VILLIN-INDUCED GROWTH OF MICROVILLI IS REVERSIBLY INHIBITED BY CYTOCHALASIN-D

Villin is an actin-binding protein that is associated with the cytoske leton of brush border microvilli. In vitro, villin nucleates, caps or severs actin filaments in a Ca2+dependent manner. In the absence of Ca 2+, villin organizes microfilaments into bundles. Transfection of a vi llin-specific cDNA into cultured cells that do not produce this protei n results in the growth of long surface microvilli and the reorganizat ion of the underlying actin cytoskeleton. Here we studied the effects of low concentrations of cytochalasin D on the induction of these plas ma membrane-actin cytoskeleton specializations. Transfected cells were treated with concentrations of cytochalasin D that prevent the associ ation of actin monomers with the fast-growing end of microfilaments in vitro. In villin-positive cells, cytochalasin D inhibited the growth of microvilli and promoted the formation of rodlet-like actin structur es, which were randomly distributed throughout the cytoplasm. The form ation of these structures was dependent on large amounts of villin and on the integrity of an actin-binding site located at the carboxy term inus of villin, which is required for microfilament bundling in vitro and for the growth of microvilli in vivo. The effect of cytochalasin D was reversible. The observation of living cells by video-imaging reve aled that when cytochalasin D was removed, rapid disassembly of actin rodlets occurred after a lag phase. The present data stress the import ant role of the plasma membrane in the organization of the actin cytos keleton and suggest that the extension of the microvillar plasma membr ane is dependent on the elongation of microfilaments at their fast-gro wing end. Inhibition of microfilament elongation near the plasma membr ane by cytochalasin D may result in the 'random' nucleation of actin f ilaments throughout the cytoplasm. On the basis of the present data, w e propose that villin is involved in the assembly of the microvillar a ctin bundle by a mechanism that does not prevent monomer association w ith the prefered end of microfilaments. For instance, villin may stabi lize actin filaments by lateral interactions. The functional importanc e of the carboxy-terminal F-actin binding site in such a mechanism is stressed by the fact that it is required for the formation of F-actin rodlets in cytochalasin D-treated cells. Finally, our data further emp hasize the observations that the effects of cytochalain D in living ce lls can be modulated by actin-binding proteins.