Profilin and formin constitute a pacemaker system for robust actin filament growth
- Max Planck Institute of Molecular Physiology, Germany
- Institut Curie UMR144 CNRS, France
- TU Dortmund University, Germany
Figures
Figure 1 with 2 supplements
Filament assembly at physiological profilin-actin concentrations.
(A) Scheme of barbed end elongation from profilin-actin alone (top) or with formins (bottom). (B) Structural model of profilin at filament barbed ends (Materials and methods). The incoming …
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Figure 1—source data 1
Data Figure 1.
- https://doi.org/10.7554/eLife.50963.005
Figure 1—figure supplement 1
Quantification of profilin-actin levels and purification of mammalian profilin-actin.
(A) Profilin-actin concentration determination in mammalian cells. Left to right: name of cell type and origin, histograms of the single cell volume from fluorescence eXclusion measurements, …
Figure 1—figure supplement 2
Western blots and graphical summary of profilin-actin levels per cell.
Determination of protein-levels in different cell types by quantitative western blot analysis. For each cell type (HT1080, B16F10, BMDC, neutrophils, HT1080/B16F10 overexpressing profilin1 and …
Figure 2 with 1 supplement
A kinetic limit to actin filament elongation from profilin-actin.
(A) Scheme of TIRFM elongation assays of surface-attached filaments from profilin-actin on functionalized coverslips. (B) TIRFM time-lapse images (top) and kymographs (bottom) of filament elongation …
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Figure 2—source data 1
Data Figure 2.
- https://doi.org/10.7554/eLife.50963.008
Figure 2—figure supplement 1
Control experiments for barbed end polymerization in TIRF-M single filament assays.
(A) Calculations of profilin-actin complex and free profilin and actin concentrations [μM] (see inset) as a function of the total profilin-actin concentration (see Materials and methods). (B) …
Figure 3 with 1 supplement
Profilin release kinetically limits filament elongation.
(A) Scheme of barbed end elongation from profilin-actin alone indicating the potential limiting kinetic steps. (B) Structural models (Materials and methods) of the actin interface of stabilizing and …
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Figure 3—source data 1
Data Figure 3.
- https://doi.org/10.7554/eLife.50963.012
Figure 3—figure supplement 1
Measurements of profilin1-actin monomer association kinetics and characterization of a ultra-tight binding profilin1 mutant that blocks filament polymerization.
(A) Amino acid logos around K125/E129 or S71, for the top 50 profilin designs obtained from the G- or F-actin complexes. The plot represents the frequency with which each amino acid was found in the …
Figure 4 with 1 supplement
ATP hydrolysis is not required for profilin release from the barbed end.
(A) Nucleotide-binding site of filamentous actin. Left: the overall structure of filamentous actin. Right: Inset of the active site (PDBID 6FHL), including the three amino acids involved in …
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Figure 4—source data 1
Data Figure 4.
- https://doi.org/10.7554/eLife.50963.016
Figure 4—figure supplement 1
Affinity measurements of profilin1 to wt β-actin and ATPase-deficient actin.
(A) Binding of profilin1 to cytoplasmic actin, either wt (black dashed) or AD (cyan) measured by fluorescence anisotropy competition assays. Fluorescence anisotropy of Atto488-WAVE1WCA [4 nM] as a …
Figure 5 with 1 supplement
Formins accelerate filament elongation at saturating profilin-actin concentrations.
(A) Scheme of TIRFM assays with formin catalyzing the elongation of a filament from profilin-actin on functionalized coverslips. (B) Top: TIRFM time-lapse images of formin-mediated actin elongation …
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Figure 5—source data 1
Data Figure 5.
- https://doi.org/10.7554/eLife.50963.019
Figure 5—figure supplement 1
Profilin release but not ATP hydrolysis is limiting for formin-mediated actin polymerization.
(A) Fits showing the relative (x-fold) enhancement of the filament growth velocity obtained derived from hyperbolic fits to the raw data (Figure 5C) by either mDia1, mDia2 or DAAM1 as indicated. …
Figure 6 with 2 supplements
Formin single molecule imaging reveals buffered elongation rates in mammalian cells.
(A) Scheme of TIRFM imaging of single formins in the actin cortex of cells. (B) Maximum intensity projection of a TIRFM time-lapse shows growth trajectories of single mNeonGreen-mDia1 molecules in …
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Figure 6—source data 1
Data Figure 6.
- https://doi.org/10.7554/eLife.50963.025
Figure 6—figure supplement 1
Control experiments for single formin imaging in vivo.
(A) Left: Still images from TIRF imaging of surface-immobilized mNeongreen-mDia2 FH1-2 molecules from serial dilutions of HT1080 cell lysate at indicated dilution factors. Intensity distributions …
Figure 6—figure supplement 2
Profilin1-actin overexpression and in vivo formin speeds at different profilin1-actin levels.
