Modulation of formin processivity by profilin and mechanical tension
- Mikael Kerleau
- Emiko L. Suzuki
- Hugo Wioland
- Sandy Jouet
- Berengere Guichard
- Martin Lenz
- Guillaume Romet-Lemonne
- Antoine Jegou
- Institut Jacques Monod, CNRS, Université Paris Diderot, France
- LPTMS, CNRS, Université Paris-Sud, Université Paris-Saclay, France
Figures
Figure 1
Single-filament microfluidics experimental configurations to measure formin processivity.
(A–C) Different experimental configurations using microfluidics for the study of formin processivity, showing sketches of the side view (top) and typical kymographs of individual filaments (bottom). …
Figure 2 with 1 supplement
Impact of salt and actin labeling fraction on mDia1 formin processivity.
(A,B) Effect of salt concentration on the survival fraction of formin-bound barbed ends (A), on the formin dissociation rates (B, log-linear scale) as well as on the barbed end elongation rates (B, …
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Figure 2—source data 1
Spreadsheet containing the data plotted in Figure 2 and Figure 2—figure supplement 1.
Units and conditions are indicated in the figure legends. The data are on separate sheets, as indicated on the thumbnails.
- https://doi.org/10.7554/eLife.34176.008
Figure 2—figure supplement 1
Variation of the formin mDia1(FH1-FH2-DAD) dissociation rate as a function of profilin concentration, for 2 µM unlabeled actin at 50 mM KCl (red) or for 1 µM unlabeled actin at 100 mM KCl (blue), showing that formin processivity is decreased by profilin with unlabeled actin, for both salt conditions.
https://doi.org/10.7554/eLife.34176.007
Figure 3 with 2 supplements
Formin dissociation is enhanced by G-actin concentration, and slowed down by profilin.
(A, C) Variation of the mDia1 (A) or mDia2 (C) formin dissociation rate as a function of the barbed end elongation rate. Each data set (N = 30–40 filaments) was obtained with a fixed profilin …
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Figure 3—source data 1
Spreadsheet containing the data plotted in Figure 3, Figure 3—figure supplement 1, and Figure 3—figure supplement 2.
Units and conditions are indicated in the figures legends. The data are on separate sheets, as indicated on the thumbnails.
- https://doi.org/10.7554/eLife.34176.012
Figure 3—figure supplement 1
Variation of the mDia1 formin-bound or free barbed end elongation rate velong as a function of actin concentration.
Each data point corresponds to an independent experiment (N = 30–40 filaments) which was conducted at 100 mM KCl. Error bars indicate standard deviations.
Figure 3—figure supplement 2
Mutant profilin-R88E has no impact on the processivity of mDia1.
Survival fraction of mDia1 formin-anchored filaments (N = 30 for each curve), exposed to negligible force (<0.1 pN) while elongating with 1 µM actin and either zero (blue), 10 µM (gray) or 20 µM …
Figure 4 with 3 supplements
Force has a great impact on formin processivity.
(A) Sketch of the experimental configuration, similar to that of Figure 1C, but where significant forces are applied using various flow rates. The applied force scales with the filament length. (B) …
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Figure 4—source data 1
Spreadsheet containing the data plotted in Figure 4, Figure 4—figure supplement 1, Figure 4—figure supplement 2, and Figure 4—figure supplement 3.
Units and conditions are indicated in the figures legends. The data are on separate sheets, as indicated on the thumbnails.
- https://doi.org/10.7554/eLife.34176.017
Figure 4—figure supplement 1
C-terminus anchored mDia1 (FH1-FH2-DAD) formin renucleation.
Nucleation of new filaments from surface-anchored mDia1 formins. The anchored mDia1(FH1-FH2-DAD) formins that participated in a pulling force experiment with 1 µM actin 4 µM profilin in a moderate …
Figure 4—figure supplement 2
mDia1 formin dissociates faster with force.
mDia1 formin dissociation rate as a function of applied force (log-linear plots): for different actin concentrations in the absence of profilin (left); for 1 µM actin with different profilin …
Figure 4—figure supplement 3
mDia2 formin elongation rate is mechanosensitive.
