Nucleated polymerization of actin from the membrane-associated ends of microvillar filaments in the intestinal brush border.

Abstract

The nucleated polymerization of actin from the 2 ends of filament that comprise the microvillus (MV) core in intestinal epithelial cells was examined by EM. Three different in vitro preparations were used to nucleate the polymerization of muscle G-actin: MV core fragments containing barbed and pointed filament ends exposed by shear during isolation, isolated, membrane-intact brush borders and brush borders demembranated with Triton-X 100. MV core fragments nucleate filament growth from both ends with a strong bias for one end. Here the barbed end of the core fragment was identified as the fast growing end by decoration with myosin subfragment 1. Both cytochalasin B (CB) and Acanthamoeba capping protein block filament growth from the barbed but not the pointed end of MV core fragments. To examine actin assembly from the naturally ocurring, membrane-associated ends of MV core filaments, isolated membrane-intact brush borders were used to nucleate the polymerization of G-actin. Addition of salt (75 mM KCl, 1 mM MgSO4) to brush borders preincubated briefly at low ionic strength with G-actin induced the formation of 0.2-0.4 .mu.m growth zones at the tips of microvilli. The dense plaque at the tip of the filaments. Filament growth was also observed from the pointed ends of core filaments in the terminal web. Filament growth was not observed at the membrane-associated ends of core filaments when the latter were in the presence of 2 .mu.M CB or if the low ionic strength incubation step was omitted. Addition of G-actin to demembranated brush borders, which retain the dense plaque on their MV tips, resulted in filament growth from both ends of the MV core. Again, 2 .mu.M CB blocked filament growth from only the barbed (tip) end of the core. The dense plaque remained associated with the tip-end of the core in the presence of CB but usually was dislodged in control preparations where nucleated polymerization from the tip-end of the core occurred. Microvillar assembly and changes in microvillar length could probably occur by actin monomer addition/loss at the barbed, membrane-associated ends of MV core filaments.