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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 binds Tpm1.A) Tpm1 binds directly to wild-type Cof1 and mutant Cof1-5 but not Cof1-22. BSA beads were used as a control. Beads coated with Tpm1 were incubated with 50 nM Cof1, Cof1-5 or Cof1-22. Bound cofilin was visualized by immunoblotting using yeast cofilin antibody [39]. B) Determination of dissociation constant Kd between Cof1 and Tpm1. Beads coated with 0 to 8 µM Tpm1 were incubated with 50 nM Cof1. Bound cofilin was visualized as in (A). C) Bound and free Tpm1 from (B) were quantified and plotted. The calculated Kd was 3.34±0.12 µM (mean±SD).
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pone-0003641-g001: Yeast Cofilin binds Tpm1.A) Tpm1 binds directly to wild-type Cof1 and mutant Cof1-5 but not Cof1-22. BSA beads were used as a control. Beads coated with Tpm1 were incubated with 50 nM Cof1, Cof1-5 or Cof1-22. Bound cofilin was visualized by immunoblotting using yeast cofilin antibody [39]. B) Determination of dissociation constant Kd between Cof1 and Tpm1. Beads coated with 0 to 8 µM Tpm1 were incubated with 50 nM Cof1. Bound cofilin was visualized as in (A). C) Bound and free Tpm1 from (B) were quantified and plotted. The calculated Kd was 3.34±0.12 µM (mean±SD).

Mentions: To test if the association between Cof1 and Tpm1 was direct, we expressed and purified yeast Cof1 from E. coli and found that it binds to Tpm1-coupled beads but not the control BSA beads (Fig. 1A). Using a quantitative Cof1 pull-down assay [25] the apparent Kd for Cof1 binding to Tpm1 was determined to be 3.34±0.12 µM (Fig. 1B). Despite a weak affinity, this interaction is likely to be specific because it was abolished by the cof1-22 mutation, which was shown previously to inhibit turnover of actin patches and cables in vivo [4], [5], but not the cof1-5 mutation (Fig. 1A). Neither mutation affected the folding stability of Cof1 [26].


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 binds Tpm1.A) Tpm1 binds directly to wild-type Cof1 and mutant Cof1-5 but not Cof1-22. BSA beads were used as a control. Beads coated with Tpm1 were incubated with 50 nM Cof1, Cof1-5 or Cof1-22. Bound cofilin was visualized by immunoblotting using yeast cofilin antibody [39]. B) Determination of dissociation constant Kd between Cof1 and Tpm1. Beads coated with 0 to 8 µM Tpm1 were incubated with 50 nM Cof1. Bound cofilin was visualized as in (A). C) Bound and free Tpm1 from (B) were quantified and plotted. The calculated Kd was 3.34±0.12 µM (mean±SD).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2572843&req=5

pone-0003641-g001: Yeast Cofilin binds Tpm1.A) Tpm1 binds directly to wild-type Cof1 and mutant Cof1-5 but not Cof1-22. BSA beads were used as a control. Beads coated with Tpm1 were incubated with 50 nM Cof1, Cof1-5 or Cof1-22. Bound cofilin was visualized by immunoblotting using yeast cofilin antibody [39]. B) Determination of dissociation constant Kd between Cof1 and Tpm1. Beads coated with 0 to 8 µM Tpm1 were incubated with 50 nM Cof1. Bound cofilin was visualized as in (A). C) Bound and free Tpm1 from (B) were quantified and plotted. The calculated Kd was 3.34±0.12 µM (mean±SD).
Mentions: To test if the association between Cof1 and Tpm1 was direct, we expressed and purified yeast Cof1 from E. coli and found that it binds to Tpm1-coupled beads but not the control BSA beads (Fig. 1A). Using a quantitative Cof1 pull-down assay [25] the apparent Kd for Cof1 binding to Tpm1 was determined to be 3.34±0.12 µM (Fig. 1B). Despite a weak affinity, this interaction is likely to be specific because it was abolished by the cof1-22 mutation, which was shown previously to inhibit turnover of actin patches and cables in vivo [4], [5], but not the cof1-5 mutation (Fig. 1A). Neither mutation affected the folding stability of Cof1 [26].

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.

Show MeSH
Related in: MedlinePlus