Formin mDia1 senses and generates mechanical forces on actin filaments

Abstract

Cytoskeleton assembly is instrumental in the regulation of biological functions by physical forces. In a number of key cellular processes, actin filaments elongated by formins such as mDia are subject to mechanical tension, yet how mechanical forces modulate the assembly of actin filaments is an open question. Here, using the viscous drag of a microfluidic flow, we apply calibrated piconewton pulling forces to individual actin filaments that are being elongated at their barbed end by surface-anchored mDia1 proteins. We show that mDia1 is mechanosensitive and that the elongation rate of filaments is increased up to two-fold by the application of a pulling force. We also show that mDia1 is able to track a depolymerizing barbed end in spite of an opposing pulling force, which means that mDia1 can efficiently put actin filaments under mechanical tension. Our findings suggest that formin function in cells is tightly coupled to the mechanical activity of other machineries.

ARTICLE

Received 4 Nov 2012 | Accepted 16 Apr 2013 | Published 21 May 2013

Cytoskeleton assembly is instrumental in the regulation of biological functions by physical forces. In a number of key cellular processes, actin laments elongated by formins such as mDia are subject to mechanical tension, yet how mechanical forces modulate the assembly of actin laments is an open question. Here, using the viscous drag of a microuidic ow, we apply calibrated piconewton pulling forces to individual actin laments that are being elongated at their barbed end by surface-anchored mDia1 proteins. We show that mDia1 is mechanosensitive and that the elongation rate of laments is increased up to two-fold by the application of a pulling force. We also show that mDia1 is able to track a depolymerizing barbed end in spite of an opposing pulling force, which means that mDia1 can efciently put actin laments under mechanical tension. Our ndings suggest that formin function in cells is tightly coupled to the mechanical activity of other machineries.

DOI: 10.1038/ncomms2888

Formin mDia1 senses and generates mechanical forces on actin laments

Antoine Jgou1, Marie-France Carlier1 & Guillaume Romet-Lemonne1

1 Laboratoire dEnzymologie et Biochimie Structurales, CNRS, avenue de la Terrasse, 91190 Gif-sur-Yvette, France. Correspondence and requests for materials should be addressed to G.R.-L. (email: mailto:[email protected]

Web End [email protected] ).

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Physical forces participate in the regulation of biological functions, from embryonic development to adult physiology, and are key factors in numerous pathological

processes1. Individual cells are sensitive to their mechanical environment, which can be used to steer stem cell differentiation2. Single-molecule approaches have provided insight into some molecular mechanisms responsible for the conversion of physical forces into biochemical signals. For example, talin, a protein involved in mechano-transduction at focal adhesions, exposes buried vinculin interaction sites when it is stretched by forces of tens of piconewtons3. Molecular motors that convert the energy from ATP hydrolysis into mechanical movement along lament tracks are also sensitive to applied forces in the piconewton range, and have been studied with extreme accuracy4,5. In stark contrast, nearly nothing is known about the regulation of cytoskeletal lament assembly by mechanical forces, besides the slowing down of polymerization by an opposing obstaclean effect observed more clearly for microtubules6 than actin laments7, which are too exible to work individually. Evidence of a mechanism where a mechanical force modulates the elongation of individual actin laments is still missing.

Formins elongate actin laments while remaining processively attached to their growing barbed ends8,9. They are responsible for the rapid generation of long actin cables which form the backbone of key cellular substructures such as lopodia10, stress bres11 and cytokinetic rings12,13. Formin elongation has been proposed to be mechano-sensitive14, as the two protomers of the FH2 (Formin Homology domain 2) dimer translocate in a rotating stair-step fashion to track the elongating barbed end15,16. From a physiological perspective, the regulation of formin activity by mechanical forces would have a central role in numerous situations where formin-elongated laments are eventually put under tension, for example, during lopodia retraction or cytokinetic ring contraction.

The aim of the present work is to establish how a formin responds to the application of mechanical tension on the lament that it is elongating. We use microuidics to apply piconewton pulling forces to individual actin laments elongated by surface-anchored formin mDia1, and show that their elongation rate can thus be increased up to two-fold. This increase in elongation rate can be understood by considering that the pulling force favours the open conformation of the FH2 dimer, in which an additional actin subunit can be added at the lament barbed end. The mechanical response of formin mDia1 is the same whether the pulling force is applied at the N-terminal side of the FH1 domain or at the C-terminal side of the FH2 domain. We also show that formin mDia1 remains processively bound to the barbed end of a depolymerizing lament which is being pulled on, and that the pulling force slows down depolymerization. This result implies that formins can put actin laments under tension.

