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Effects of mechanical stimuli on profilin/formin-mediated actin polymerization Miao Yu, Shimin Le, Artem K. Efremov, Xiangjun Zeng, Alexander D. Bershadsky, and Jie Yan Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b02211 • Publication Date (Web): 06 Jul 2018 Downloaded from http://pubs.acs.org on July 8, 2018

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Effects of mechanical stimuli on profilin/formin-mediated actin polymerization Miao Yu1, 2, #, Shimin Le1, 2, #, Artem K. Efremov1, Xiangjun Zeng2, Alexander Bershadsky1, 3 and Jie Yan1, 2, 4, * 1

Mechanobiology Institute, National University of Singapore, Singapore 117411; Department of Physics, National University of Singapore, Singapore 117542; 3Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel; 4Centre for Bioimaging Sciences, National University of Singapore, Singapore 117546. 2

#

These two authors contributed equally to this work. To whom the correspondence may be addressed: [email protected] (YJ).

*

ACS Paragon Plus Environment

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ABSTRACT Self-assembling actin filaments not only form the basis of the cytoskeleton network in cells but also are utilized as nano building blocks to make novel active matters, where dynamic polymerization and depolymerization of actin filaments play a key role. Formins belong to a main family of actin nucleation factors that bind to the barbed end of actin filament and regulate actin polymerization through an interaction with profilin. Due to actomyosin contractility and relative rotation between formin and actin filament, formin-dependent actin polymerization is subject to force and rotation constraints. However, it remains unclear how force and rotation constraints affect formin-dependent actin polymerization in the presence of profilin. Here, we show that for rotation-unconstrained actin filaments, elongation is accelerated by both force and profilin. The combined effect leads to surprisingly fast actin elongation that can approach the diffusion limited rate at forces of a few pN. The elongation of rotation-constrained filaments is also accelerated by profilin, while it is insensitive to applied force. We show that FH2, the main actin binding domain plays the primary mechanosensing role. Together, the findings not only significantly advance our understanding of the mechano-chemical regulation of formin-mediated actin polymerization in cells, but also can potentially be utilized to make novel actin based active matters.

Keywords: Formin, actin, profilin, force, rotation, magnetic tweezers

The actin cytoskeleton is involved in various physiological and pathological functions, such as cell migration, differentiation, embryo development, and cancer metastasis 1, 2. The highly dynamic organization of the actin cytoskeleton is tightly controlled by a variety of proteins that regulate actin nucleation, polymerization, de-polymerization, branching, bundling, and localization

3, 4

. Besides the crucial roles in vivo, the actin networks mediated by these


2

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remodeling proteins have also been inspired to design nano-scale building blocks to make actin-based active matters whose structure and stiffness can be actively modified by controlling these proteins 5-9.

The formin family proteins play critical roles in promoting nucleation and regulating actin polymerization. Formin forms a homodimer through its FH2 homology domains, and these dimerized FH2 domains form a ring-like shape, which encircles the barbed end of an actin filament 10. This FH2 ring slows down the recruitment of G-actin at the encircled barbed end compared with free barbed ends

10, 11

. The N-terminus of each FH2 monomer is linked to an

FH1 domain containing multiple polyproline tracks that interact with the actin-binding protein profilin with an affinity of ~16.5 µM 12, 13. Although the FH1 domain is believed to be intrinsically disordered, this has not been confirmed directly in experiments. Profilin has a higher affinity for G-actin (Kd ~ 0.21 µM

14-16

); therefore, the two FH1 domains of a formin

homodimer has been proposed to enrich the local concentration of G-actin near the vicinity of the barbed end of actin filaments, facilitating actin polymerization

11

. Actin polymerization

depends on the complex interplay between the formin FH1 domain that promotes actin polymerization and the FH2 ring that suppresses actin polymerization. In addition, previous studies revealed that actin polymerization in the presence of profilin biphasically depends on the profilin:G-actin ratio 11, 13, 17-21.

Actin filaments are subject to mechanical stretch generated by actomyosin contraction in vivo 22

, and the resulting tensile force is transmitted to formin in the case of formin dependent

actin polymerization 23. Previous structural studies have predicted that formin rotates relative to the actin filament around the filament axis 10, and this has been demonstrated in the case of mDia1∆N3-dependent polymerization 24. Actin filaments in vivo are highly cross-linked and


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therefore unable to freely rotate 25, 26. Therefore, if formin is linked to a cellular structure that prevents formin rotation, then a torsion stress could build up during actin polymerization. In contrast, if formin is linked to a cellular structure that allows formin rotation, such torsion stress would not accumulate.

