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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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Severing of Tpm1-bound yeast F-actin by Cof1 but not Cof1-22.A) Representative confocal images of F-actin (5 µM), assembled without (upper panels) or with Tpm1 (lower panels), after incubation with 50 nM Cof1 for lengths of time as indicated (see Materials and Methods for more details). B) Representative confocal images of F-actin (5 µM), assembled without (upper panels) or with 5 µM Tpm1 (lower panels), after incubation with 50 nM Cof1-5 (left panels) or Cof1-22 (right panels) for 2 min. C) Measurements of actin filaments length from images recorded in experiments in (A) and (B). Shown are averages of filament length measurements from three fields per sample with error bars representing standard deviations. D) Representative confocal images of F-actin (5 µM), assembled with 5 µM Tpm1, after incubation with 50 nM Cof1-22 for lengths of time as indicated.
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pone-0003641-g005: Severing of Tpm1-bound yeast F-actin by Cof1 but not Cof1-22.A) Representative confocal images of F-actin (5 µM), assembled without (upper panels) or with Tpm1 (lower panels), after incubation with 50 nM Cof1 for lengths of time as indicated (see Materials and Methods for more details). B) Representative confocal images of F-actin (5 µM), assembled without (upper panels) or with 5 µM Tpm1 (lower panels), after incubation with 50 nM Cof1-5 (left panels) or Cof1-22 (right panels) for 2 min. C) Measurements of actin filaments length from images recorded in experiments in (A) and (B). Shown are averages of filament length measurements from three fields per sample with error bars representing standard deviations. D) Representative confocal images of F-actin (5 µM), assembled with 5 µM Tpm1, after incubation with 50 nM Cof1-22 for lengths of time as indicated.

Mentions: Cofilin possesses both actin depolymerization and severing activities, and the latter requires much lower concentrations of cofilin than the former [29]. We used a microscopy-based assay to test the effect of Tpm1 on actin severing by substoichiometric amounts of Cof1 (see Materials and Methods). Pre-polymerized yeast actin was incubated with Cof1 for various lengths of time. The reactions were stopped by dilution into a buffer containing Alexa488-phalloidin, and the lengths of F-actin were quantified using fluorescence microscopy. Under our experimental conditions, Tpm1 increased the average F-actin length from 9.81±4.56 µm to 15.6±3.45 µm (>100 filaments measured). After addition of 50 nM cofilin to 5 µM F-actin, the average F-actin length decreased rapidly in the presence or absence of Tpm1 (Fig. 5A). Even though Tpm1-bound filaments started longer, the rate of filament severing by Cof1 was similar with or without Tpm1 bound to F-actin. Cof1-5 exhibited an only slightly reduced rate of actin severing, which was not affected by Tpm1 binding (Fig. 5B,C). Interestingly, Cof1-22 only exhibited a moderate defect severing naked F-actin, however, the severing defect was much more enhanced toward Tpm1-bound F-actin, and long filaments were observable even after 40 min of incubation with Cof1-22 (Fig. 5B,C,D).


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)

Severing of Tpm1-bound yeast F-actin by Cof1 but not Cof1-22.A) Representative confocal images of F-actin (5 µM), assembled without (upper panels) or with Tpm1 (lower panels), after incubation with 50 nM Cof1 for lengths of time as indicated (see Materials and Methods for more details). B) Representative confocal images of F-actin (5 µM), assembled without (upper panels) or with 5 µM Tpm1 (lower panels), after incubation with 50 nM Cof1-5 (left panels) or Cof1-22 (right panels) for 2 min. C) Measurements of actin filaments length from images recorded in experiments in (A) and (B). Shown are averages of filament length measurements from three fields per sample with error bars representing standard deviations. D) Representative confocal images of F-actin (5 µM), assembled with 5 µM Tpm1, after incubation with 50 nM Cof1-22 for lengths of time as indicated.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003641-g005: Severing of Tpm1-bound yeast F-actin by Cof1 but not Cof1-22.A) Representative confocal images of F-actin (5 µM), assembled without (upper panels) or with Tpm1 (lower panels), after incubation with 50 nM Cof1 for lengths of time as indicated (see Materials and Methods for more details). B) Representative confocal images of F-actin (5 µM), assembled without (upper panels) or with 5 µM Tpm1 (lower panels), after incubation with 50 nM Cof1-5 (left panels) or Cof1-22 (right panels) for 2 min. C) Measurements of actin filaments length from images recorded in experiments in (A) and (B). Shown are averages of filament length measurements from three fields per sample with error bars representing standard deviations. D) Representative confocal images of F-actin (5 µM), assembled with 5 µM Tpm1, after incubation with 50 nM Cof1-22 for lengths of time as indicated.
Mentions: Cofilin possesses both actin depolymerization and severing activities, and the latter requires much lower concentrations of cofilin than the former [29]. We used a microscopy-based assay to test the effect of Tpm1 on actin severing by substoichiometric amounts of Cof1 (see Materials and Methods). Pre-polymerized yeast actin was incubated with Cof1 for various lengths of time. The reactions were stopped by dilution into a buffer containing Alexa488-phalloidin, and the lengths of F-actin were quantified using fluorescence microscopy. Under our experimental conditions, Tpm1 increased the average F-actin length from 9.81±4.56 µm to 15.6±3.45 µm (>100 filaments measured). After addition of 50 nM cofilin to 5 µM F-actin, the average F-actin length decreased rapidly in the presence or absence of Tpm1 (Fig. 5A). Even though Tpm1-bound filaments started longer, the rate of filament severing by Cof1 was similar with or without Tpm1 bound to F-actin. Cof1-5 exhibited an only slightly reduced rate of actin severing, which was not affected by Tpm1 binding (Fig. 5B,C). Interestingly, Cof1-22 only exhibited a moderate defect severing naked F-actin, however, the severing defect was much more enhanced toward Tpm1-bound F-actin, and long filaments were observable even after 40 min of incubation with Cof1-22 (Fig. 5B,C,D).

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