Regulation of microvillus structure: calcium-dependent solation and cross-linking of actin filaments in the microvilli of intestinal epithelial cells.

Abstract

The bundle of filaments within microvilli of intestinal epithelial cells contains 5 major proteins including actin, calmodulin, and subunits of 105-, 95-, and 70-kdaltons. Brush-border the addition of Ca2+ (> 10-6 M) to microvillus cores causes a rapid, drastic, but at least partially reversible disruption of this actin filament bundle [Howe et al, 1980]. High-speed centrifugation of microvillus cores treated with Ca2+ indicates that several core proteins are solubilized, including 30-50% of the actin and calmodulin, along with much of the 95- and 70-kdalton subunits. Gel filtration of such Ca2+ extracts in the presence and absence of Ca2+ indicates that microvillar actin solated by Ca2+ is in an oligomeric state probably complexed with the 95-kdalton subunit. Removal of Ca2+ results in the reassembly of F-actin, probably still complexed with 95-kdalton subunit, as determined by gel filtration, cosedimentation, viscometry, and EM. The 95-kdalton subunit (95K) was purified from Ca2+ extracts by DEAE-Sephadex chromatography and its interaction with actin characterized by viscometry, cosedimentation, and EM in the presence and absence of Ca2+. In the presence, but not absence, of Ca2+, 95K inhibits actin assembly (50% inhibition at 1:50-60 95K to actin) and also reduces the viscosity of F-actin solutions. Sedimentation of actin is inhibited by 95K, but a small, presumably oligomeric actin- 95K complex formed in the presence of Ca2+ is pelletable after long-term centrifugation. In the absence of Ca2+, 95K cosediments with F-actin. EM of 95K-actin mixtures reveals that 95K breaks actin into small, filamentous fragments in the presence of Ca2+. Reassembly of filaments occurs once Ca2+ is removed. In the absence of Ca2+, 95K has no effect on filament structure and, at relatively high ratios (1:2-6) of 95K to actin, this core protein will aggregate actin filaments into bundles.