In our previous work

27

, we investigated the effects of force applied to formin on the actin

polymerization rate in the absence of profilin in order to understand the mechanosensing function of the FH2 domain. Using mDia1 lacking the DAD and DID domains (mDia1∆N3), we observed a force-sensitive species and a force-insensitive species, which correspond to rotation-unconstrained actin filaments and rotation-constrained actin filaments, respectively. Due to the absence of the profilin in that experiment, the result indicates that the FH2 domain can sense force and torque during actin polymerization. However, in vivo formin-dependent actin polymerization relies on profilin that binds the polyproline tracks in the FH1 domains, while

how force

and

rotation

constraints

affect

formin/profilin-dependent actin

polymerization is unclear. Previous studies reported that force of a few pN overall accelerates formin/profilin-dependent actin polymerization

17, 18, 28

, but it remains unclear whether it is

through force-sensing by FH2, or force-dependent binding between FH1 and profilin, or both.

In order to provide a deeper understanding of formin/profilin-dependent actin polymerization under force and rotation constraints, we tethered a single actin filament encircled by a mDia1∆N3 between a coverslip surface and a superparamagnetic bead, and recorded the extension change of the actin filament under different forces with or without rotational constraint using magnetic tweezers, in different profilin:G-actin ratios. In addition, in order to determine the potential mechanosensing role of FH1, we also investigated the force-response of the FH1 domain and probed its binding to profilin under force.


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We find that in the presence of profilin, actin polymerization can still be categorized into two species, a force-insensitive species for rotation-constrained actin filaments and a forcesensitive species for rotation-unconstrained actin filaments. In both cases, the actin polymerization rate biphasically depends on the profilin:G-actin ratio. Notably, for the forcesensitive species, introduction of 2 µM profilin to 500 nM G-actin results in surprisingly fast actin polymerization with rates > 600 subunit s-1 at forces above 2 pN. Furthermore, we developed a theoretical framework on the effects of profilin on the local concentration of Gactin near the formin-capped barbed end of an actin filament, and how it affects the net rate of G-actin recruited to the barbed end. The results show that weak profilin binding to FH1 is capable of affecting the G-actin local concentration and the actin elongation rate in a biphasic manner, consistent with the experimental observations. In addition, our result shows that the conformation of the FH1 domain can be described as a randomly coiled disordered peptide. Together, these results provide further insights into the mechano-chemical regulations of formin-dependent actin polymerization.

mDia1∆N3-mediated actin polymerization under mechanical constraints in the presence of profilin. Tensile forces were applied to actin filaments tethered in several different ways using a transverse magnetic tweezers setup (Fig. 1a). The actin filaments are linked to an immobilized NEM-NMII coated 3-µm polystyrene bead on the coverslip surface via a region somewhere near the tipped end of the actin filament. The barbed end is associated with either a GST-mDia1∆N3 homodimer or a biotin-mDia1∆N3 homodimer (1-µm-diameter MyOne bead or 2.8-µm-diameter M-280 Dynal bead (Methods)). The GST-mDia1∆N3 homodimer is linked to an anti-GST coated superparamagnetic bead, while the biotin-mDia1∆N3 homodimer is linked to a streptavidin coated superparamagnetic bead, or to an anti-


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digoxigenin coated superparamagnetic bead through a rotationally unconstrained DNA linker. For both cases, force is applied to mDia1∆N3 and actin through the superparamagnetic bead. The elongation of an actin filament as a result from actin polymerization at the barbed end, indicated by the displacement of the bead, was tracked at a nanometer resolution in real-time.

Fig. 1. mDia1∆N3 mediated actin polymerization under force in the presence of profilin. (a) Schematics of experimental design. The actin filament is tethered to an immobilized NEM-NMII coated polystyrene-bead on a coverslip through a point on the actin filament near the tipped end. The barbed end of the actin filament is attached to an anti-GST-Protein A coated superparamagnetic bead through a GSTtagged mDia1∆N3 (I and II), or to a streptavidin coated superparamagnetic bead through a biotin-tagged mDia1∆N3 without (III) or with (IV) a torsion-relaxing DNA linker. The actin filament is stretched at around 6o - 10o above the surface using transverse magnetic tweezers. (b&c) Representative elongation time courses of rotationally constrained (b) and unconstrained (c) actin filaments in 500 nM G-actin and 2 µM profilin. The applied forces and resulting elongation rates are color-indicated.

Each mDia1∆N3 dimer carries two tags at the N-termini of the two FH1 domains, therefore, either one or both of the FH1 domains are linked to an anti-GST-Protein A (in the case of GST-mDia1∆N3) or to a streptavidin (in the case of biotin-mDia1∆N3). Previous studies have suggested that a polymerizing actin filament should rotate relative to the FH2 ring encircled at the barbed end of the filament

10, 24

. Since the rotation of the actin filament is


6

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inhibited on the NEM-NMII coated bead surface and since the rotation of the superparamagnetic bead is also inhibited by the magnetic field, torsion stress will build up when the rotation of mDia1∆N3 relative to the actin filament is inhibited. Therefore, torsion stress may build up during actin polymerization in the case of double-FH1 tethering for both GSTmDia1∆N3 and biotin-mDia1∆N3 that are directly linked the superparamagnetic bead. In contrast, accumulation of torsion stress does not occur in the case of single-FH1 tethering, or in the presence of a rotationally unconstrained DNA-linker between biotin-mDia1∆N3 and the bead for both single-FH1 and double-FH1 tethering geometry. As suggested in our previous studies of mDia1∆N3 mediated actin polymerization in the absence of profilin