ResultsApplying forces to individual laments with microuidics. In order to apply pulling forces to individual laments, we have taken advantage of a yet unexploited mechanical feature of our recently developed microuidics setup17,18. In this experimental conguration, laments grow anchored by one end to the glass coverslip surface at the bottom of a ow chamber. The laments are aligned and maintained close to the coverslip surface by the ow (Fig. 1b). The viscous drag exerted by the owing liquid on the anchored lament results in a pulling force, which is maximal at the anchoring point. Using a bead in an optical trap to measure this force, we have veried that it was proportional to the local uid velocity times the length of the lament, and thereby determined the longitudinal friction coefcient for an actin lament (Fig. 1a, Methods).

Filaments anchored at the bottom of the ow cell are in a region where the ow velocity increases linearly with distance away from the surface (Supplementary Fig. S1). As the anchored lament uctuates in the owing uid, it explores different distances away from the surface. Using Total Internal Reection Fluorescence (TIRF) microscopy with a calibrated penetration depth (Supplementary Fig. S2) we have measured the contour prole of individual laments, which uctuate around an average position 250 nm above the surface for the range of ow velocities and lament lengths used in this work (Supplementary Fig. S3). The force exerted on the anchoring point is computed by integrating the viscous drag over the whole-lament contour, which experiences different local ow velocities as it explores different distances above the surface. Within a 10% error, the average force is equal to the viscous drag the lament would experience if it remained entirely 250 nm above the surface. The force uctuates within 30% of its average value, at a frequency far superior to that of data acquisition (Fig. 1b, Supplementary Fig. S3, Methods).

When a lament grows from a surface-anchored spectrin-actin seed, no signicant force is exerted on a formin interacting with its freely growing barbed end (Fig. 2a). In this situation, as in the absence of formin17, the ow velocity has no impact on the elongation rate.

Here, we elongate laments from surface-anchored formins on which we thus exert a pulling force. We have used a formin construct mDia1(FH1FH2), which comprises the functional FH1 and FH2 domains, as well as the DAD domain which has no impact on lament elongation19, an N-terminal GST tag and a C-terminal His tag (Fig. 2). Proper passivation of the coverslip surface and functionalization with streptavidin ensured the specic anchoring of either the N-ter-FH1 or the C-ter-FH2 domain of mDia1(FH1FH2) pre-incubated with biotinylated anti-GST or anti-His antibodies (Fig. 2), without affecting their activity (Methods). As uorescent labelling can give rise to unexpected artifacts20, we monitor the elongation of laments from unlabelled actin monomers, by rst nucleating and

Optical trap

Flow

Flow

550 m

350 nm

Force

Actin filament

mDia1(FH1FH2)

150 nm

Actin filament

Glass slide

12

10

8

6

4

Force (pN)

Number of events

90 80 70 60 50 40 30 20

2

10

0

00.4

0.8

1.2

1.6

22.4

2.8

3.2

3.6

4

0 0 5,000 10,000 15,000 20,000

Force (pN)

Figure 1 | Pulling on actin laments with the viscous drag of a owing uid. (a) The force exerted by the owing uid on an actin lament is measured with a microbead in an optical trap, a few micrometres above the surface. Data points and error bars show the averages and s.d. of 47 measurements, performed with different lament lengths and different ow velocities. (b) Filaments anchored to the bottom of the microuidic chamber uctuate around an average position located 250 nm above the surface, resulting in rapid uctuations of the force exerted on the anchoring point. Histograms show the force distributions for a 14-mm long lament in a ow velocity gradient of 194 s 1 (red bars, force 0.400.16 pN)

and for a 20-mm long lament in a ow velocity gradient of 565 s 1 (blue bars, force 1.630.53 pN).

