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.
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24 March 1997
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March 24 1997
Xenopus Actin Depolymerizing Factor/Cofilin (XAC) Is Responsible for the Turnover of Actin Filaments in Listeria monocytogenes Tails
Jody Rosenblatt,
Jody Rosenblatt
*Department of Biochemistry, University of California, San Francisco, San Francisco, California 94143; and ‡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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Brian J. Agnew,
Brian J. Agnew
*Department of Biochemistry, University of California, San Francisco, San Francisco, California 94143; and ‡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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Hiroshi Abe,
Hiroshi Abe
*Department of Biochemistry, University of California, San Francisco, San Francisco, California 94143; and ‡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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James R. Bamburg,
James R. Bamburg
*Department of Biochemistry, University of California, San Francisco, San Francisco, California 94143; and ‡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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Timothy J. Mitchison
Timothy J. Mitchison
*Department of Biochemistry, University of California, San Francisco, San Francisco, California 94143; and ‡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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Jody Rosenblatt
*Department of Biochemistry, University of California, San Francisco, San Francisco, California 94143; and ‡Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523; and §Department of Biology, Chiba University, Chiba 263, Japan
Brian J. Agnew
*Department of Biochemistry, University of California, San Francisco, San Francisco, California 94143; and ‡Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523; and §Department of Biology, Chiba University, Chiba 263, Japan
Hiroshi Abe
*Department of Biochemistry, University of California, San Francisco, San Francisco, California 94143; and ‡Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523; and §Department of Biology, Chiba University, Chiba 263, Japan
James R. Bamburg
*Department of Biochemistry, University of California, San Francisco, San Francisco, California 94143; and ‡Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523; and §Department of Biology, Chiba University, Chiba 263, Japan
Timothy J. Mitchison
*Department of Biochemistry, University of California, San Francisco, San Francisco, California 94143; and ‡Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523; and §Department of Biology, Chiba University, Chiba 263, Japan
This work is dedicated to Michael Redd and our baby, Nadja.
Address all correspondence to Jody Rosenblatt, Department of Biochemistry, University of California, San Francisco, San Francisco, CA 94143. Tel.: (415) 476-4002. Fax: (415) 476-5233.
Received:
September 11 1996
Revision Received:
December 12 1996
Online ISSN: 1540-8140
Print ISSN: 0021-9525
1997
J Cell Biol (1997) 136 (6): 1323–1332.
Article history
Received:
September 11 1996
Revision Received:
December 12 1996
Citation
Jody Rosenblatt, Brian J. Agnew, Hiroshi Abe, James R. Bamburg, Timothy J. Mitchison; Xenopus Actin Depolymerizing Factor/Cofilin (XAC) Is Responsible for the Turnover of Actin Filaments in Listeria monocytogenes Tails. J Cell Biol 24 March 1997; 136 (6): 1323–1332. doi: https://doi.org/10.1083/jcb.136.6.1323
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