27

,

rotationally constrained polymerization is insensitive to force, while the rotationally unconstrained polymerization is sensitive to force. For rotationally unconstrained polymerization, increasing force applied to mDia1∆N3 from near zero to above 5 pN resulted in an increase the polymerization speed of ~ 7 times. Our previous results suggest that in the case of GST-mDia1∆N3, both single-FH1 tethering and double-FH1 tethering occur with significant fractions; while in the case of biotin-mDia1∆N3, double-FH1 tethering to streptavidin is predominant.

Fig.1 b&c show representative time traces of elongation of actin filaments in 500 nM G-actin and 2 µM profilin, for rotationally constrained cases (Fig. 1b) and rotationally unconstrained cases (Fig. 1c), respectively. The slope of the elongation at each force indicates the rate of net recruitment of G-actin to the barbed end. The force-dependence of polymerization rate for both cases was obtained from multiple independent experiments (Fig. 2, blue). The results clearly show that the actin polymerization rate is force-insensitive for rotationally constrained filaments, while it is force-sensitive for rotationally unconstrained filaments. The observed trend of force-dependence is similar to that observed in the absence of profilin, suggesting


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that profilin does not alter the nature of the response of mDia1∆N3-mediated actin polymerization to force and rotation constraints through the FH2 domain.

Effects of profilin on the force-dependent mDia1∆N3-mediated actin polymerization. While the results in the previous section show that profilin does not alter the nature of the response of mDia1∆N3-mediated actin polymerization to force and rotational constraints, the polymerization rates for both rotationally constrained and unconstrained cases in 2 µM profilin are around six folds faster than the corresponding values observed in the absence of profilin

27

. In order to understand the effects of profilin concentration on mDia1∆N3-

mediated actin polymerization under force and rotational constraints, we performed experiments in 500 nM G-actin at several different profilin concentrations.

Fig. 2a shows the polymerization rates obtained for rotationally unconstrained actin filaments in 500 nM G-actin together with 0 nM (red), 500 nM (light blue), 2 µM (blue) or 5 µM profilin (black), respectively. A significant increase in the polymerization rate was observed at profilin concentrations of 500 nM and 2 µM profilin, while the polymerization rate with 5 µM profilin was similar to that observed in the absence of profilin. The results indicate a biphasic dependence of profilin:G-actin concentration ratio on the actin polymerization rate. A similar biphasic dependence of profilin:G-actin concentration ratio on mDia1∆N3mediated actin polymerization was also observed for rotationally constrained actin filaments, although all the rates with profilin are faster than that without (Fig. 2b).


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Fig. 2. Force-dependent F-actin elongation at different profilin:G-actin concentration ratios. The data show the actin elongation rate as a function of force for rotationally unconstrained (a) and constrained (b) filaments in 500 nM G-actin, at four different profilin concentrations: 0 nM (red), 500 nM (light blue), 2µM (blue) and 5µM (black). Vertical error bars represent standard errors obtained from multiple independent experiments at corresponding forces. Horizontal error bars represent the 20% uncertainty in force calibration. The number labelled on each data point indicates the number of independent experiments carried out at the corresponding force.

It is possible that the observed effects of profilin can be explained by a profilin-dependent modulation of the effective local G-actin concentration near the barbed end. This explanation implies that the effect of a given profilin concentration in 500 nM G-actin on the actin filament polymerization rate should be similar to a different G-actin concentration in the absence of profilin. This is supported by the results in Fig. 3, which show that the force dependent mDia1∆N3- mediated actin polymerization rate in 2 µM G-actin in the absence of profilin is similar to that observed in 500 nM G-actin and 2 µM profilin. This result suggests that introducing 2 µM profilin in 500 nM G-actin is equivalent to increasing the effective local concentration of G-actin to 2 µM in terms of promoting actin filament polymerization.


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Fig. 3. F-actin elongation at different G-actin concentrations. The force-dependent actin elongation rate for rotationally unconstrained (a) and constrained (b) actin filaments at 500 nM G-actin in the presence (blue), or in the absence (red) of 2 µM profilin, or at 2 µM G-actin in the absence of profilin (dark red). Vertical error bars represent standard errors obtained from multiple independent experiments at corresponding forces. Horizontal error bars represent the 20% uncertainty in force calibration. The number labelled on each data point indicates the number of independent experiments carried out at the corresponding force.