Filament length flow velocity (m2 s1)

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mDia1(FH1FH2)

6xHis

Unlabelled G-actin

Labelled F-actin segment

Flow

GST

FH1

FH2

DAD

Flow

Force

Force

FH1 FH2

Anti-His

10 m

Anti-GST

1

where d 5.4 nm is the actin monomer size, k is Boltzmanns

constant and T is temperature. The elongation rate can hence be written as

velong F

Time

1 1 p0p0 e Fd=kT

CONTROL

Spectrinactin

No force

Force

Flow

Figure 2 | Applying forces to surface-anchored formins as they elongate actin laments. (a) The mDia1(FH1FH2) construct comprises the DAD domain, as well as a C-terminal His-tag and an N-terminal GST-tag. The use of biotinylated antibodies and a streptavidin-coated surface allows the specic anchoring of mDia1(FH1FH2) dimers by their FH1 or FH2 domains. Control experiments where the formin feels no force are performed by growing laments from surface-anchored spectrin-actin seeds. (b) An anchored mDia1(FH1FH2) elongates a lament from its barbed end using unlabelled monomers, following a brief elongation from uorescently labelled actin, which formed the uorescent segment visible at the pointed end. (c) Epiuorescence image of the situation sketched in (b) with 1 mM unlabelled ATP-actin and 4 mM prolin. The time sequence corresponds to the area indicated by the yellow frame in the larger image, and to

Supplementary Movie 1. The interval between two images is 20 s.

The triangles indicate the location of two anchored mDia1(FH1FH2) dimers. The ow velocity 250 nm above the surface is 101 mm s 1.

konC Cc1 1 p0p0 e Fd=kT

elongating uorescent lament segments, and subsequently owing a solution of unlabelled actin in the microchamber (Fig. 2b). Elongation is monitored by tracking the position of the uorescent lament segment as it moves away from the anchored mDia1(FH1FH2).

Pulling forces accelerate elongation by formin mDia1. At a given ow rate, the force exerted on the anchored mDia1(FH1FH2) increases progressively as the lament elongates, and the elongation rate increases with this force (Fig. 3). The pulling force can also be varied rapidly by changing the ow rate. The relation between force and elongation rate does not depend on the method by which the force is varied, and the elongation rate drops upon a decrease of the pulling force (Fig. 3a,b and Supplementary Fig. S4). Forces hence modulate formin activity in a reversible fashion.

To understand what causes this increase in elongation rate upon the application of a pulling force, we have measured this effect at different prolinactin concentrations (Fig. 3c). Pulling forces increase the elongation rate to a plateau value that is roughly twice the value in the absence of force, with a half-effect reached for a force of B0.6 pN. The same behaviour is observed whether mDia1(FH1FH2) is anchored via anti-His antibodies, thereby pulling on the FH2 domain alone, or via anti-GST antibodies, thereby pulling on the FH1 domain as well (Figs 2a,3c).

The pulling force exerted on the FH2 dimer should favour its translocation14. The linear increase of elongation rate with prolinactin concentration in the absence of force (Fig. 3c inset) indicates that the translocation of the FH2 dimer is not

rate-limiting and that we can consider the FH2 dimer to be in rapid equilibrium between a closed conformation, in which no subunit can be added, and an open conformation, in which subunits can be added with rate constant kon to the

barbed end15,21 (Fig. 4). The elongation rate can be written as velong p0kon(C Cc), where C is the prolinactin

concentration, Cc is the critical concentration and p0 is the probability to nd the FH2 dimer in the open conformation. Based on a simple description of translocation using transition state theory22,23, we can write that upon application of a pulling force F, the probability p0 becomes

p F

1

2

Fits of our data by this simple model, with p0 and kon(C Cc)

as free parameters for each curve, are shown in Fig. 3c. We can hence interpret our results by considering that a pulling force accelerates lament elongation by favouring the open conformation of the FH2 dimer. In the conditions of our measurements (that is, with a 3 mM excess of prolin), we nd kon 84.8 mM 1 s 1 and Cc 0.07 mM (Fig. 3c, inset). In the

absence of force, the FH2 dimer is in the open conformation only 56% of the time (p0 0.560.06), resulting in a slower

elongation, with an apparent on-rate of 48.1 mM 1 s 1.