(A) Profilin-actin concentration determination in mammalian HT1080 cells overexpressing profilin1 and β-actin. Left to right: name of cell type and origin, histograms of the single cell volume from …
Figure 7
Profilin release controls the speed of actin filament growth.
Kinetic scheme of the filament elongation cycle from profilin-actin either in the absence (top) or the presence (bottom) of formins. Reaction 1 and 2 are very fast at physiological profilin-actin …
Appendix 1—figure 1
Four reaction model for actin filament growth from actin monomers in the presence of profilin.
1)binding of profilin-actin complex to the terminal protomer, 2) profilin release from the terminal protomer, 3) binding of monomeric actin to the terminal protomer, 4) binding of profilin and …
Appendix 1—figure 2
Fit of growth velocity data as a function of [AP] for β-actin-profilin one from Figure 2C using either the Michaelis-Menten approximation (5) with vmax and KM as fit parameters (dashed blue) and its linear low-[AP] approximation (solid blue), the full result (3) with k1, k-1, k2, and k-2 as fit parameters (solid yellow) and with the additional constraint of detailed balance on the fit parameters (dotted green) and the low-[AP] approximations to these results (dashed yellow, solid green).
https://doi.org/10.7554/eLife.50963.034Videos
Video 1
Polymerization of actin filaments from different profilin1-actin concentrations.
Filaments were visualized with 10 nM Cy5-UTRN261 in TIRF-M. Polymerization from increasing profilin1-actin concentrations from left to right: 2.5 μM, 40 μM, 125 μM, 175 μM.
Video 2
Polymerization of actin filaments from different profilin1 mutant-actin complexes at 125 μM.
Filaments were visualized with 10 nM Cy5-UTRN261 in TIRF-M. Polymerization was performed from the following profilin1 mutant-actin complexes, left to right: profilin1-K125E + E129K, -wt, -E82A, -R88K.
Video 3
Polymerization of actin filaments from profilin1-actin at different concentrations in presence of mDia1 FH1-FH2.
Filaments were acquired in TIRF-M (filaments with 10 nM Cy5-UTRN261 - green; 0.7 nM TMR-mDia1 FH-FH2 – magenta). mDia1-mediated actin filament barbed end polymerization was performed at different …
Video 4
Polymerization of actin filaments from 75 μM profilin1-actin in presence/absence of formins.
Filaments were visualized with 10 nM Cy5-UTRN261 in TIRF-M. All filament barbed ends were saturated with 15 nM formin FH1-FH2. Polymerization was performed in presence of different formins, left to …
Video 5
mDia1 and mDia2 formin single molecule movement in HT1080 cells under conditions with either wt or overexpression of profilin1–actin.
mNeonGreen–mDia1/2 FH1-FH2 single molecules were visualized in TIRF-M. To indicate the cell shape, HT1080 cells were masked. Top: mDia1 (left) and mDia2 (right) molecules in wt HT1080 cells. Bottom: …
Video 6
In vivo mDia2 single molecule movement in absence/presence of latrunculinB, JASP and y27632.
To indicate the cell shape, HT1080 cells were masked. mNeonGreen-mDia2 FH1-FH2 single molecules were visualized in TIRF-M. mDia2 molecules were monitored without and after 10 min of drug treatment. …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | BL21 Star pRARE | EMBL Protein Expression Facility | Chemically competent cells | |
| Strain, strain background (Escherichia coli) | BL21 Rosetta | Novagen | Cat# 70954 | Chemically competent cells |
| Cell line (S. frugiperda) | SF9 | A. Musacchio, MPI Dortmund | RRID:CVCL_0549 | Cell line for virus generation |
| Cell line (T. ni) | TnaO38 | A. Musacchio, MPI Dortmund | RRID:CVCL_Z252 | Cell line for protein expression from baculovirus system |
| Cell line (Homo-sapiens) | HT1080 | ATCC | Cat# CCL-121, RRID:CVCL_0317 | Profilin and actin quantifications by WB, formin single molecule transfection |
| Cell line (Homo-sapiens) | B16F10 | ATCC | Cat# CRL-6475, RRID:CVCL_0159 | Profilin and actin quantifications by WB, formin single molecule transfection |