The force response of formin FH1 domain. The proline-rich FH1 domain plays a critical role in the formin-dependent actin polymerization, which is predicted to be unstructured based on previous bioinformatics studies

4, 29, 30

. However, whether it assumes a compact

folded conformation that suppresses profilin binding or takes an entropic randomly coiled polymer conformation that allows better accessibility to profilin remains unclear. The FH1 domain (583 a.a.-764 a.a., from human DIAPH1) has 14 proline rich tracks, which provide binding sites for profilin. It has been proposed that these profilin binding sites on the FH1 domain play a role in enriching the local G-actin concentration through an interaction between profilin and G-actin11, 19-21, facilitating formin-mediated actin filament elongation. In vivo, the FH1 domain is also subject to mechanical stretching. However, how FH1 responds to force and how its interaction with profilin is affected by force remains unclear. In this


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section, we sought to provide some insights into these questions by investigating the forceextension curve of the FH1 domain in the absence and presence of profilin.

Fig. 4a shows the schematics of the experimental design where the FH1 domain is subject to forces applied using a vertical magnetic tweezers setup. The FH1 domain is spanned between two titin I27 domains at each side which serve as molecular spacers and as a positive control based on its characteristic force-dependent unfolding tethered

bead

were

obtained

using

both

31, 32

. The force-height curves of the

2.8-µm-diameter

and

1-µm-diameter

superparamagnetic beads. The larger bead is capable of applying larger force, but it suffers from greater influence of the bead rotation due to an off-center attachment which varies significantly among different tethers (Fig. S1). While the smaller bead has a limited force range up to ~ 8 pN, the resulting force-height data are less influenced by bead rotation, better reflecting the force-extension curve of the tether.

Representative force-height data obtained using the 1-µm-diameter superparamagnetic bead measured in KMEI buffer (Methods) are shown in Fig. 4b. No abrupt extension changes were observed within the force range. Similar data were obtained from 14 independent tethers. In addition, the force-height data can be fitted by the worm-like-chain (WLC) polymer model with a certain bending persistence length. Best fitting of the WLC model to the data estimates the number of residues of the unstructured region to be 184±20 a.a., with a bending persistence length of 0.8±0.1 nm, where the errors are standard deviations from 14 independent measurements. These results indicate that the FH1 domain does not contain detectable folded units over this force range, which is consistent with a previous prediction that FH1 is unstructured

4, 29, 30

. Overall, this result suggests that the force response of FH1

within the physiological force range can be described by simple polymer models.


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Fig. 4. Force-response of the FH1 domain. (a) Schematic of the FH1 domains in a mechanically stretched formin (left panel) and the construct (I272FH1I272) that contains a single FH1 constructed for the single molecule manipulation study (middle panel). The two titin I27 domains at each side of the FH1 domain serve as a molecular spacer and positive control. Schematic of a mechanically stretched FH1 domain using magnetic tweezers (right panel). The height of bead from the surface was recorded in real time at a nanometer resolution. (b) A representative force-height curve of a I272FH1I272 tether during force-increase/decrease scans with a loading rate of 0.1 pN s-1 (-0.1 pN s-1) using a 1-µm-diameter superparamagnetic bead. The blue and cyan lines represent the best fitting of the data (>3 pN) using the WLC model, with A and N as free parameters (blue), or by fixing N=182 a.a. (cyan), respectively. The average and standard deviation of the persistence length and number of residues obtained from the WLC fitting with A and N as free parameters (14 independent experiments) are shown in the panel. The WLCfitting was done for data >3 pN, and was extrapolated to lower forces. (c) A representative force-height curve of an I272FH1I272 during force-increase scans (magenta and red), and a force-decrease scan (orange) with loading rates of ± 2.7 pN s-1 using a 2.8-µm-diameter superparamagnetic bead. Small unfolding steps (arrows) with step-sizes ~ 3.5 nm are observed at forces of ~30-40 pN. Top right inset shows an example


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curve of unfolding of the four I27 at high forces (~ 100 pN) during force-increase scan; Top left inset shows zoom-in of small step fluctuations during force-increase scans; Bottom inset shows the normalized step-size histogram of the small steps (N=258). (d) Representative force-height curves of an I272FH1I272 tether obtained using a 2.8-µm-diameter superparamagnetic bead before and after introducing profilin at a concentration of 5 µM or 20 µM. Similar experiments of FH1 stretching before and after profilin introduction were repeated six times (Fig. S3).

In order to probe the force-response of FH1 at larger forces, the force-extension data of the FH1 were also obtained at greater forces using a 2.8-µm-diameter superparamagnetic bead. Although the measurement is typically influenced by bead rotation during force change, it is possible to search for tethers that are not significantly affected by bead rotation (Fig. S1). As shown in the representative data in Fig. 4c from such a tether, at forces above ~30 pN, small stepwise extension increases with step-sizes of ∆ ~ 3.5 nm were observed. Repeating the experiments on more than ten independent tethers reveals that such stepwise extension change is highly characteristic in both step size and the force range where they occur (Fig. S2), which suggest the existence of a regular structural domain in FH1. Dynamic fluctuation between the two conformations of this potential structural domain were investigated at constant forces, from which a critical force of Fc ~ 42 pN corresponding to 50% probability of folding is determined. A folding energy of this structure can be roughly estimated by

× ∆~30 kBT. While the nature of these small unfolding signals is unclear, we suspect that they might be from the transition of polyproline helices of the proline-rich tracks into a disordered peptide chain.