Formin mDia1 generates tension on depolymerizing laments. Recent work on individual actin laments has shown that mDia1(FH1FH2) is able to track a depolymerizing barbed end16. Using our control conguration, where no force is applied to mDia1(FH1FH2), we conrm this observation. We have previously reported that free barbed ends depolymerize faster in the presence of prolin17 and we now show that this effect is further amplied by the presence of mDia1(FH1FH2) at the barbed end (Fig. 5b). This effect is reminiscent of the acceleration of barbed end elongation from prolinactin by formins. Future experiments should reveal whether this effect is due to the action of FH1 alone or also involves an alteration of the barbed end by the FH2 dimer.

The fact that a formin can track a depolymerizing barbed end suggests that it could perhaps generate pulling forces on the disassembling lament. To address this question, we have triggered the depolymerization of laments under mechanical force in our microuidic setup, by owing in a solution of prolin. We observe that mDia1(FH1FH2) remains bound to the depolymerizing lament, pulling it upstream against piconewton forces, thus showing that formins are able to generate mechanical forces (Fig. 5). This activity was observed on laments elongated from ATP-actin and depolymerized in standard F-buffer (Supplementary Fig. S5) as well as laments elongated from ADP-actin and depolymerized in ATP-free buffer (Fig. 5).

To further characterize the mechanical response of mDia1(FH1FH2) as a force-generating motor, we have measured the depolymerization rate of ADP-actin laments against different loads (Fig. 5). We nd that a pulling force exerted on the laments slows down depolymerization. As for elongation experiments (Fig. 3), we have veried that this effect is reversible, and that the depolymerization rates measured against a

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16,000 265

240

215 300 250 200 150 100

50

00 0.5 1 1.5

[Profilin-actin] (M)

2 2.5 3 3.5

14,000

12,000

10,000

8,000

6,000

4,000

2,000

Filament length (subunits)

Elongation rate (sub. s1 )

190

165

140

115

90

65

40

15 0 0.5 1 1.5 2 2.5 3

0

0

50 100 150 200 250

Force (pN)

3.5 3

2.5 2

1.5 1

0.5 0

Elongation rate (sub. s1)

0

50 100 150

Time (s)

200 250

Elongation rate (sub. s1)

100

90

80

70

60

50

40

30

20

10

0 0 0.5 1 1.5 2

Force (pN) Force (pN)

2.5 3 3.5

Figure 3 | Pulling forces increase the elongation rate of laments from mDia1(FH1FH2). (a) Traces for two different laments, elongating from1 mM ATP-actin and 4 mM prolin. One lament is exposed to a ow velocity of 101 mm s 1 (blue diamonds) while the other is exposed to a ow velocity of 54 mm s 1 (red open circles) with a temporary increase to 215 mm s 1 (red bullets). Each actin subunit contributes to 2.7 nm of the lament length. (b) Resulting elongation rate versus force for each lament. (c) Average data from traces of 814 laments for each prolinactin concentration:0.5 mM (blue diamonds, n 9 laments), 1 mM (triangles, n 8), 2 mM (squares, n 14) and 3 mM (bullets, n 11), each with a 3-mM excess of prolin.

Error bars are s.d. All data were obtained with mDia1(FH1FH2) anchored by the FH2 domain, except grey data points where the anchor was on the FH1 domain. Open symbols correspond to control experiments with unanchored formins at the barbed end of laments grown from anchored spectrin-actin seeds. Lines are ts of the data by equation (2), performed with p0 and the plateau velocity kon(C Cc) as free parameters for each

set of data. Inset: plot of the resulting plateau values (black symbols) at high force, as a function of prolinactin concentration, compared with elongation rates in the absence of force (open symbols). Lines are linear ts.

very low pulling force are similar to control measurements in the absence of force.

Following a similar reasoning as for elongation, we can assume that the terminal actin subunit can depart from the barbed end, with a rate constant koff, only when the FH2 dimer is in the closed conformation (Fig. 4). The depolymerization rate can then be written as

vdepol F

koff

1

Also, the computed depolymerization rates are nearly zero at 3 pN, whereas the experimental data seem to indicate a higher stall force. Indeed, our experimental data show that depolymerization remains signicant for forces as high as2.4 pN. Under these conditions, mDia1 produces a mechanical work of B13 pN nmE3.2 kT for every departing actin subunit.

