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Single-molecule visualization of a formin-capping protein 'decision complex' at the actin filament barbed end.

Bombardier JP, Eskin JA, Jaiswal R, Corrêa IR, Xu MQ, Goode BL, Gelles J - Nat Commun (2015)

Bottom Line: Here we use multi-wavelength single-molecule fluorescence microscopy to observe the fully reversible formation of a long-lived 'decision complex' in which a CP dimer and a dimer of the formin mDia1 simultaneously bind the barbed end.Quantitative kinetic analysis reveals that the CP-mDia1 antagonism that we observe in vitro occurs through the decision complex.Our observations suggest new molecular mechanisms for the control of actin filament length and for the capture of filament barbed ends in cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454-9110, USA.

ABSTRACT
Precise control of actin filament length is essential to many cellular processes. Formins processively elongate filaments, whereas capping protein (CP) binds to barbed ends and arrests polymerization. While genetic and biochemical evidence has indicated that these two proteins function antagonistically, the mechanism underlying the antagonism has remained unresolved. Here we use multi-wavelength single-molecule fluorescence microscopy to observe the fully reversible formation of a long-lived 'decision complex' in which a CP dimer and a dimer of the formin mDia1 simultaneously bind the barbed end. Further, mDia1 displaced from the barbed end by CP can randomly slide along the filament and later return to the barbed end to re-form the complex. Quantitative kinetic analysis reveals that the CP-mDia1 antagonism that we observe in vitro occurs through the decision complex. Our observations suggest new molecular mechanisms for the control of actin filament length and for the capture of filament barbed ends in cells.

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Decision complex kinetics and mechanism.(a) Cumulative lifetime distribution of decision complexes containing 549-mDia1 and 649-CP (black; N=171), and a maximum likelihood fit (see Methods) of the measured lifetimes to a single-species (exponential) kinetic model (red; shading indicates 95% CI). Fit yielded a characteristic lifetime μ=359 s (s.e.: 41 s; 95% CI: 290–448 s); this quantity includes contributions from both 549-mDia1 and 649-CP dissociation and from photobleaching of both dyes. (b) Deduced kinetic schemes and rate constants (±s.e.) for the interaction of actin filament barbed ends with CP only (red box), mDia1 only (green box) or both (magenta box). Green and red arrows mark structures that can or cannot, respectively, add actin monomers to the filament end. Values of k−1′ and k2′ are corrected for dye photobleaching (see Methods). The value of k2 is not corrected for photobleaching; it is an estimated upper limit since only a single 549-mDia1 disappearance event was observed, and this may have been caused by photobleaching rather than dissociation.
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f5: Decision complex kinetics and mechanism.(a) Cumulative lifetime distribution of decision complexes containing 549-mDia1 and 649-CP (black; N=171), and a maximum likelihood fit (see Methods) of the measured lifetimes to a single-species (exponential) kinetic model (red; shading indicates 95% CI). Fit yielded a characteristic lifetime μ=359 s (s.e.: 41 s; 95% CI: 290–448 s); this quantity includes contributions from both 549-mDia1 and 649-CP dissociation and from photobleaching of both dyes. (b) Deduced kinetic schemes and rate constants (±s.e.) for the interaction of actin filament barbed ends with CP only (red box), mDia1 only (green box) or both (magenta box). Green and red arrows mark structures that can or cannot, respectively, add actin monomers to the filament end. Values of k−1′ and k2′ are corrected for dye photobleaching (see Methods). The value of k2 is not corrected for photobleaching; it is an estimated upper limit since only a single 549-mDia1 disappearance event was observed, and this may have been caused by photobleaching rather than dissociation.

Mentions: To more fully understand the nature of the decision complex, we investigated its kinetic behaviour. The N=171 complexes we observed had an exponential lifetime distribution (Fig. 5a). The mean lifetime of the decision complexes that ended by CP dissociation (199±36 s (s.e.m.); N=51) was the same within experimental uncertainty as those that ended by mDia1 dissociation (171±24 s; N=67). These data are consistent with the hypothesis that the same, single decision complex species produces both outcomes. This model is corroborated by our observations that mDia1 could return to a 649-CP-capped barbed end (thus re-forming the decision complex), wait for 649-CP to dissociate, and then recommence elongation (Fig. 4a,b and Supplementary Movie 6). These observations show that the formation of the decision complex can occur from either direction. Thus, multiple lines of evidence suggest that the decision complex is the single kinetically significant intermediate in the CP/mDia1 antagonism mechanism, can be formed by starting from either a CP-capped, static barbed end or an mDia1-bound, growing barbed end, and can yield either of these barbed-end complexes as an outcome (Fig. 5b, magenta).