C-terminus anchored mDia2 (FH1-FH2-DAD) formin elongation rate as a function of the applied tension for 2 µM unlabeled actin in absence (left) or in presence of 4 µM profilin (right), at 100 mM KCl. …
Figure 5
Mean Filament Length generated by mDia1 or mDia2 formins.
Mean flament length (A) as a function of actin concentration, in the absence of profilin, normalized by its value at 1 µM actin (which equals 0.65 µm for mDia1 and 8.22 µm for mDia2); (B) mean …
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Figure 5—source data 1
Spreadsheet containing the data plotted in Figure 5.
Units and conditions are indicated in the figures legends. The data are on separate sheets, as indicated on the thumbnails.
- https://doi.org/10.7554/eLife.34176.019
Figure 6
Modeling formin dissociation, in the absence of profilin.
(A) Sketch summarizing the conformations adopted by the FH2 dimer and the actin filament barbed end in our model, in the absence of profilin (for a complete description of the model, see Appendix …
Appendix 1—figure 1
Model of formin function based on transitions between discrete states, as described in the text.
Unlike those of the main text, the schematics presented here only picture one actin protofilament for simplicity, without any implications for the model itself. In both panels, formin dissociation …
Appendix 1—figure 2
Predictions for the elongation velocity (a) and the formin dissociation rate (b) as functions of the profilin concentration within the approximation of strong actin-profilin binding of Eq.(A13).
While the exact position of the curves is dependent on the choice of parameters as indicated on the figure, their qualitative shapes are a robust prediction of the model, and agree well with the …
Videos
Video 1
Actin filaments elongating with 1 µM 15% Alexa 488-labeled actin and 5 µM profilin, at 100 mM KCl, are transiently exposed to a solution of 20 nM mDia1(FH1-FH2-DAD) for 20 s (during frames 27–30).
Images were acquired in TIRF. Full field of view is 137 × 137 µm. Interval between images is 5 s (movie is accelerated 75x). The solution flows from left to right. Corresponds to Figure 1A of the …
Video 2
Actin filaments are exposed to a periodic alternation of a solution of unlabeled actin (0.3 µM actin, 50 mM KCl) for 100 s and a solution of 15% Alexa 488-labeled actin (0.5 µM actin +2 µM profilin, 50 mM KCl) for 20 s.
The filaments are transiently exposed to a solution of 11 nM mDia1(FH1-FH2-DAD) for 5 s, after frame number 5. Images were acquired in epifluorescence while exposing to unlabeled actin. Full field …
Video 3
Actin filaments were nucleated from surface-anchored formins mDia1(FH1-FH2-DAD) with 15% Alexa 488-labeled actin, and elongate with 0.3 µM unlabeled actin, at 50 mM KCl.
Full field of view is 221 × 221 µm. Images were acquired in epifluorescence. Interval between images is 10 s (movie is accelerated 70x). A minimal flow is applied. The solution flows from left to …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (E. coli) | BL21(DE3) | Thermo Fischer | Cat# C600003 | |
| Biological sample (Rabbit) | Rabbit muscle | INRA Jouy-en-Josas | N/A | |
| Peptide, recombinant protein | Mouse mDia1(FH1-FH2-DAD) | Uniprot | O08808 | seq. 552–1255 aa |
| Peptide, recombinant protein | Mouse mDia2(FH1-FH2-DAD) | Uniprot | Q9Z207 | seq. 521–1171 aa |
| Peptide, recombinant protein | Human profilin-1 | Uniprot | P07737 | |
| Antibody | PentaHis Biotin conjugate | Qiagen | 34440 | |
| Chemical compound, drug | Alexa Fluor 488 succinimidyl ester | Life Technologies | Cat#A20000 | |
| Commercial assay or kit | Protino Ni-NTA Agarose beads | Macherey-Nagel | Cat#745400.25 | |
| Commercial assay or kit | HiLoad 16/60 Superdex 200 gel filtration column | GE Healthcare | Cat#28-9893-35 | |
| Software, algorithm | numpy/scipy packages | Python |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.34176.021