DiscussionTo describe the conformation changes of the FH2 dimer during lament elongation, one can use the gating factor, which is dened as the ratio of the elongation rate of barbed ends with and without FH2, in the absence of prolin21. The gating factor can be written as p0kFH2on=kon, where kFH2on and kon are the monomer on-rate constants in the presence and absence of an FH2 dimer, respectively. For mDia1, the gating factor is of the order of 0.9. If we assume that the translocation of the FH2 domain is not affected by prolin, our estimated value of p0 0.56 leads to

kFH2on 1:6kon. Our results therefore indicate that the mDia1 FH2

dimer, when present at the lament barbed end in the open conformation, increases the on-rate constant of actin monomers by 60%.

Formin mechano-sensitivity has been proposed by Kozlov and Bershadsky14 to stem from FH2 elasticity. We could not t our data with this model where an activation energy, associated to a

p01 p0 eFd=kT

3

Fits of our data by this simple model, with p0 0.56

(determined by the ts of the elongation data by equation (2)), d 5.4 nm, and leaving koff as a free parameter for each curve, are

shown in Fig. 5. As for elongation, the interpretation here is that the pulling force favours the open conformation of the FH2 dimer. This simple two-state model accounts for the observed force-induced decrease of the depolymerization rate, but misses some of its features. For instance, our data indicate that the force-depolymerization prole becomes less sensitive to prolin concentration as force increases, more drastically than what is computed theoretically, suggesting that the interaction of prolin with the formin-bound barbed end may also be affected by force.

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60

50

40

30

20

10

Anchored mDia1(FH1FH2)

Flow

Closed

Open

Pointed ends

Depolymerization rate (sub. s1 )

Time

Closed

E

Open

[afii9829] = 5.4 nm

Force

5 m

Actin monomer

FH2 dimer

Figure 4 | Schematic representation of FH2 dimer translocation at the barbed end of an actin lament. The lament barbed end is in a non-polymerizing state when the FH2 dimer is in the closed conformation, while it can bind an actin monomer (not shown) when the FH2 dimer is in the open conformation15. When analysing lament depolymerization,we consider that the FH2 dimer in the open conformation prevents the departure of the terminal actin subunit from the barbed end. Applying a pulling force tilts the energy landscape of the FH2 dimer at the barbed end, favouring the open conformation. In the frame of reference of the microchamber, the FH2 dimer is in a xed location and the lamentis displaced as it elongates or depolymerizes.

0

0

0.5

80 70 60 50 40 30 20 10

0 0 5 10 15 20 25[Profilin] (M)

1 1.5 2 2.5 3

Force (pN)

Depolymerization rate

(sub. s1 )

rate-limiting formin deformation step (translocation), is reduced by the pulling force. Nonetheless, it is possible that formin elasticity contributes to the energy landscape sketched in Fig. 4.

The FH1 domain is often presented as a exible chain that captures and delivers prolinactin to the barbed end21. The FH1 domain of mDia1 comprises n 14 polyproline domains of

length dE3 nm, separated by linkers. Assuming these linkers are exible, we can model the FH1 domain as a exible chain, with an entropic spring stiffness of 3 kT/nd2E0.1 pN nm 1. A 1 pN force should then be able to stretch the FH1 domain, keeping its extremities 10 nm apart. This is a signicant constraint on the possible conformations that the FH1 chain, which has a full contour length of about 42 nm, is able to explore. If the FH1 domain delivers prolinactin to the barbed end by taking advantage of its exibility to rapidly explore different conformations, the elongation rate of mDia1(FH1FH2)-bound laments should decrease drastically when piconewton forces are applied on the FH1 domain, compared with the situation where the pulling force is applied to the FH2 domain alone. Beyond this capture and delivery mechanism, it seems likely that during FH1-assisted elongation the newly added actin subunit is transiently bound to prolin-FH1 and to the FH2-lament barbed end simultaneously. This conguration should become extremely difcult to achieve when polyproline regions of the FH1 domain are pulled away from the barbed end by pN forces. Our results therefore indicate that the exible FH1 domain is able to remain in the vicinity of the FH2 dimer and the barbed end, in spite of a pulling force applied on its N-terminal extremity. This could be achieved by interactions of FH1 polyproline helices with FH2 alpha-helices.24 Such a connection between the FH1 and FH2 domains could lead to a situation where the N-terminal region of the FH1 domain is maintained close to the C-terminal

region of the FH2 domain, and pulling on one or the other would have very similar consequences. Elucidating the detailed function of FH1 in rapid processive growth from prolinactin will require the combination of mechanical experiments like the present ones and of biochemical approaches25, which by themselves have not brought denite answers.

