments into filament bundles as detected in the electron microscope. At high free Ca++(>10-6M), there was a decrease in the apparent viscosity in the villin-actin mixtures to a level measured for the buffer. Furthermore, these Ca++ effects were correlated with the loss of protein sedimented, the disappearance of filament bundles, and the appearance of short fragments of filaments. Bundle formation is also pH-sensitive, being favored at mildly acidic pH. A decrease in the pH from 7.6 to 6.6 results in an increase in sedimentable protein and also a transformation of loosely associated actin filaments into compact actin bundles. These results are consistent with the suggestions that villin is a bundling protein in the microvillus and is responsible for the Ca++-sensitive disassembly of the microvillar cytoskeleton. Thus villin may function in the cytoplasm as a major cytoskeletal element regulation microvillar shape.