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 MW polypeptide, villin. The purification and characterization of villin as a Ca2+-dependent, actin bundling/depolymerizing protein are described. The 100,000 g supernatant from a Ca2+ extract of isolated brush borders is composed of 3 polypeptides of 95,000 (villin), 68,000 (fimbrin) and 42,000 MW (actin). Villin, following purification from this extract by differential ammonium sulfate precipitation and ion-exchange chromatography, was mixed with skeletal muscle F-actin. EM 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 Ca2+ concentration in solution. At low free Ca2+ (< 10-6 M), 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 EM. At high free Ca2+ (> 10-6 M), there was a decrease in the apparent viscosity in the villin-actin mixtures to a level measured for the buffer. These Ca2+ 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. Villin is apparently a bundling protein in the microvillus and could be responsible for the Ca2+-sensitive disassembly of the microvillar cytoskeleton. Villin may function in the cytoplasm as a major cytoskeletal element regulating microvillar shape.