If profilin binding can cause a conformational change to FH1, then it would likely lead to changes in the force-extension curve of the tether, which may be detected experimentally. To explore this possibility, we measured the force-height data of FH1 in different concentrations


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of profilin. Apparent changes in the force-height data were only observed at concentrations greater than 10 µM (Fig. 4d, Fig. S3), where the bead height significantly increases from the level recorded on the same tether before introducing the profilin at low forces (< 7 pN) but not at higher forces. Such behaviour can be explained by ligand binding induced stiffening of a flexible polymer without changing the contour length

33, 34

. Due to the profilin binding

induced stiffening of FH1, one may expect that force may increase the binding affinity between FH1 and profilin to a certain extent.

Effects of FH1 and profilin on the local concentration of G-actin and actin elongation rate. Previous experimental studies in the absence or presence of force applied to formin/actin filament revealed that at given G-actin concentrations, the profilin dependent promotion of actin polymerization biphasically depends on the profilin concentration 11, 13, 1721

. A similar biphasic dependence on the profilin concentration was also revealed in our

experiments regardless of whether the rotation of actin filament relative to formin is constrained. A previous Monte Carlo kinetics simulation of actin polymerization based on association and dissociation rates of profilin/G-actin/FH1 interactions and the transfer rate of G-actin to the barbed end of actin filament suggests that this phenomenon can be explained by these interactions11. We hypothesize that, among these interactions, the biphasic dependence of actin filament polymerization on profilin:G-actin ratio is a result from profilin-influenced local G-actin concentration near the barbed end and profilin-influenced rate of association of local G-actin to the barbed end of actin filament.

We developed a theoretical framework to calculate the effective local concentration of Gactin near the barbed end in given bulk concentrations of G-actin and profilin based on equilibrium chemical reaction theory. In this model, a profilin binding site on FH1 can


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undergo equilibrium transitions among the following three states: 1) unbound, 2) profilin bound and 3) profilin/G-actin complex bound. In solution, profilin and G-actin in given concentrations undergo equilibrium association or dissociation between each other. The kinetic scheme of these transitions is sketched in Fig. 5a. For simplicity of the derivation, we assume that G-actin binding to profilin does not affect the affinity of profilin binding to the profilin binding sites on FH1, and FH1 binding to profilin does not affect the affinity of Gactin binding to profilin. A simple solution of the probability of profilin/G-actin complex bound state of a profilin-binding site on FH1, P is obtained as

=

where = 50 µM and = 0.21 µM

16

(1)

are the dissociation constants of profilin binding to

one proline rich track on FH1, and G-actin monomer binding to profilin, respectively. , , and are the respective concentrations of free G-actin, free profilin, and profilin/G-actin complexes in solution. The total concentration of G-actin and profilin, = + and

= + , remain constant during the reaction.

The local G-actin concentration is defined to be the average number of G-actin molecules that can be found near the barbed end through profilin-mediated association with a FH1 domain divided by a characteristic volume v. The FH1 domain has 164 or 182 residues corresponding to a contour length range of 62-70 nm, for mDia1 or DIAPH1, respectively. Under a physiological level of forces of a few pN, the extension is around 10-20 nm, which defines a characteristic length scale over which the local G-actin concentration can be affected by the interaction between the FH1 domain and profilin. Taking the extension as the

radius r of a sphere centered at the barbed end, v is estimated to be . Each FH1 domain

contains N = 14 proline rich tracks, therefore the local G-actin concentration in Molar units is


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estimated by =

where &' = 6.02 × 10

!" !#

%$ (2)

is the Avogadro constant. More details can be found in

supplementary note 1.

Fig. 5. Theoretical model of profilin-dependent local G-actin concentration and polymerization rate in formin dependent actin polymerization. (a) Schematics of profilin-dependent local G-actin

concentration in formin dependent actin polymerization. (b) Theoretical calculation of the effective local

concentration of G-actin and the net actin elongation rate as a function of profilin concentration. and denote dissociation constants of profilin binding to one proline rich track on FH1 and G-actin monomer

binding to profilin, respectively. denotes the G-actin concentration in our experiments. Note the values of the net actin elongation rate are scaled by an arbitrary factor to the order of hundreds of subunits per second at the peak profilin concentration, which corresponds to the measured polymerization rate at ~ 1 pN in 500 nM G-actin and 2 µM profilin.

