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Intrinsic capability of budding yeast cofilin to promote turnover of tropomyosin-bound actin filaments.

Fan X, Martin-Brown S, Florens L, Li R - PLoS ONE (2008)

Bottom Line: Yeast cells contain two prominent actin structures, cables and patches, both of which are rapidly assembled and disassembled.Using a variety of assays, we show that yeast cofilin can efficiently depolymerize and sever yeast actin filaments decorated with either Tpm1 or mouse tropomyosins TM1 and TM4.Our results suggest that yeast cofilin has the intrinsic ability to promote actin cable turnover, and that the severing activity may rely on its ability to bind Tpm1.

View Article: PubMed Central - PubMed

Affiliation: The Stowers Institute for Medical Research, Kansas City, MO, USA.

ABSTRACT
The ability of actin filaments to function in cell morphogenesis and motility is closely coupled to their dynamic properties. Yeast cells contain two prominent actin structures, cables and patches, both of which are rapidly assembled and disassembled. Although genetic studies have shown that rapid actin turnover in patches and cables depends on cofilin, how cofilin might control cable disassembly remains unclear, because tropomyosin, a component of actin cables, is thought to protect actin filaments against the depolymerizing activity of ADF/cofilin. We have identified cofilin as a yeast tropomyosin (Tpm1) binding protein through Tpm1 affinity column and mass spectrometry. Using a variety of assays, we show that yeast cofilin can efficiently depolymerize and sever yeast actin filaments decorated with either Tpm1 or mouse tropomyosins TM1 and TM4. Our results suggest that yeast cofilin has the intrinsic ability to promote actin cable turnover, and that the severing activity may rely on its ability to bind Tpm1.

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Yeast cofilin but not mouse cofilin 1 depolymerizes F-actin decorated with tropomyosin.A) Yeast F-actin (10 µM) polymerized with or without 10 µM tropomyosin was incubated with 0, 10 or 20 µM Cof1 (Tpm1-containing sample), or with 0 or 10 µM Cof1 (TM1 or TM4-containing sample). The supernatants and pellets after ultracentrifugation (see Materials and Methods) were analyzed on an SDS-PAGE gel. B) F-actin (10 µM) polymerized with or without 10 µM Tpm1p was incubated with 0, 2.5, 5, 10, 20 and 40 µM Cof1. Subsequent analysis was done as in (A). C) Rabbit muscle actin (10 µM) polymerized with or without 10 µM TM1 was incubated with 0, 5, 10, 20 and 40 µM mouse cofilin 1. Subsequent analysis was done as in (A). D, E) Quantification by densitometry of actin in pellet fractions (as % of the total actin) from experiments in (B) and (C), respectively.
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pone-0003641-g002: Yeast cofilin but not mouse cofilin 1 depolymerizes F-actin decorated with tropomyosin.A) Yeast F-actin (10 µM) polymerized with or without 10 µM tropomyosin was incubated with 0, 10 or 20 µM Cof1 (Tpm1-containing sample), or with 0 or 10 µM Cof1 (TM1 or TM4-containing sample). The supernatants and pellets after ultracentrifugation (see Materials and Methods) were analyzed on an SDS-PAGE gel. B) F-actin (10 µM) polymerized with or without 10 µM Tpm1p was incubated with 0, 2.5, 5, 10, 20 and 40 µM Cof1. Subsequent analysis was done as in (A). C) Rabbit muscle actin (10 µM) polymerized with or without 10 µM TM1 was incubated with 0, 5, 10, 20 and 40 µM mouse cofilin 1. Subsequent analysis was done as in (A). D, E) Quantification by densitometry of actin in pellet fractions (as % of the total actin) from experiments in (B) and (C), respectively.

Mentions: The observation of a direct interaction between Cof1 and Tpm1 led us to test whether Cof1 impacts the stability of yeast F-actin decorated with Tpm1 using a co-sedimentation assay. Yeast actin, purified as described [27], was polymerized in the presence of Tpm1 and then incubated with or without an equimolar or excess cofilin for 20 min. In the absence of Cof1, Tpm1 co-sedimented with F-actin in the pellet fraction (Fig. 2A). Upon incubation with Cof1, most Tpm1 no longer sedimented in the pellet, but instead moved with F-actin to the supernatant fraction. Cof1 had a similar effect when incubated with yeast F-actin bound to either the mouse high-molecular-weight tropomyosin, TM1, or low-molecular-weight tropomyosin, TM4. The extents to which actin was solublized by cofilin were identical with or without tropomyosin bound. This is best observed in an experiment where increasing concentrations of Cof1 was used (Fig. 2B). The two yeast cofilin mutants, Cof1-5 and Cof1-22, showed a similar effect in this assay to that of wild-type Cof1 (Fig. 3). In contrast, mouse non-muscle cofilin 1, which is more similar than mouse cofilin 2 to yeast cofilin, showed diminished ability to depolymerize muscle actin bound to TM1 (Fig. 2C,E), TM4 or Tpm1, as expected (Table 2). This result raised the question of whether tropomyosin could indeed protect F-actin against yeast cofilin.


