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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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mDia1/CP/actin filament barbed-end complex (N=171 complexes observed).(a) Merged three-colour TIRF image of the actin filament (blue) in Supplementary Movie 3, showing 549-mDia1 (yellow) and 649-CP (red) molecules bound simultaneously to the barbed end. Inset: magnified and filtered view of the indicated barbed end. (b) Fluorescence intensity and filament length record of the filament in a. Colour ribbon indicates the time intervals in which the barbed end is occupied by 549-mDia1 only (green), 649-CP only (red) or both proteins (magenta). Insets: three-colour merged images of the barbed end using the same colour scheme as in a, taken at the indicated times. Scale bars in a and b, 2 μm. Frame interval: 5 s. (c) Possible mechanisms for CP antagonism of mDia1-catalysed filament elongation. mDia1 (orange) elongates actin filaments (blue) by incorporating actin subunits (green arrows). The dissociative pathway requires the formin to first dissociate before CP (red) can bind the barbed end; the associative pathway allows CP to bind a formin-occupied barbed end before formin dissociation. (d,e) Example filament length records from actin polymerization experiments containing 549-mDia1 alone (d) or 549-mDia1 plus 649-CP (e). Traces are coloured to indicate which proteins are visible at the barbed end, using the same colour scheme as in the ribbon in b. Percentages indicate the fraction of 549-mDia1/649-CP/barbed-end complexes (out of N=171) that ended by loss of 549-mDia1 fluorescence (top) or loss of 649-CP fluorescence (bottom). Percentages do not sum to 100 because some the dissociation of some complexes could not be unambiguously scored due to overlap with the images of other filaments. Inset: mean elongation rate in filament record segments classified by which barbed-end proteins were present. The experiments in this and all following figures used 0.5–1 μM actin in the presence of 1.5–3 μM profilin.
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f3: mDia1/CP/actin filament barbed-end complex (N=171 complexes observed).(a) Merged three-colour TIRF image of the actin filament (blue) in Supplementary Movie 3, showing 549-mDia1 (yellow) and 649-CP (red) molecules bound simultaneously to the barbed end. Inset: magnified and filtered view of the indicated barbed end. (b) Fluorescence intensity and filament length record of the filament in a. Colour ribbon indicates the time intervals in which the barbed end is occupied by 549-mDia1 only (green), 649-CP only (red) or both proteins (magenta). Insets: three-colour merged images of the barbed end using the same colour scheme as in a, taken at the indicated times. Scale bars in a and b, 2 μm. Frame interval: 5 s. (c) Possible mechanisms for CP antagonism of mDia1-catalysed filament elongation. mDia1 (orange) elongates actin filaments (blue) by incorporating actin subunits (green arrows). The dissociative pathway requires the formin to first dissociate before CP (red) can bind the barbed end; the associative pathway allows CP to bind a formin-occupied barbed end before formin dissociation. (d,e) Example filament length records from actin polymerization experiments containing 549-mDia1 alone (d) or 549-mDia1 plus 649-CP (e). Traces are coloured to indicate which proteins are visible at the barbed end, using the same colour scheme as in the ribbon in b. Percentages indicate the fraction of 549-mDia1/649-CP/barbed-end complexes (out of N=171) that ended by loss of 549-mDia1 fluorescence (top) or loss of 649-CP fluorescence (bottom). Percentages do not sum to 100 because some the dissociation of some complexes could not be unambiguously scored due to overlap with the images of other filaments. Inset: mean elongation rate in filament record segments classified by which barbed-end proteins were present. The experiments in this and all following figures used 0.5–1 μM actin in the presence of 1.5–3 μM profilin.

Mentions: Significantly, the transition from a growing, mDia1-associated end to a non-growing CP-associated end proceeded through a characteristic sequence of events. We never (0% of N=40 mDia1-bound barbed ends) observed 549-mDia1 dissociation followed by continuing filament growth and subsequent 649-CP binding. Instead, we almost always (93%) saw that capping occurred through the formation of an unexpected long-lived ternary complex between 549-mDia1, 649-CP and the barbed end (Fig. 3a,b and Supplementary Movies 3 and 4). (In the remaining 7% of events, the transition of the barbed end from 549-mDia1 bound to 649-CP bound happened too quickly to discern the order of the dissociation and binding events.) The high proportion of events in which CP was observed to bind before departure of mDia1 from the barbed end suggested that CP usually or always acts on barbed ends being elongated by mDia1 through an ‘associative competition' mechanism (Fig. 3c, bottom), rather than the long-presumed dissociative mechanism (Fig. 3c, top). Differentiating between these two mechanisms is critical, because they have profoundly different implications for cellular function, including the duration of formin ‘runs' on filament ends, the regulation of filament length distribution in actin networks and the ability of localized CP to capture the barbed ends of growing filaments (see Discussion).