The black curve in Fig. 5b and Fig. S4 show the effective local concentration of G-actin and its ratio over the total G-actin concentration ( = 0.5 µM in our experiments) as a function of profilin concentration. The curve reveals a biphasic dependence of the effective local Gactin concentration on the profilin concentration with the peak located at ~ 3 µM. Below this peak, the local G-actin concentration increases as profilin concentration increases, and then it


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decreases after the peak position as the profilin concentration increases further. However, the local G-actin concentration is always higher than the bulk G-actin concentration over this wide range of profilin concentration. Importantly, at the optimized profilin concentration of ~3 µM, the effective local concentration of G-actin reaches two orders of magnitude higher than the total G-actin concentration of 0.5 µM.

The ternary FH1:profilin:G-actin complex can dissociate, and the freed G-actin can be recruited into the barbed end of actin filament. Therefore, the mDia1/profilin-dependent actin polymerization is expected to be facilitated by the drastically increased local concentration of G-actin. However, the actin polymerization rate also depends on the association rate of Gactin to the barbed end, which may also be influenced by profilin. It has been known that profilin can bind the barbed end of actin filament

35

; therefore, the association rate may

depend on whether the barbed end is occupied with a profilin or not. It is reasonable to assume that a barbed end occupied with profilin may result in a slower association rate. Through this mechanism, the net actin polymerization speed is determined by a tug-of-war between increased local G-actin concentration that speeds up the actin polymerization, and the decreased association rate of G-actin due to profilin-occupied barbed end of the actin filament that slows down the actin polymerization. The competition between the two factors could result in a steeper dependence of the net actin polymerization speed on profilin concentration, as shown by the blue curve in Fig. 5b (further details can be found in supplementary note). As shown by the results, the model is able to explain the general trend of the observed facilitating effect of profilin on actin filament polymerization and the biphasic dependence of profilin concentration observed in experiments.

Taken together, these results suggest that an intricate interplay of the weak FH1:profilin


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interaction, the moderate profilin:G-actin interaction, and the number of profilin binding sites on FH1 combine to dramatically influence the local G-actin concentration and the actin filament elongation rate.

In this study, we show that the mDia1∆N3-mediated actin filament polymerization rate is regulated by force, rotation constraints, and profilin. We also investigated the conformation of the FH1 domain, its interaction with profilin under force, and how the interplay of FH1:profilin:G-actin affects the local concentration of G-actin monomers near the formincapped barbed end of actin filaments and how it regulate actin elongation rate.

Our results reveal that, at each given profilin concentration, the elongation rates of rotationally unconstrained filaments are accelerated by around 3-5 folds at forces above the saturating force (~ 5 pN), compared to the elongating filaments at lower forces of ~1 pN. Such force-dependent promotion of actin filament elongation is not observed if the relative rotation between the filament and mDia1∆N3 is constrained. Our previous study shows that, in the absence of profilin, forces in the range of a few pN also accelerate actin filament elongation by a similar fold increase

27

. In the absence of profilin, the force-sensing

mechanism is known to be the force-dependent conformational change of FH2 dimer encircled on the barbed end of the filament

27

. The similar trend of force-sensing in the

presence of profilin suggests that the FH2 domain remains as the main force-sensing component.

Notably, in the presence of profilin, surprisingly fast actin polymerization is observed when the filament is subjected to forces in the range of a few pN. As shown in Fig. 2, at forces above 2 pN, introduction of 2 µM profilin and 500 nM G-actin results in elongation speeds >


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600 subunit s-1, which corresponds to an apparent association rate of ~109 M-1 s-1. For comparison, in 500 nM G-actin without profilin, at the same force the association rate is about six times slower

27

. The association rate at this order of magnitude is close to the

expected diffusion limited association rate based on the binary collision model

36, 37

. It has

been known that the diffusion limited association rate can be exceeded in the presence of a long-range attractive interaction between the ligand and substrate

38-40

. In the case of the

formin-dependent actin polymerization, the interplay of FH1, profilin, and G-actin monomers can introduce an effective attractive potential for profilin:G-actin, resulting in a much higher local concentration of G-actin. As shown by data in Fig. 5, the complex interplay of FH1:profilin:G-actin can enrich the local concentration of G-actin around the barbed end to a level of 10-100 µM in 2 µM profilin and 500 nM G-actin. Therefore, the observed fast actin elongation speed compared that in the absence of profilin can be explained by the increased local concentration of G-actin.

Such force-sensitive fast growing filaments are likely to be physiologically important, as it permits sensitive response of actin filaments to force changes in the cytoskeleton during cell migration. Since formin is often tethered to cytoplasm membrane through adapter proteins24, 41-43

, it is likely to be able to rotate on the membrane during actin filament elongation, which

would avoid accumulation of torsion stress at the interface between formin and the actin filament. The sensitivity to the applied force and the fast elongation speed of such forminmediated filaments may also help to prevent fast tension accumulation in the filament during actomyosin contraction, thereby contributing to the maintenance of the actin cytoskeleton integrity. This also implies that formin could serve as an effective force buffer to maintain forces in the filament within a few pN at very fast elongation speeds. This quick response of formin-mediated actin polymerization to force may also contribute to the timely


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rearrangement and adaptation of the actin cytoskeleton to a rapidly changing environment.

