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First published online 28 March 2006
doi: 10.1242/jcs.02860

Journal of Cell Science 119, 1547-1557 (2006)
Published by The Company of Biologists 2006
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Twinfilin is an actin-filament-severing protein and promotes rapid turnover of actin structures in vivo

James B. Moseley1, Kyoko Okada1, Heath I. Balcer1, David R. Kovar2, Thomas D. Pollard2 and Bruce L. Goode1,*

1 Department of Biology and The Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454, USA
2 Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA


Figure 1
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  		Fig. 1. S. cerevisiae twinfilin (Twf1) binds to and depolymerizes actin filaments in a pH-dependent manner. (A) Coomassie-stained SDS-polyacrylamide gel of purified Twf1. Positions of molecular mass markers (kDa) are indicated. (B,C) Time course of actin filament fluorescence (rabbit skeletal muscle actin; 30% pyrene labeled) after dilution at time zero (from 5 µM to 0.1 µM) alone or in the presence of Cof1, Twf1 or Lat-A at pH 5.0 (B) or pH 5.8 (C). The inset legend for B also applies to C. (D) Initial rate of actin filament fluorescence decrease induced by 250 nM Twf1 (reaction conditions as in B,C) as a function of pH in the reaction. The rate of fluorescence decrease for actin alone was set at 1 to reference Twf1 effects. (E,F) Time course of actin filament fluorescence (S. cerevisiae actin; 30% pyrene labeled) after dilution at time zero (from 5 µM to 0.1 µM) alone or in the presence of Cof1, Twf1 or Lat-A at pH 5.0 (E) or pH 7.5 (F).

 

Figure 2
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  		Fig. 2. Concentration-dependent effects of Twf1 on rates of dilution-induced actin filament disassembly. (A) Time course of actin filament fluorescence upon dilution (as in Fig. 1) in the presence of a range of concentrations of Twf1 at pH 5.0. (B) Concentration-dependent effects of Twf1 on the initial rate of decrease of actin filament fluorescence at different pH values. Rates were obtained from fluorescence curves in A and data not shown. (C) Comparison of Twf1 and Cof1 effects on the initial rate of actin filament fluorescence decrease at pH 5.0. Rates were obtained from fluorescence curves in A and data not shown. F.A.U., fluorescence arbitrary units.

 

Figure 3
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  		Fig. 3. Twf1 and Cof1 promote actin filament disassembly in dilution-induced assays. (A,B) Time course of unlabeled actin filament light scattering (monitored at 400 nm) after dilution at time zero, from 5 µM to 0.25 µM, in the presence of indicated proteins at pH 5.0 (A) or pH 7.5 (B). A.U., arbitrary units. (C) Fluorescence micrographs of actin filaments incubated alone or with 100 nM Cof1 or Twf1 as in Fig. 1B. At the indicated time points, samples were removed from dilution-induced reactions, incubated with Alexa Fluor 488-congugated phalloidin, and visualized by light microscopy. Bar, 5 µm.

 

Figure 4
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  		Fig. 4. Time-lapse TIRF microscopy of Twf1 severing actin filaments. (A-C) 1.5 µM monomeric actin (30% Alexa Fluor 488 labeled) was assembled into filaments in a flow cell, and then control buffer at pH 5.0 (A), 10 µM Lat-A at pH 5.0 (B), or 2 µM Twf1 at pH 5.0 (C) was applied at time zero. The time in seconds following addition of buffer/proteins is shown in the upper right corner of each panel. (D) Following filament severing by Twf1 in C, the immobilized filaments were incubated with fresh 1.5 µM actin monomers (30% Alexa Fluor 488 labeled) at time zero. Yellow arrowheads track the growth of barbed ends in consecutive frames (barbed end growth rate 8.9 µM–1 second–1, 10 filaments measured). Blue arrows mark the original positions of barbed ends (at time zero) for frame of reference. The field in D is the same field as that shown in C. Movies of these TIRF microscopy experiments are provided in supplemental materials (Movies 1-3, supplementary material).

 

Figure 5
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  		Fig. 5. Effects of Twf1 and Cof1 on the fluorescence of actin filaments capped at their barbed ends with gelsolin. Time course of gelsolin-capped actin filament (G-A) fluorescence (40% pyrene labeled) at pH 5.0 after dilution (from 5 µM to 0.25 µM) at time zero in the presence of the indicated proteins. Actin (5 µM) was polymerized in the presence of 25 nM gelsolin. 1 µM CP had no effect on the depolymerization rate, verifying that these filaments were capped efficiently at their barbed ends by gelsolin. A.U., arbitrary units.

