Abstract
We characterized the properties of Drosophila melanogaster DAAM-FH2 and DAAM-FH1-FH2 fragments and their interactions with actin and profilin by using various biophysical methods and in vivo experiments. The results show that although the DAAM-FH2 fragment does not have any conspicuous effect on actin assembly in vivo, in cells expressing the DAAM-FH1-FH2 fragment, a profilin-dependent increase in the formation of actin structures is observed. The trachea-specific expression of DAAM-FH1-FH2 also induces phenotypic effects, leading to the collapse of the tracheal tube and lethality in the larval stages. In vitro, both DAAM fragments catalyze actin nucleation but severely decrease both the elongation and depolymerization rate of the filaments. Profilin acts as a molecular switch in DAAM function. DAAM-FH1-FH2, remaining bound to barbed ends, drives processive assembly of profilin-actin, whereas DAAM-FH2 forms an abortive complex with barbed ends that does not support profilin-actin assembly. Both DAAM fragments also bind to the sides of the actin filaments and induce actin bundling. These observations show that the D. melanogaster DAAM formin represents an extreme class of barbed end regulators gated by profilin.
Highlights
The actin cytoskeleton fulfills its various biological functions under the tight and well controlled balance of regulatory systems
To characterize the D. melanogaster DAAM formin, we analyzed the interactions of its FH2 and FH1-FH2 fragments with actin and profilin by using various biophysical methods
We investigated the properties of D. melanogaster DAAM-FH2 and DAAM-FH1-FH2 formin fragments
Summary
Actin was prepared from rabbit skeletal muscle [33]. The concentration of G-actin was determined spectrophotometrically using a Shimadzu UV-2100 spectrophotometer, with the absorption coefficient of 0.63 mgϪ1 ml cmϪ1 at 290 nm [34]. The concentration of the fluorescence dye in the protein solution was determined using the absorption coefficient of 2.2 ϫ 104 MϪ1 cmϪ1 at 344 nm for pyrenyl-actin. We used the same dDAAM subfragments as above, inserted into a pGEX-2T vector (Amersham Biosciences). The cells were incubated for 30 min at room temperature, followed by the addition of 2 ml of Schneider’s medium (Sigma) containing fetal bovine serum (Sigma). The cells were incubated for an additional 3 days and transfected with pAVW-FH1-FH2, and 15 g of dsRNA was added . These cells were incubated in Drosophila Schneider’s medium (with fetal bovine serum) for 24 h before fixation. When following the same protocol, cells were treated with 15 g of dsRNA each day; most cells died by the time of fixation
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