IC generates power over a range of PNAS PLUS loads via a new tension-sensing mechanism

Michael J. Greenberg, Tianming Lin, Yale E. Goldman, Henry Shuman, and E. Michael Ostap1

The Pennsylvania Muscle Institute and Department of Physiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104-6085

Edited by Steven M. Block, Stanford University, Stanford, CA, and approved August 3, 2012 (received for review May 8, 2012)

Myosin IC (), a widely expressed motor that links the fine the myo1c -activated ATPase pathway in solution are actin to cell membranes, has been associated with very similar to (10, 12, 16, 19), a closely related myo- numerous cellular processes, including insulin-stimulated transport sin-I isoform that has actin-detachment kinetics that are exqui- of GLUT4, mechanosensation in sensory hair cells, endocytosis, sitely sensitive to tension (20). Myo1b is suited to function as of DNA in the nucleus, exocytosis, and membrane a tension-sensitive anchor, transforming from a low duty-ratio trafficking. The molecular role of myo1c in these processes has not motor to a high duty ratio-motor with forces resisting its working been defined, so to better understand myo1c function, we utilized stroke >0.5 pN. Moreover, the rate of actin translocation in the ensemble kinetic and single-molecule techniques to probe myo1c’s in vitro motility assay is similar for both myo1b (21) and myo1c biochemical and mechanical properties. Utilizing a myo1c construct (16) (when corrected for the differences in length of the light containing the motor and regulatory domains, we found the force chain–binding domain). Given the similarity of their unloaded dependence of the actin-attachment lifetime to have two distinct biochemical properties, one may have expected myo1c to share regimes: a force-independent regime at forces <1 pN, and a highly the force-sensing properties of myo1b (22, 23). force-dependent regime at higher loads. In this force-dependent To better define the molecular role of myo1c in the cell, we regime, forces that resist the working stroke increase the actin- examined the mechanical and kinetic properties of a myo1c con- 3 attachment lifetime. Unexpectedly, the primary force-sensitive struct containing the motor and regulatory domains (myo1c IQ) transition is the isomerization that follows ATP binding, not ADP at the ensemble level and the single-molecule level in the pre- BIOPHYSICS AND release as in other slow . This force-sensing behavior is sence and absence of force. We found that, compared to myo1b,

3 COMPUTATIONAL BIOLOGY unique amongst characterized myosins and clearly demonstrates myo1c IQ displays a more modest change in attachment lifetime 3 mechanochemical diversity within the myosin family. Based on in response to force. In fact, myo1c IQ’s actin-attachment lifetime these results, we propose that myo1c functions as a slow transpor- is insensitive to low forces (<1 pN), but very sensitive to higher ter rather than a tension-sensitive anchor. loads (>1 pN) that resist the powerstroke where increasing force on the myosin increases the attachment lifetime. Furthermore, 3 mechanochemistry ∣ optical tweezers ∣ transient kinetics the primary force-sensitive transition in myo1c IQ appears not to be associated with ADP release, as has been observed in many yosin IC (myo1c) is a widely expressed myosin-I isoform other slow myosins [including myo1b (20, 24), myosin-V (25), Mthat has been associated with several important cellular and smooth muscle myosin (26, 27)], but rather with an isomer- processes, including endocytosis (1), exocytosis (2) (including in- ization that follows ATP binding. This force-sensing behavior is sulin-stimulated GLUT4 translocation to the ; unique amongst characterized myosin isoforms. The response of 3IQ refs. 3–5), membrane ruffling (6), transcription of DNA in the myo1c to force implies that it is able to generate power over a nucleus (7, 8), and mechanosensing in sensory hair cells (9–13). range of loads, more consistent with its serving a role as a trans- Although it is known that myo1c links cell membranes to the actin porter than as a tension-sensitive anchor. Furthermore, these cytoskeleton (14, 15), its molecular role in these cellular pro- results clearly demonstrate mechanochemical diversity within the cesses has not been determined. For example, in its proposed role myosin-I family, possibly shedding light on the evolutionary im- in exocytosis, it is not known if myo1c acts as a motor for trans- peratives that resulted in the retention of eight distinct myosin-I port, moving vesicles into position for plasma membrane fusion in higher vertebrates. and/or as a tension-sensitive anchor that docks exocytic vesicles to the actin cytoskeleton and plasma membrane. Results Most members of the myosin family share the same kinetic Actomyo1c3IQ ATPase Ensemble Solution Kinetics. To c o r r e l a t e m e - pathway for ATP hydrolysis, in which force-generating structural chanical transitions in the optical trap with biochemical transitions, 1 3IQ changes are linked to release of inorganic phosphate and ADP, rate and equilibrium constants for key steps of the actomyo c but different myosin isoforms have evolved different biochemical ATPase pathway (Scheme 1) were determined at 20 °C using SI Text reaction rates and force-dependent kinetics to suit their cellular stopped-flow kinetic techniques (Table 1 and ). Some of functions. For example, in myosins that are thought to act as ten- these rates have already been measured under different buffer sion-sensitive anchors, the kinetic steps that limit actomyosin de- (10, 12) and temperature conditions (16); however, they were tachment are highly sensitive to load. In contrast, myosins that repeated here to ensure consistency between the ensemble and are thought to act as transporters have actin-detachment kinetics single-molecule experiments. The steady-state actin-activated that are less sensitive to load, allowing work to be performed over ATPase rate increased linearly with the actin concentration, a range of forces. Thus, insight into the molecular role of myosin