The generation of force by proteins tracking depolymerizing lament ends has been proposed theoretically26,27 but has only

Figure 5 | mDia1(FH1FH2) can generate piconewton pulling forces on depolymerizing actin laments. (a) Effect of force on the depolymerization rate of ADP-actin laments bound to mDia1(FH1FH2) anchored by their FH2 domain, in the presence of 1 mM (red squares, n 4 laments),

4 mM (green diamonds, n 8) and 10 mM (blue bullets, n 4) prolin.

Open symbols correspond to control experiments with unanchored formins at the barbed end of laments grown from anchored spectrin-actin seeds. Error bars are s.d.. Lines are t of the data by equation (3). Inset: epiuorescence time sequence of an ADP-actin lament bound to a surface-anchored mDia1(FH1FH2), depolymerizing in the presence of4 mM prolin, while exposed to a ow velocity of 52 mm s 1 (Supplementary Movie 2). Only the uorescent segment near the pointed end is visible. The interval between images is 20 s.(b) Depolymerization rate of ADP-actin lament barbed ends by mDia1(FH1FH2) in the absence of force, as a function of prolin concentration (data points), compared with the depolymerization rate without mDia1 (dashed line, determined in Jegou et al.17).

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been observed so far for microtubules, with either depolymerizing motors28 or passive couplers29. Formin mDia1 bears more similarities with the latter as it does not hydrolyse ATP, and converts energy from actin conformational changes into mechanical work. However, even though mDia1 does not directly induce depolymerization, it largely amplies the depolymerizing action of prolin (Fig. 5b).

The pulling forces that we apply to the laments also detach formins from the surface (Supplementary Fig. S6), thereby limiting the collection of data at high forces, and precluding the study of the force dependence of formin departure from the barbed end (processivity). With a stronger anchoring of formins to the surface, future experiments should allow the study of the release of the lament by mDia1(FH1FH2) and provide accurate measurements of the stall force of depolymerization.

Nonetheless, the pulling forces generated by mDia1(FH1FH2) that we measure are large enough to play a role in cells. Very recent evidence from cell studies indicates that this could indeed be the case. Romero et al.30 have shown that the stall force of lopodium retraction strongly depends on the molecular details of lopodium tip adhesion. This result suggests that the proteins located at the lopodium tip, including formins, actively participate in the generation of the pulling forces responsible for retraction. Two other very recent studies in cells31,32 report that cytokinetic ring contraction requires actin depolymerization, and still takes place when myosin motor activity is suppressed. This implies that actomyosin contraction is not the only way to generate mechanical tension in cells. Our ndings show that formins anchored to depolymerizing laments are a molecular alternative to produce the required pulling forces.

Methods

Proteins and buffers. Actin was puried from rabbit muscle. Recombinant Prolin I from mouse was expressed in E. Coli and puried. Spectrin-actin seeds were puried from human erythrocytes. Fluorescent actin was labelled with Alexa488 succinimidyl ester. ADP-actin was made from Ca-ATP-actin, using hexokinase and glucose, converted into Mg-ADP-actin by adding MgCl2 and

EGTA at the beginning of each experiment, and kept on ice for a maximumof 2 h. Formin mDia1(GST-FH1-FH2-DAD-His6), simply referred to as mDia1(FH1FH2), was expressed in E. Coli and puried9. Biotinylated mDia1(FH1FH2) was obtained by incubating mDia1(FH1FH2) with either biotinylated anti-His antibody (mouse monoclonal IgG, AnaSpec) or biotinylated anti-GST antibody (mouse monoclonal IgG, Santa Cruz Biotechnology) for at least 1 h on ice. The antibody-to-mDia1FH1FH2 ratio was varied between 0.046 and2.3 and no differences were observed in the resulting force-velocity curves. Standard elongation and depolymerization of laments was done in F-buffer

(5 mM TRIS pH 7.8, 0.2 mM ATP, 0.1 mM CaCl2, 0.01% NaN3, 100 mM KCl,1 mM MgCl2, 0.2 mM EGTA) supplemented with 10 mM DTT and 1 mM DABCO to limit photobleaching. ATP was replaced with ADP 10 mM Ap5A in

experiments with ADP-actin. Nucleation of laments by mDia1(FH1FH2) was carried out at a lower ionic strength, using 50 mM KCl. All measurements were carried out at 25 C.