| Cell line (Homo-sapiens) | BMDC | Lab of M. Piel, Institut Curie, Paris | Profilin and actin quantifications by WB | |
| Cell line (M. musculus) | neutrophils | Lab of M. Piel, Institut Curie, Paris | Profilin and actin quantifications by WB | |
| Cell line (M. musculus) | EL4, T-lymphocytes | Lab of M. Taylor, MPI Berlin | Profilin and actin quantifications by WB | |
| Transfected construct | pΔCMV-mNeongreen-mDia1FH1-2 | This paper | Uniprot: O08808 | transfected construct, can be obtained in the lab of P. Bieling, MPI Dortmund |
| Transfected construct | pΔCMV-mNeongreen-mDia2FH1-2 | This paper | Uniprot: Q9Z207 | transfected construct, can be obtained in the lab of P. Bieling, MPI Dortmund |
| Transfected construct | pPBCAG-β-actin-P2A-mScarletI-T2A-profilin1 | This paper | transfected construct, can be obtained in the lab of P. Bieling, MPI Dortmund | |
| Antibody | anti-actin (mouse monoclonal) | ThermoFisher | Cat# MA5-11869, RRID:AB_11004139 | WB (1:1000) |
| Antibody | anti-profilin1 (mouse monoclonal) | Sigma Aldrich | Cat# 061M4892 | WB (1:20000) |
| Antibody | anti-profilin2 (mouse monoclonal) | Santa Cruz | Cat# sc-100955, RRID:AB_2163221 | WB (1:20000) |
| Antibody | anti-GAPDH (14C10) (rabbit monoclonal) | Cell Signaling | Cat# 2118, RRID:AB_561053 | WB (1:5000) |
| Antibody | anti-mouse (donkey polyclonal) | Licor | Cat# 925–32212, RRID:AB_2716622 | WB (1:10000) |
| Antibody | anti-rabbit (donkey polyclonal) | Licor | Cat# 926–68073, RRID:AB_10954442 | WB (1:10000) |
| Recombinant DNA reagent | pFL-h.s. β-actin_wt-linker-T4b (plasmid) | This paper | Uniprot: P60709 | β-actin insect cell expression, can be obtained in the lab of P. Bieling, MPI Dortmund |
| Recombinant DNA reagent | pFL-h.s. β-actin_Q137A_D154A_H161A-linker-T4b (plasmid) | This paper | β-actin insect cell expression, can be obtained in the lab of P. Bieling, MPI Dortmund | |
| Peptide, recombinant protein | Streptavidin | Sigma Aldrich | Cat. #: 189730 | For filament attachment |
| Chemical compound, drug | Latrunculin B | Sigma Aldrich | Cat. #: L5288 | For actin arrest |
| Chemical compound, drug | Y-27632 dihydrochloride | Sigma Aldrich | Cat. #: Y0503 | For actin arrest |
| Chemical compound, drug | jasplakinolide | Sigma Aldrich | Cat. #: J4580 | For actin arrest |
| Chemical compound, drug | phalloidin | Sigma Aldrich | Cat. #: P2141 | For actin arrest |
| Chemical compound, drug | 1,5-IAEDANS | Thermo Fisher | Cat. #: I14 | For actin labeling |
| Chemical compound, drug | EZ-Link Maleimide-PEG2-Biotin | Thermo Fisher | Cat. #: A39261 | For actin labeling |
| Chemical compound, drug | γ–32P–ATP (3000 Ci/mmol) | PerkinElmer | Cat. #: NEG002A | For ATPase assay |
| Chemical compound, drug | HO-PEG-NH2 and Biotin-CONH-PEG-O-C3-H6-CONHS | Rapp Polymere | # 103000–20 and # 133000-25-35 | For glass surface functionalization |
Appendix 1—table 1
Model parameters.
https://doi.org/10.7554/eLife.50963.031| kinetic parameter | value | reference |
|---|---|---|
| k3 | 11 μM-1s-1 | Pollard, 1986 and this work |
| k-3 | 1 s-1 | Pollard, 1986 |
| 0.58 s-1 | this work | |
| k4 | 40 μM-1s-1 | this work |
| k-4 | 0.75 s-1 | this work |
| k1 | 11 μM-1s-1 | Courtemanche and Pollard, 2013 |
| k-1 | 50 s-1 | Courtemanche and Pollard, 2013 |
| 5 s-1 | Pernier et al., 2016 |
Appendix 1—table 2
Results for rates k-2, k1 and KM from Michaelis-Menten fits of the growth velocity data in Figure 2C, Figure 3E (mutant profilin), and Figure 5C (with formins) in the main text.
https://doi.org/10.7554/eLife.50963.033| k-2 [s-1] | KM [μM] | k1 [μM-1s-1] | |
|---|---|---|---|
| β-actin-profilin | 558 ± 24 | 66 ± 3 | 8.4 ± 0.1 |
| β,γ-actin-profilin1 | 478 ± 31 | 54 ± 5 | 8.9 ± 0.3 |
| wt | 495 ± 15 | 57 ± 2 | 8.7 ± 0.1 |
| K125EE129K | 53 ± 7 | 26 ± 7 | 2.0 ± 0.3 |
| E82A | 609 ± 7 | 26.8 ± 0.7 | 22.7 ± 0.4 |
| R88K | 746 ± 21 | 35.7 ± 1.3 | 20.9 ± 0.3 |
| mdia1 | 1450 ± 167 | 22 ± 4 | 66 ± 5 |
| mdia2 | 820 ± 87 | 26 ± 4 | 31 ± 2 |
| daam1 | 573 ± 30 | 36 ± 3 | 16.1 ± 1.0 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.50963.029