Intrinsic capability of budding yeast cofilin to promote turnover of tropomyosin-bound actin filaments.

Fan X, Martin-Brown S, Florens L, Li R - PLoS ONE (2008)

Yeast cofilin but not mouse cofilin 1 depolymerizes F-actin decorated with tropomyosin.A) Yeast F-actin (10 µM) polymerized with or without 10 µM tropomyosin was incubated with 0, 10 or 20 µM Cof1 (Tpm1-containing sample), or with 0 or 10 µM Cof1 (TM1 or TM4-containing sample). The supernatants and pellets after ultracentrifugation (see Materials and Methods) were analyzed on an SDS-PAGE gel. B) F-actin (10 µM) polymerized with or without 10 µM Tpm1p was incubated with 0, 2.5, 5, 10, 20 and 40 µM Cof1. Subsequent analysis was done as in (A). C) Rabbit muscle actin (10 µM) polymerized with or without 10 µM TM1 was incubated with 0, 5, 10, 20 and 40 µM mouse cofilin 1. Subsequent analysis was done as in (A). D, E) Quantification by densitometry of actin in pellet fractions (as % of the total actin) from experiments in (B) and (C), respectively.
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Related In: Results  -  Collection

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pone-0003641-g002: Yeast cofilin but not mouse cofilin 1 depolymerizes F-actin decorated with tropomyosin.A) Yeast F-actin (10 µM) polymerized with or without 10 µM tropomyosin was incubated with 0, 10 or 20 µM Cof1 (Tpm1-containing sample), or with 0 or 10 µM Cof1 (TM1 or TM4-containing sample). The supernatants and pellets after ultracentrifugation (see Materials and Methods) were analyzed on an SDS-PAGE gel. B) F-actin (10 µM) polymerized with or without 10 µM Tpm1p was incubated with 0, 2.5, 5, 10, 20 and 40 µM Cof1. Subsequent analysis was done as in (A). C) Rabbit muscle actin (10 µM) polymerized with or without 10 µM TM1 was incubated with 0, 5, 10, 20 and 40 µM mouse cofilin 1. Subsequent analysis was done as in (A). D, E) Quantification by densitometry of actin in pellet fractions (as % of the total actin) from experiments in (B) and (C), respectively.
Mentions: The observation of a direct interaction between Cof1 and Tpm1 led us to test whether Cof1 impacts the stability of yeast F-actin decorated with Tpm1 using a co-sedimentation assay. Yeast actin, purified as described [27], was polymerized in the presence of Tpm1 and then incubated with or without an equimolar or excess cofilin for 20 min. In the absence of Cof1, Tpm1 co-sedimented with F-actin in the pellet fraction (Fig. 2A). Upon incubation with Cof1, most Tpm1 no longer sedimented in the pellet, but instead moved with F-actin to the supernatant fraction. Cof1 had a similar effect when incubated with yeast F-actin bound to either the mouse high-molecular-weight tropomyosin, TM1, or low-molecular-weight tropomyosin, TM4. The extents to which actin was solublized by cofilin were identical with or without tropomyosin bound. This is best observed in an experiment where increasing concentrations of Cof1 was used (Fig. 2B). The two yeast cofilin mutants, Cof1-5 and Cof1-22, showed a similar effect in this assay to that of wild-type Cof1 (Fig. 3). In contrast, mouse non-muscle cofilin 1, which is more similar than mouse cofilin 2 to yeast cofilin, showed diminished ability to depolymerize muscle actin bound to TM1 (Fig. 2C,E), TM4 or Tpm1, as expected (Table 2). This result raised the question of whether tropomyosin could indeed protect F-actin against yeast cofilin.

Bottom Line: Yeast cells contain two prominent actin structures, cables and patches, both of which are rapidly assembled and disassembled.Using a variety of assays, we show that yeast cofilin can efficiently depolymerize and sever yeast actin filaments decorated with either Tpm1 or mouse tropomyosins TM1 and TM4.Our results suggest that yeast cofilin has the intrinsic ability to promote actin cable turnover, and that the severing activity may rely on its ability to bind Tpm1.

View Article: PubMed Central - PubMed

Affiliation: The Stowers Institute for Medical Research, Kansas City, MO, USA.

ABSTRACT
The ability of actin filaments to function in cell morphogenesis and motility is closely coupled to their dynamic properties. Yeast cells contain two prominent actin structures, cables and patches, both of which are rapidly assembled and disassembled. Although genetic studies have shown that rapid actin turnover in patches and cables depends on cofilin, how cofilin might control cable disassembly remains unclear, because tropomyosin, a component of actin cables, is thought to protect actin filaments against the depolymerizing activity of ADF/cofilin. We have identified cofilin as a yeast tropomyosin (Tpm1) binding protein through Tpm1 affinity column and mass spectrometry. Using a variety of assays, we show that yeast cofilin can efficiently depolymerize and sever yeast actin filaments decorated with either Tpm1 or mouse tropomyosins TM1 and TM4. Our results suggest that yeast cofilin has the intrinsic ability to promote actin cable turnover, and that the severing activity may rely on its ability to bind Tpm1.

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