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 NO 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 M r (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" (<10-6 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 electron microscope . At high free Ca" (>10-6 M), 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 regulating microvillar shape .The brush border microvillus is a model biological system for investigations aimed at understanding how a cell maintains its shape. Ultrastructural studies have shown that the microvillus contains a core of bundled microfilaments which extend into the underlying terminal web (44,45) . This core bundle is associated with the overlying plasma membrane by two types of attachments. The first type laterally connects the membrane along the length of the bundle by means of cross filaments (35,39,44,45). In the second type of membrane attachment, the microfilaments insert end-on into the electron-dense cap at the tips of the microvilli. Earlier studies had suggested that both types of attachments as well as the bundling protein in the microvillus core were composed of alpha-actinin (3,44,48) . However, recent studies have shown instead that alpha-actinin is not a structural component of the microvillus but rather is a component of the te...

Villin Function in the Organization of the Actin Cytoskeleton

“…1B) also affect bundling activity of villin in vitro. Experiments were carried out at physiological ionic strength (150 mM KCl) at pH 7, conditions under which villin efficiently bundles actin filaments (43). In addition, because subtle variations of villin:actin molar ratios have important consequences on bundle formation, we analyzed bundling activity at saturating quantities of villin relative to actin (villin:actin 1:2; Ref.…”

“…In addition, because subtle variations of villin:actin molar ratios have important consequences on bundle formation, we analyzed bundling activity at saturating quantities of villin relative to actin (villin:actin 1:2; Ref. 43), in the presence of EGTA (Fig. 4).…”

Organization of the actin filament cytoskeleton in the intestinal brush border: a quantitative and qualitative immunoelectron microscope study.

“…The actin filament core bundle is extensively cross-linked by two proteins, fimbrin (68 kD) and villin (95 kD; 2,4,5,6,25,55). In the presence of Ca ++ (>10 -6 M), villin causes fragmentation of the core bundle whereas the bundling activity of fimbrin appears to be independent of Ca++ (9,17,48,49,55). Villin is also present in the rootlets (23).…”

Studies on the spectrin-like protein from the intestinal brush border, TW 260/240, and characterization of its interaction with the cytoskeleton and actin.

“…The apical brush border surface of intestinal epithelial cells consists of a tightly-packed array of microvilli, each of which contains a supporting bundle of actin filaments, cross-linked by at least two bundling proteins, fimbrin (15)(16)(17) and villin (18)(19)(20)(21)(22). The basal or "rootlet" ends of these bundles descend below the plasma membrane into the apical cytoplasm of the cell.…”