Sample preparation. Glass coverslips were cleaned by sonication in Hellmanex followed by ethanol, rinsed in water and blow dried. Microuidic chambers were built by assembling clean glass coverslips to PDMS structures composed of three entry channels and one exit channel (more details below). The microchambers were placed on the microscope stage, and connected to the microuidic system17. For control experiments where lament barbed ends grow freely, the microchambers were incubated with spectrin-actin seeds, followed by a solution of BSA. For experiments with anchored mDia1(FH1FH2), the chamber was incubated repeatedly with biotinylated BSA, BSA and Streptavidin, before incubation with biotinylated mDia1(FH1FH2). The specicity of the anchoring of mDia1(FH1FH2) via its biotin tag was tested by owing mDia1(FH1FH2) pre-incubated with biotinylated antibodies in one half of the microuidic chamber, and mDia1(FH1FH2) alone in the other half, and verifying that laments were subsequently nucleated in the region exposed to biotinylated mDia1(FH1FH2) only. The elongation rates of laments growing from anchored mDia1(FH1FH2) in the absence of mechanical tension were identical to the ones measured on laments growing from adsorbed spectrin-actin seeds with mDia1(FH1FH2) at their barbed end.

Microscopes. TIRF and epiuorescence observations were carried out on an Olympus IX71 inverted microscope, with a 60 TIRF objective, and a 473 nm

laser (Cobolt). Images were acquired using a cascade II EMCCD camera (Photometrics). The angles of incidence of the laser beam were determined by mounting a triangular prism on the microscope stage.33 As the refractive indices of the glass coverslip and buffer solution are known with limited accuracy33, we have measured the penetration depth of the TIRF excitation eld by monitoring uorescent microbeads in the ow chamber (Supplementary Fig. S2).

The optical trap setup was built on a Nikon eclipse TE2000-U inverted microscope, using an 1064 nm laser, and opto-actoustic deectors (AA opto-electronics) for attenuation and deection. Polystyrene microbeads (Polysciences) of 2 mm diameter were used for trapping. The optical trap stiffness was calibrated using the Stokes force on microbeads in a microuidic ow, and conrmed by the roll-off frequency technique applied in glass microchambers. These measurements were carried out 5 or 10 mm above the coverslip surface, and the same results were obtained at both positions, conrming that the surface was far enough not to perturb these measurements.34 Images were acquired using an Orca Flash camera (Hamamatsu).

Microuidic system. PDMS microchambers were made from photoresist moulds (Stanford Microuidics Foundry), and mounted on clean glass coverslips. The chambers are composed of three inlets and one outlet. Observations were carried out in the main channel, a few hundred micrometres downstream of the inlets junction. The main channel is 1 mm wide and 42 mm high. The ow of solutions in the chamber is controlled and monitored using a MAESFLO system (MFCS and Flowell, from Fluigent). We have veried that the ow rates indicated by the ow metres on the three inlets of our microuidic apparatus were accurate, within a few per cent.

Force calibration and control experiments. The friction coefcient of actin laments was determined using uorescent laments growing from spectrin-actin seeds adsorbed on microbeads held in an optical trap, as depicted in Fig. 1a. Force and length were measured within 5 s, for different lament lengths. Force was calibrated for each bead, before growing the lament. The ow velocity at the position of the bead was measured by releasing the bead and monitoring its movement. These measurements allowed us to verify that the force is proportional to lament length times ow velocity, and that the uctuations of the lament did not bias the measurement. These measurements were repeated at different heights, 510 mm above the surface, and in different microchambers, 40100 mm high, hence with different local ow velocity gradients. This allowed us to verify that the application of a ow velocity gradient had no measurable impact on the force.