Single-molecule visualization of a formin-capping protein 'decision complex' at the actin filament barbed end.

Bombardier JP, Eskin JA, Jaiswal R, Corrêa IR, Xu MQ, Goode BL, Gelles J - Nat Commun (2015)

Decision complex kinetics and mechanism.(a) Cumulative lifetime distribution of decision complexes containing 549-mDia1 and 649-CP (black; N=171), and a maximum likelihood fit (see Methods) of the measured lifetimes to a single-species (exponential) kinetic model (red; shading indicates 95% CI). Fit yielded a characteristic lifetime μ=359 s (s.e.: 41 s; 95% CI: 290–448 s); this quantity includes contributions from both 549-mDia1 and 649-CP dissociation and from photobleaching of both dyes. (b) Deduced kinetic schemes and rate constants (±s.e.) for the interaction of actin filament barbed ends with CP only (red box), mDia1 only (green box) or both (magenta box). Green and red arrows mark structures that can or cannot, respectively, add actin monomers to the filament end. Values of k−1′ and k2′ are corrected for dye photobleaching (see Methods). The value of k2 is not corrected for photobleaching; it is an estimated upper limit since only a single 549-mDia1 disappearance event was observed, and this may have been caused by photobleaching rather than dissociation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660045&req=5

f5: Decision complex kinetics and mechanism.(a) Cumulative lifetime distribution of decision complexes containing 549-mDia1 and 649-CP (black; N=171), and a maximum likelihood fit (see Methods) of the measured lifetimes to a single-species (exponential) kinetic model (red; shading indicates 95% CI). Fit yielded a characteristic lifetime μ=359 s (s.e.: 41 s; 95% CI: 290–448 s); this quantity includes contributions from both 549-mDia1 and 649-CP dissociation and from photobleaching of both dyes. (b) Deduced kinetic schemes and rate constants (±s.e.) for the interaction of actin filament barbed ends with CP only (red box), mDia1 only (green box) or both (magenta box). Green and red arrows mark structures that can or cannot, respectively, add actin monomers to the filament end. Values of k−1′ and k2′ are corrected for dye photobleaching (see Methods). The value of k2 is not corrected for photobleaching; it is an estimated upper limit since only a single 549-mDia1 disappearance event was observed, and this may have been caused by photobleaching rather than dissociation.
Mentions: To more fully understand the nature of the decision complex, we investigated its kinetic behaviour. The N=171 complexes we observed had an exponential lifetime distribution (Fig. 5a). The mean lifetime of the decision complexes that ended by CP dissociation (199±36 s (s.e.m.); N=51) was the same within experimental uncertainty as those that ended by mDia1 dissociation (171±24 s; N=67). These data are consistent with the hypothesis that the same, single decision complex species produces both outcomes. This model is corroborated by our observations that mDia1 could return to a 649-CP-capped barbed end (thus re-forming the decision complex), wait for 649-CP to dissociate, and then recommence elongation (Fig. 4a,b and Supplementary Movie 6). These observations show that the formation of the decision complex can occur from either direction. Thus, multiple lines of evidence suggest that the decision complex is the single kinetically significant intermediate in the CP/mDia1 antagonism mechanism, can be formed by starting from either a CP-capped, static barbed end or an mDia1-bound, growing barbed end, and can yield either of these barbed-end complexes as an outcome (Fig. 5b, magenta).

Bottom Line: Here we use multi-wavelength single-molecule fluorescence microscopy to observe the fully reversible formation of a long-lived 'decision complex' in which a CP dimer and a dimer of the formin mDia1 simultaneously bind the barbed end.Quantitative kinetic analysis reveals that the CP-mDia1 antagonism that we observe in vitro occurs through the decision complex.Our observations suggest new molecular mechanisms for the control of actin filament length and for the capture of filament barbed ends in cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454-9110, USA.

ABSTRACT
Precise control of actin filament length is essential to many cellular processes. Formins processively elongate filaments, whereas capping protein (CP) binds to barbed ends and arrests polymerization. While genetic and biochemical evidence has indicated that these two proteins function antagonistically, the mechanism underlying the antagonism has remained unresolved. Here we use multi-wavelength single-molecule fluorescence microscopy to observe the fully reversible formation of a long-lived 'decision complex' in which a CP dimer and a dimer of the formin mDia1 simultaneously bind the barbed end. Further, mDia1 displaced from the barbed end by CP can randomly slide along the filament and later return to the barbed end to re-form the complex. Quantitative kinetic analysis reveals that the CP-mDia1 antagonism that we observe in vitro occurs through the decision complex. Our observations suggest new molecular mechanisms for the control of actin filament length and for the capture of filament barbed ends in cells.

Show MeSH
Related in: MedlinePlus