Our results show that, for both rotationally constrained (force-insensitive) and unconstrained (force-sensitive) actin filaments, in a given G-actin concentration, mDia1∆N3-mediated actin filament polymerization rates are biphasically dependent on the concentration of profilin. Such biphasic dependence of profilin concentration was also observed previously by a bulk biochemistry assay of formin-mediated actin filament polymerization in the absence of force 11, 13, 19-21

and from single-molecule assays when the actin filaments are mechanically

stretched 17, 18. Our theoretical model based on equilibrium interactions of FH1, profilin, and G-actin predicts a mild biphasic dependence of G-actin local concentration on profilin concentration for formin-mediated actin filament. In addition, a steeper biphasic dependence of actin elongation rate on profilin concentration is predicted based on a tug-of-war between increased local G-actin concentration that speeds up the actin polymerization, and the decreased association rate of G-actin due to profilin-occupied barbed end of the actin filament that slows down the actin polymerization. Thus, the theoretical model provides further insights into the physical mechanism underlying the observed biphasic dependence of the actin elongation rate on profilin concentration.

The potential involvement of FH1 in the force sensing of formin-mediated actin polymerization has been proposed in several recent studies

17, 18, 21, 28-30

. Our results (Fig. 4)

reveal that the force-response of FH1 domain can be described as a disordered flexible peptide chain, suggesting that the profilin binding sites are readily accessible to profilin without the need for mechanical activation. On the other hand, we show that binding of profilin results in stiffening of the FH1 domain at a few pN forces, suggesting that force may further increase the binding affinity of profilin to FH1 domain, which makes FH1 another


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potential force sensor in addition to FH2. However, based on our observation that force accelerates the actin elongation rate by a similar fold in both the presence and absence of profilin, we reason that FH1 plays a minor role in mechanosensing compared to the FH2 domain.

Prior to this work, formin-dependent actin polymerization in the presence of profilin was investigated in several studies where the actin filaments were stretched by flow 17, 28 or using optical tweezers

18

. At similar stoichiometric ratios for profilin to G-actin, the apparent

association rate measured in our experiments at a force range of a few pN is at least one order of magnitude faster than the values obtained in those studies. While the reason behind this difference is unclear, there are several possibilities that can provide an explanation.

In a paper by Jégou et al 28, mDia1 was immobilized on a coverslip through the FH2 domains by His/anti-His interaction, or through the FH1 domains by GST/anti-GST interaction. In the former case, the rotation of the FH2 ring relative to the coverslip surface is likely inhibited. While the actin filament can rotate relative to formin during its elongation, it is not completely unconstrained because of the rotation drag applied to actin in a viscous medium. For mDia1 immobilized through the FH1 domains by GST/anti-GST interaction, either only one or both GST-tagged FH1 domains could be engaged in the interaction, resulting in a rotation-constrained and rotation-unconstrained mDia1 species relative to the surface. In the above cases, only actin elongation mediated by the rotation-unconstrained formin species is not subject to negative torque and therefore can be compared with our data obtained for actin filaments mediated by rotation-unconstrained mDia1. However, if the data obtained under different tethering conditions were combined it could lead to an overall slower average elongation rate, which might provide an explanation to the different apparent association


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rates measured in the two studies.

In another study, Courtemanche et al. investigated the effects of force on budding yeast formin Bni1p dependent actin polymerization 17. In the presence of profilin, the authors found that force could weakly accelerate actin polymerization in a force range up to 1.5 pN. In that experiment, biotin tagged Bni1p molecules were anchored to streptavidin molecules on a lipid bilayer constrained within areas with rigid diffusion barriers, and an actin filament elongating from Bni1p was stretched by flow. However, the flow might press the filament against the diffusion barrier and the coverslip surface outside the barrier, resulting in potential friction between the actin filaments and barriers as well as between the actin filaments and the coverslip surface. As a result, while the Bni1p on the lipid bilayer is likely to be rotationally unconstrained relative to the surface, the force applied to the elongating filament by the flow could be partially offset by friction. Therefore, the difference in the elongation rates between Courtemanche et al. and our study could be explained by the use of different formin, a different profilin:G-actin stoichiometric ratio, and the potential friction in the flow-stretching experiment.

In a more recent paper, Kubota et al.18 investigated force-dependent acceleration of mDia1∆N3 mediated actin polymerization using a dual-trap optical tweezers setup, where the actin filament is spanned between two optically trapped beads. Compared to our results, they observed a much weaker force dependence of actin polymerization in the presence of profilin in a similar profilin:G-actin stoichiometric ratio. In their experiment, the bead-to-bead distance was increased manually to maintain a roughly constant force, and the rate of the bead-to-bead distance increase was interpreted as the actin elongation speed at the corresponding forces. Such measurement would depend on how fast the bead-to-bead


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distance was changed. The typical pulling rate in that experiment is in the range of 20-30 nm s-1 (Fig. S2 from Kubota et al. 18), at which only the slow elongating filaments could generate forces of a few pN. To detect actin filaments elongating at much faster speeds, much faster pulling rates would be needed to allow the fast-elongating filaments to generate similar level of forces. Due to this reason, that experiment could have missed the detection of much faster elongating actin filaments.