By monitoring the movement of polystyrene microbeads in the microchamber, we have mapped the velocity prole of the microuidic ow along the vertical axis. Data were in very good agreement with the theoretical parabolic prole (Supplementary Fig. S1). Thus, the ow prole near the surface is well approximated by a linear function, with a slope that can be computed fromthe global incoming ow rate and the microchamber dimensions.

Using TIRF microscopy, we have taken advantage of the exponential decay of the evanescent excitation eld to monitor the vertical position and the uctuations of the anchored laments in the uid ow (Supplementary Fig. S3). These measurements were done on highly labelled laments (3070% Alexa488) in order to have a homogeneous uorescence. We nd that uctuations are larger near the free end of the lament, and larger over the whole lament for lower ow rates. Nevertheless, except for a short region (less than 2 mm long) near the anchoring point, the different portions of the laments are located 250 nm above the surface on average, and uctuate within 30% of this plane. For a given lament prole (that is, a set of z-positions for different portions of the lament), we can compute the force exerted by the owing uid on the lament and transmitted to the anchoring point, by integrating the viscous drag exerted on each portion of the lament. We have veried that the result was not affected by the size and number of the lament portions that we considered. By acquiring TIRF images of the uctuating lament with a high frame rate (down to 17 ms between images) we can monitor the force uctuations over time. For the present range of ow velocities (up to 200 mm s 1) and lament lengths (up to 50 mm), taking the ow velocity 250 nm above the surface and the full-length of the lament reliably gives the average of the force distribution exerted on the anchoring point. Fluctuations are very rapid: the full breadth of the force distribution is explored in less than a second. This ensures that the average force exerted on the anchoring point between two images in our experiments (typically 5 s) is the average force of the distribution. The effect of force uctuations was estimated by a convolution of the theoretical curves, and found to be negligible.

The local ow velocity experienced by surface-anchored laments was conrmed by exposing highly uorescent laments to continuous TIRF illumination in order to fragment them, and monitoring the velocity of detaching fragments witha high camera frame rate. The measured velocities are in good agreement with the height positions estimated by TIRF, with the expected velocity prole near the surface of the ow cell.

Control experiments with laments grown from surface-anchored spectrin-actin seeds were performed in order to measure the elongation and depolymerization rates in the absence of force. These experiments were done with 512%

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Alexa488-labelled G-actin. These experiments also showed that the activity of mDia1(FH1FH2) bound to free barbed ends was not affected by the lengthof the lament or the microuidic ow rate.

Image analysis. Images were analysed using ImageJ. Contrast was enhanced using the KymoToolBox plugin (available from [email protected]).

The uorescent segment of actin laments was tracked using the snake t programme35. Very short uorescent segments were tracked using the spot tracker 2D0 plugin in Image J (available at http://bigwww.epfl.ch/spottracker/

Web End =http://bigwww.ep.ch/spottracker/ ). We consider that each actin subunit contributes to 2.7 nm of the lament length. We thus obtained length-versus-time data, which we could t to extract elongation or depolymerization rates. Knowing the ow rate (measured by the MAESFLO microuidic apparatus during the experiment) and the length of the lament (full-length, including both the unlabelled and the uorescently labelled segment), we can compute the force exerted on the surface-anchored mDia1(FH1FH2).

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Acknowledgements

GRL acknowledges support from the Human Frontier Science Program (grant RGY0067/ 2008). MFC acknowledges support from the European Research Council (advanced grant ERC 2009-249982-Forcefulactin) and the European Union Seventh Framework Program (MitoSys, Grant 241548). The group is part of the CNRS consortium GDR CellTiss. We thank Dominique Didry and Brengre Guichard for help with protein purication, Astrid van der Horst and Jacob Kerssemakers for advice on optical trapping, Christian Hubert (ERROL) for help with the optical trap setup and TIRF lasers, Olivia du Roure for advice on microuidics, Christophe Le Clainche and Pierre Montaville for discussions on formins and molecular biology.

Author contributions

A.J. and G.R.-L. designed the research and performed the experiments; A.J., M.-F.C. and G.R.-L. Analysed the data; G.R.-L. wrote the paper.

Additional information

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How to cite this article: Jgou, A. et al. A Formin mDia1 senses and generates mechanical forces on actin laments. Nat. Commun. 4:1883 doi: 10.1038/ncomms2888 (2013).

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