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)

mDia1/CP/actin filament barbed-end complex (N=171 complexes observed).(a) Merged three-colour TIRF image of the actin filament (blue) in Supplementary Movie 3, showing 549-mDia1 (yellow) and 649-CP (red) molecules bound simultaneously to the barbed end. Inset: magnified and filtered view of the indicated barbed end. (b) Fluorescence intensity and filament length record of the filament in a. Colour ribbon indicates the time intervals in which the barbed end is occupied by 549-mDia1 only (green), 649-CP only (red) or both proteins (magenta). Insets: three-colour merged images of the barbed end using the same colour scheme as in a, taken at the indicated times. Scale bars in a and b, 2 μm. Frame interval: 5 s. (c) Possible mechanisms for CP antagonism of mDia1-catalysed filament elongation. mDia1 (orange) elongates actin filaments (blue) by incorporating actin subunits (green arrows). The dissociative pathway requires the formin to first dissociate before CP (red) can bind the barbed end; the associative pathway allows CP to bind a formin-occupied barbed end before formin dissociation. (d,e) Example filament length records from actin polymerization experiments containing 549-mDia1 alone (d) or 549-mDia1 plus 649-CP (e). Traces are coloured to indicate which proteins are visible at the barbed end, using the same colour scheme as in the ribbon in b. Percentages indicate the fraction of 549-mDia1/649-CP/barbed-end complexes (out of N=171) that ended by loss of 549-mDia1 fluorescence (top) or loss of 649-CP fluorescence (bottom). Percentages do not sum to 100 because some the dissociation of some complexes could not be unambiguously scored due to overlap with the images of other filaments. Inset: mean elongation rate in filament record segments classified by which barbed-end proteins were present. The experiments in this and all following figures used 0.5–1 μM actin in the presence of 1.5–3 μM profilin.
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f3: mDia1/CP/actin filament barbed-end complex (N=171 complexes observed).(a) Merged three-colour TIRF image of the actin filament (blue) in Supplementary Movie 3, showing 549-mDia1 (yellow) and 649-CP (red) molecules bound simultaneously to the barbed end. Inset: magnified and filtered view of the indicated barbed end. (b) Fluorescence intensity and filament length record of the filament in a. Colour ribbon indicates the time intervals in which the barbed end is occupied by 549-mDia1 only (green), 649-CP only (red) or both proteins (magenta). Insets: three-colour merged images of the barbed end using the same colour scheme as in a, taken at the indicated times. Scale bars in a and b, 2 μm. Frame interval: 5 s. (c) Possible mechanisms for CP antagonism of mDia1-catalysed filament elongation. mDia1 (orange) elongates actin filaments (blue) by incorporating actin subunits (green arrows). The dissociative pathway requires the formin to first dissociate before CP (red) can bind the barbed end; the associative pathway allows CP to bind a formin-occupied barbed end before formin dissociation. (d,e) Example filament length records from actin polymerization experiments containing 549-mDia1 alone (d) or 549-mDia1 plus 649-CP (e). Traces are coloured to indicate which proteins are visible at the barbed end, using the same colour scheme as in the ribbon in b. Percentages indicate the fraction of 549-mDia1/649-CP/barbed-end complexes (out of N=171) that ended by loss of 549-mDia1 fluorescence (top) or loss of 649-CP fluorescence (bottom). Percentages do not sum to 100 because some the dissociation of some complexes could not be unambiguously scored due to overlap with the images of other filaments. Inset: mean elongation rate in filament record segments classified by which barbed-end proteins were present. The experiments in this and all following figures used 0.5–1 μM actin in the presence of 1.5–3 μM profilin.
Mentions: Significantly, the transition from a growing, mDia1-associated end to a non-growing CP-associated end proceeded through a characteristic sequence of events. We never (0% of N=40 mDia1-bound barbed ends) observed 549-mDia1 dissociation followed by continuing filament growth and subsequent 649-CP binding. Instead, we almost always (93%) saw that capping occurred through the formation of an unexpected long-lived ternary complex between 549-mDia1, 649-CP and the barbed end (Fig. 3a,b and Supplementary Movies 3 and 4). (In the remaining 7% of events, the transition of the barbed end from 549-mDia1 bound to 649-CP bound happened too quickly to discern the order of the dissociation and binding events.) The high proportion of events in which CP was observed to bind before departure of mDia1 from the barbed end suggested that CP usually or always acts on barbed ends being elongated by mDia1 through an ‘associative competition' mechanism (Fig. 3c, bottom), rather than the long-presumed dissociative mechanism (Fig. 3c, top). Differentiating between these two mechanisms is critical, because they have profoundly different implications for cellular function, including the duration of formin ‘runs' on filament ends, the regulation of filament length distribution in actin networks and the ability of localized CP to capture the barbed ends of growing filaments (see Discussion).

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