The results reported in this study and in previous studies 17, 18, 27, 28, 44 reveal a highly complex regulation of formin-mediated actin polymerization by profilin and mechanical constraints. Profilin facilitates actin polymerization not only by increasing the local G-actin concentration but also by increase lifetime of formin on the barbed end of F-actin

44

. Force on one hand

speeds up polymerization of rotation-unconstrained actin filament, on the other hand it decrease the lifetime of formin bound on the barbed end

44, 45

. Working together, these

factors allow cells to regulation actin cytoskeleton network by quickly responding to environmental changes.

Finally, we stress that the findings in this study could be harnessed to make novel actin-based active materials. Current actin-based active matters are mainly built by elements including actin filaments, actin filament crosslinking proteins, actin filament branching proteins and myosin motor proteins

5, 7-9

. Introducing formin and profilin into the reaction system, the

unique responses of formin/profilin-mediated actin filament polymerization to force and rotation constraints may bring new mechanical properties to the actin based active materials.

Protein sample preparation - Actin, GST-mDia1∆N3(GST-avi-FH1-FH2) and BiotinylatedmDia1∆N3 was obtained as described previously

27

. NEM-NMII is from Hypermol


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(cat.8316-01). Profilin is expressed as described in

Page 24 of 28

46

. The DNA of Formin mDia1 FH1

fragment (182 a.a.) spanned between four titin I27 domain (Fig. 4a) was synthesized by geneArt, and sub-cloned into an expression vector (pET151-TOPO) with an avi-tag at the Nterminal and a spy-tag at the C-terminal; The plasmid was co-transformed with a BirA plasmid and expressed in Escherichia coli BL21 (DE3), cultured in Luria-Bertani (LB) media, and then affinity purified through his-tag purification. Preparation of coverslips, seed actin filaments, and superparamagnetic beads - Three different tethering methods were used in our experiments (Fig. 1a), which required different preparations of coverslips, seed actin filaments, and superparamagnetic beads. The details of the sample preparation have been described previously 27. Single actin filament manipulation assay - The transverse magnetic tweezers used in this study were made in-house, and controlled as described in

47, 48

. In order to perform the

measurements, the seeding actin filaments were incubated in the channel for 10 minutes to allow actin filaments seeds to bind to the surface. After removal of free actin filaments by washing the channel with KMEI buffer (10 mM imidazole, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 400 µM ATP, 0.5 mM DTT), superparamagnetic beads were introduced into the channel, and incubated for 5 minutes to allow them to bind to actin filaments. Finally, Gactin and profilin were flowed into the channel in KMEI buffer for actin elongation. Force was applied to the tether through the attached superparamagnetic beads. The elongation of the filament was indicated by the movement of the tethered superparamagnetic bead in real time. Details of force calibration and solution exchange methods are described in 27, 49. Single-protein manipulation assay - A vertical magnetic tweezers setup (built in-house) was combined with a disturbance-free, rapid solution-exchange flow channel

49

50, 51

for protein

stretching experiments. A FH1 fragment spanned between four titin I27 domain constructs is tethered between a spy-catcher-coated coverslip and a streptavidin coated paramagnetic bead


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through N-terminus avi-biotin-tag and C-terminus spy tag. More details of single-protein manipulation can be found in previous publications 31, 32. Author Contributions JY, MY and SL designed the study. MY and SL performed the experiments and data analysis. JY and AY developed the theoretical model, MY and SL performed the calculation. MY, SL and JY interpreted the data. SL and XZ developed the solution exchange membrane well for transverse magnetic tweezers. JY and AB supervised the research. MY, SL and JY wrote the manuscript. Acknowledgement The authors thank the protein expression facility and science communication core of the Mechanobiology Institute. The research is funded by the National Research Foundation, Prime Minister’s Office, Singapore, under its NRF Investigatorship Programme (NRF Investigatorship Award NRF-NRFI2016-03 to JY), the National Research Foundation, Prime Minister’s Office, Singapore and the Ministry of Education under the Research Centres of Excellence programme (to JY and AB), and Human Frontier Science Program (RGP00001/2016 to JY).

Supporting Information Figure S1, S2 and S3 shows the supplementary data of formin FH1 domain force response.

Figure S4 represent the effect of on effective local concentration of G-actin and its ratio over the total G-actin concentration as a function of profilin concentration. Supplementary note 1 expounds the theoretical framework of local G-actin concentration calculation. Supplementary note 2 describes theoretical framework of the net recruitment rate of G-actin to the barbed end of formin-capped actin filament. References


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