 

Figure 6
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  		Fig. 6. S. cerevisiae capping protein (CP) inhibits Twf1 binding to and severing actin filaments. (A) Time course of actin filament fluorescence (40% pyrene labeled) after dilution at time zero (from 5 µM to 0.1 µM) in the presence of the indicated concentrations of CP at pH 5.0. A.U., arbitrary units. (B) Fluorescence time course, as in A, in the presence and absence of 250 nM Twf1 and a range of concentrations of CP. (C) Concentration-dependent effects of CP on the initial rate of decrease of actin filament fluorescence in the absence (black) and presence (green) of 250 nM Twf1. Rates were obtained from fluorescence curves in A and B. The theoretical curve (gray) was calculated, as described in the text, for direct inhibition of Twf1 by a range of CP concentrations. (D) TIRF microscopy of filaments incubated with Twf1 and CP. 1.5 µM monomeric actin (30% Alexa Fluor 488 labeled) was assembled into filaments in a flow cell, then 2 µM Twf1 with 3 µM CP in buffer pH 5.0 was applied at time zero, and filaments were monitored for 800 seconds. The time in seconds following addition of proteins is shown in the upper right corner of each panel. White arrows indicate the sites of the only two severing events observed in the field of view. (E) TIRF microscopy of filament growth after incubation with Twf1 and CP. Following incubation of filaments with Twf1 and CP in D, the immobilized filaments were incubated with fresh 1.5 µM actin monomers (30% Alexa Fluor 488 labeled) at time zero. Yellow arrowheads track the growth of barbed ends in consecutive frames. Blue arrows mark the original positions of barbed ends (at time zero) for frame of reference. The field in E is the same field as that shown in D. Movies of these TIRF microscopy experiments are provided in supplemental materials (Movie 4, supplementary material).

 

Figure 7
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  		Fig. 7. CP inhibition of Twf1 filament binding and severing requires their direct interaction. (A) Coomassie-stained SDS-polyacrylamide gel of purified Twf1-10. Positions of molecular mass markers (kDa) are shown on the left. (B) Time course of actin filament fluorescence (30% pyrene labeled) after dilution at time zero (from 5 µM to 0.1 µM) in the presence of different concentrations of Twf1, Twf1-10 and/or CP at pH 5.0. A.U., arbitrary units. (C) TIRF microscopy of actin filaments incubated with Twf1-10. 1.5 µM monomeric actin (30% Alexa Fluor 488 labeled) was assembled into filaments in a flow cell, then 2 µM Twf1-10 in buffer pH 5.0 was applied at time zero. The time in seconds following addition of proteins is shown in the upper right corner of each panel. (D) Following filament severing by Twf1-10 in C, the immobilized filaments were incubated with fresh 1.5 µM actin monomers (30% Alexa Fluor 488 labeled) at time zero. Yellow arrowheads track the growth of barbed ends in consecutive frames. Blue arrows mark the original positions of barbed ends (at time zero) for frame of reference. The field in D is the same field as that shown in C. (E) TIRF microscopy of filaments incubated with both Twf1-10 and CP. Actin filaments were assembled as in C, then a mixture of 2 µM Twf1-10 and 3 µM CP in buffer pH 5.0 was applied at time zero. (F) Following filament severing in E, the immobilized filaments were incubated with fresh 1.5 µM actin monomers (30% Alexa Fluor 488 labeled) at time zero. Symbols are as in D. The field in F is the same field as that shown in E. Movies of these TIRF microscopy experiments are provided in supplemental materials (Movies 5 and 6, supplementary material).

 

Figure 8
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  		Fig. 8. PtdIns(4,5)P2 inhibits Twf1 interactions with actin filaments. Time course of actin filament fluorescence (30% pyrene labeled) after dilution at time zero (from 5 µM to 0.1 µM) in the presence of the indicated concentrations of Twf1, Cof1 and/or PtdIns(4,5)P2 (PIP2) at pH 5.0. A.U., arbitrary units.

 

Figure 9
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  		Fig. 9. Twf1 accelerates actin assembly seeded by pre-formed filaments. 3 µM monomeric actin (2% pyrene labeled) and 10 µM S. cerevisiae profilin were pre-mixed and added at time zero to 0.3 µM pre-formed actin filaments (F-actin) and 500 nM Cof1, 500 nM Twf1, or control buffer (3 µM actin) at pH 5.0. A.U., arbitrary units.

 

Figure 10
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  		Fig. 10. Twf1 promotes actin filament disassembly in vivo. (A) Wild-type (WT), twf1{Delta} and cof1-22 yeast cells were grown in parallel to OD600=0.3, then treated with 50 µM Lat-A. Samples of cells were removed 0 and 5 minutes after addition of Lat-A, fixed in 5% formaldehyde, and stained for filamentous actin using Alexa Fluor 568-congugated phalloidin. Cells were visualized by fluorescence light microscopy and scored for the presence of visible actin patches and/or cables. >200 cells were counted for each data point, and similar results were obtained in two independent experiments. Treatment of cells with control buffer caused no loss of actin structures (not shown). (B) Representative wild-type (WT), twf1{Delta} and cof1-22 cells stained with Alexa Fluor 568-congugated phalloidin 0 minutes and 5 minutes after treatment with Lat-A.

 

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© The Company of Biologists Ltd 2006