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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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Effects of tropomyosin on actin depolymerization and actin binding by yeast Cof1 or mouse cofilin 1.A) Actin filament depolymerization was followed for 4 min at 25°C by the decrease in light-scattering at 400 nm. 5 µM yeast F-actin assembled in the presence or absence of Tpm1 was diluted to reactions containing final concentrations of 0.5 µM Cof1 or Cof1-22 and 0.5 µM F-actin. The spontaneous depolymerization of F-actin (no cofilin), with or without Tpm1 bound was monitored in parallel. B) Decrease in pyrene actin fluorescence was followed for 4 min at 25°C after dilution of F-actin (5 µM with 8% pyrene-labelled, assembled in the presence or absence of Tpm1) to a final concentration 0.5 µM F-actin with or without of 0.5 µM Cof1, Cof1-5 or Cof1-22. C) The same experiment as in (B) was carried out with muscle F-actin assembled with or without TM1, and in the presence or absence of mouse cofilin 1.
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pone-0003641-g004: Effects of tropomyosin on actin depolymerization and actin binding by yeast Cof1 or mouse cofilin 1.A) Actin filament depolymerization was followed for 4 min at 25°C by the decrease in light-scattering at 400 nm. 5 µM yeast F-actin assembled in the presence or absence of Tpm1 was diluted to reactions containing final concentrations of 0.5 µM Cof1 or Cof1-22 and 0.5 µM F-actin. The spontaneous depolymerization of F-actin (no cofilin), with or without Tpm1 bound was monitored in parallel. B) Decrease in pyrene actin fluorescence was followed for 4 min at 25°C after dilution of F-actin (5 µM with 8% pyrene-labelled, assembled in the presence or absence of Tpm1) to a final concentration 0.5 µM F-actin with or without of 0.5 µM Cof1, Cof1-5 or Cof1-22. C) The same experiment as in (B) was carried out with muscle F-actin assembled with or without TM1, and in the presence or absence of mouse cofilin 1.

Mentions: Because the above assay only assessed the steady-state distribution of F- versus G-actin, we next used two assays in which pyrene-labeled F-actin was diluted (see Materials and Methods) to determine the effect of Cof1 on the kinetics of actin depolymerization with or without bound tropomyosin. Light scattering showed that Cof1 drastically increased the rate of F-actin depolymerization after dilution, and Tpm1 binding to F-actin had no effect on the Cof1-induced actin depolymerization (Fig. 4A). Interestingly, measurements of pyrene actin fluorescence in these experiments suggest that binding of Cof1 to F-actin, as indicated by fluorescence quenching [28], was not affected by the presence of Tpm1 (Fig. 4B), On the other hand, mouse tropomyosin 1 reduced the binding of mouse cofilin 1 to F-actin (Fig. 4C), which is consistent with competitive binding of tropomyosin and cofilin to F-actin, as demonstrated by previous studies [16]. Cof1-22 exhibited reduced ability to promote depolymerization of naked actin compared to wild-type Cof1, confirming a previous report [4], yet again, Tpm1 did not alter the depolymerization kinetics in the presence of Cof1-22 (Fig. 4A).


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)

Effects of tropomyosin on actin depolymerization and actin binding by yeast Cof1 or mouse cofilin 1.A) Actin filament depolymerization was followed for 4 min at 25°C by the decrease in light-scattering at 400 nm. 5 µM yeast F-actin assembled in the presence or absence of Tpm1 was diluted to reactions containing final concentrations of 0.5 µM Cof1 or Cof1-22 and 0.5 µM F-actin. The spontaneous depolymerization of F-actin (no cofilin), with or without Tpm1 bound was monitored in parallel. B) Decrease in pyrene actin fluorescence was followed for 4 min at 25°C after dilution of F-actin (5 µM with 8% pyrene-labelled, assembled in the presence or absence of Tpm1) to a final concentration 0.5 µM F-actin with or without of 0.5 µM Cof1, Cof1-5 or Cof1-22. C) The same experiment as in (B) was carried out with muscle F-actin assembled with or without TM1, and in the presence or absence of mouse cofilin 1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2572843&req=5

pone-0003641-g004: Effects of tropomyosin on actin depolymerization and actin binding by yeast Cof1 or mouse cofilin 1.A) Actin filament depolymerization was followed for 4 min at 25°C by the decrease in light-scattering at 400 nm. 5 µM yeast F-actin assembled in the presence or absence of Tpm1 was diluted to reactions containing final concentrations of 0.5 µM Cof1 or Cof1-22 and 0.5 µM F-actin. The spontaneous depolymerization of F-actin (no cofilin), with or without Tpm1 bound was monitored in parallel. B) Decrease in pyrene actin fluorescence was followed for 4 min at 25°C after dilution of F-actin (5 µM with 8% pyrene-labelled, assembled in the presence or absence of Tpm1) to a final concentration 0.5 µM F-actin with or without of 0.5 µM Cof1, Cof1-5 or Cof1-22. C) The same experiment as in (B) was carried out with muscle F-actin assembled with or without TM1, and in the presence or absence of mouse cofilin 1.
Mentions: Because the above assay only assessed the steady-state distribution of F- versus G-actin, we next used two assays in which pyrene-labeled F-actin was diluted (see Materials and Methods) to determine the effect of Cof1 on the kinetics of actin depolymerization with or without bound tropomyosin. Light scattering showed that Cof1 drastically increased the rate of F-actin depolymerization after dilution, and Tpm1 binding to F-actin had no effect on the Cof1-induced actin depolymerization (Fig. 4A). Interestingly, measurements of pyrene actin fluorescence in these experiments suggest that binding of Cof1 to F-actin, as indicated by fluorescence quenching [28], was not affected by the presence of Tpm1 (Fig. 4B), On the other hand, mouse tropomyosin 1 reduced the binding of mouse cofilin 1 to F-actin (Fig. 4C), which is consistent with competitive binding of tropomyosin and cofilin to F-actin, as demonstrated by previous studies [16]. Cof1-22 exhibited reduced ability to promote depolymerization of naked actin compared to wild-type Cof1, confirming a previous report [4], yet again, Tpm1 did not alter the depolymerization kinetics in the presence of Cof1-22 (Fig. 4A).

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