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* Department of Biochemistry, University of California, San Francisco, San Francisco, California 94143; and In contrast to the slow rate of depolymerization of pure actin in vitro, populations of actin filaments
in vivo turn over rapidly. Therefore, the rate of actin
depolymerization must be accelerated by one or more
factors in the cell. Since the actin dynamics in Listeria
monocytogenes tails bear many similarities to those in
the lamellipodia of moving cells, we have used Listeria as a model system to isolate factors required for regulating the rapid actin filament turnover involved in cell
migration. Using a cell-free Xenopus egg extract system
to reproduce the Listeria movement seen in a cell, we
depleted candidate depolymerizing proteins and analyzed the effect that their removal had on the morphology of Listeria tails. Immunodepletion of Xenopus
actin depolymerizing factor (ADF)/cofilin (XAC) from
Xenopus egg extracts resulted in Listeria tails that were
approximately five times longer than the tails from undepleted extracts. Depletion of XAC did not affect the
tail assembly rate, suggesting that the increased tail
length was caused by an inhibition of actin filament depolymerization. Immunodepletion of Xenopus gelsolin
had no effect on either tail length or assembly rate. Addition of recombinant wild-type XAC or chick ADF
protein to XAC-depleted extracts restored the tail length to that of control extracts, while addition of mutant ADF S3E that mimics the phosphorylated, inactive
form of ADF did not reduce the tail length. Addition of
excess wild-type XAC to Xenopus egg extracts reduced
the length of Listeria tails to a limited extent. These observations show that XAC but not gelsolin is essential for depolymerizing actin filaments that rapidly turn
over in Xenopus extracts. We also show that while the
depolymerizing activities of XAC and Xenopus extract
are effective at depolymerizing normal filaments containing ADP, they are unable to completely depolymerize actin filaments containing AMPPNP, a slowly hydrolyzible ATP analog. This observation suggests that
the substrate for XAC is the ADP-bound subunit of actin and that the lifetime of a filament is controlled by its
nucleotide content.
Department of
Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523; and § Department of Biology,
Chiba University, Chiba 263, Japan
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