in the cell can be gained from evaluating the kinetic and mechan- Author contributions: M.J.G., Y.E.G., H.S., and E.M.O. designed research; M.J.G. performed ical properties of the motor. research; M.J.G., T.L., and H.S. contributed new reagents/analytic tools; M.J.G. analyzed Previous biochemical analyses have shown that myo1c is a low- data; and M.J.G., Y.E.G., H.S., and E.M.O. wrote the paper. duty ratio motor (i.e., it spends most of its biochemical cycle de- The authors declare no conflict of interest. tached from actin) (10, 12, 16), albeit with an actin-attachment This article is a PNAS Direct Submission. lifetime that is approximately 500-fold longer than fast skeletal 1To whom correspondence should be addressed. E-mail: [email protected]. muscle myosin-II (17) and approximately 10-fold longer than This article contains supporting information online at www.pnas.org/lookup/suppl/ the processive motor, myosin-V (18). The rate constants that de- doi:10.1073/pnas.1207811109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1207811109 PNAS Early Edition ∣ 1of8 Downloaded by guest on September 27, 2021 ADP Release Limits Actomyo1c3IQ Detachment at Low Forces in the Optical Trap. Single actomyosin interactions were detected using the three-bead assay, in which a single actin filament, suspended between two beads held by separate optical traps, is brought close to the surface of a pedestal bead that is sparsely coated with myo1c3IQ (20, 30, 31). Myo1c3IQ was attached specifically to streptavidin-coated pedestals by a biotinylation tag positioned directly C-terminal to the light chain–binding domain. Bead– Scheme 1. Pathway for the actomyosin ATPase cycle. actin–bead assemblies were constructed using a unique actin- attachment strategy. We could not use N-ethylmaleimide modi- fied myosin for these assemblies as we had done previously (20, yielding an apparent second-order rate constant (va ¼ 0.0046 30) because N-ethylmaleimide–treated myosin retains some 0.0006 μM−1 s−1)(Fig. S1A). The ATPase rate did not reach a ATP sensitivity that becomes problematic at the high ATP con- plateau within the actin concentrations tested, indicating that centrations used in the experiments described below. Further- the K (i.e., the concentration of actin at which half-maximal ATPase more, we could not use a biotin–streptavidin linkage because this activation is achieved) value is high (>150 μM) and the actin interferes with the biotin–streptavidin linkage used to attach the affinity is low (16). The apparent rate of ATP cleavage is −1 myosin site-specifically to the pedestal bead. Instead, we utilized 33 2 s (Fig. S1B). 3 the actin-binding domain of α- fused to the HaloTag ATP binding to actomyo1c IQ occurs in two steps (12). A rapid 0 product, enabling the experiments to be conducted at saturating collision complex with ATP (1∕K ¼ 97 15 μM) is followed 1 ATP concentrations while still utilizing streptavidin to attach the by a slower and effectively irreversible conformational change k 0 ¼ 26 0 8 −1 myosin specifically to the pedestal bead (for details, see Materials ( þ2 . s ) that precedes rapid dissociation of and Methods A myo1c3IQ from actin (Fig. S2). In the absence of nucleotide, ). Actomyosin-attachment events (Fig. 1 ) were actin-bound myo1c3IQ transits between a state that is able to bind identified by analyzing the force covariance of the trapped beads ’ (20) (for details, see Materials and Methods). ATP (AM ) and a state that cannot bind ATP (AM) (28). The 1 3IQ equilibrium constant for this transition favors the ATP-insensitive Actomyo c -attachment durations were first determined at low trap stiffness (approximately 0.02 pN∕nm) to minimize (AM) state (Kα ¼ 0.33 0.03) (Table 1). The rate of the transi- 3 −1 the force on actomyo1c IQ during the working stroke. The distri- tion from AM to AM’ (kþα ¼ 4.0 0.03 s ) is similar to the rate 3IQ 0 −1 bution of attachment durations acquired in the presence of 5 mM of ADP release from actomyo1c (kþ5 ¼ 3.9 0.06 s ) 1 3IQ K 0 ¼ ATP could be well fit by a single exponential function (Fig. 1B), (Fig. S3). Actomyo c has a high affinity for ADP ( 5 k ¼ 3 6 0 2 −1 0.22 0.05 μM). The affinity of myo1c3IQ for actin in the ab- yielding an actin-detachment rate ( det . . s ) that is similar to the measured rate of ADP release (k 0 ¼ 3.9 sence of nucleotide is tight (K6 ¼ 0.29 nM), and it is weakened þ5 0.06 s−1), consistent with ADP release limiting actomyosin by the addition of ADP (K10 ¼ 1.6 nM) (Fig. S3). The ratio of affinities (K10∕K6 ¼ KDA∕KA) has been termed the coupling detachment in the absence of force.

constant (22, 23), and the calculated value of 5.5 is similar to 40 previously published values for myo1b (28, 29); however, it is A B 35 larger than a previously reported value for myo1c (10). Based 30 on the solution kinetics, ADP release is expected to be rate limit- 25 20

ing for actomyosin detachment at saturating ATP concentrations; Events 15 -1 kdet = 3.6 ± 0.2 s however, a slower transition (likely associated with phosphate 10 nm 10 5 release) (16, 19) limits the rate of the overall ATPase cycle. The 10 s 3IQ 0 effective duty ratio of myo1c at 100 μM actin and saturating 00.511.522.5 ATP concentrations is 0.11. Time (s) C D 60 k Table 1. Rate and equilibrium constants for key steps of the reverse-fast 50 3 k 2.0 nm myo1c IQ ATPase cycle at 20 °C reverse-slow 40 = 7.8 nm kforward 30 = 13 nm ATP binding ADP release State 2 State 1 5.8 nm Events 20 0 0 ‡ 0.5 s 1∕K1 (μM) 97 ± 15* Kþ5 (μM) 0.22 ± 0.05 10 k 0 −1 k 0 −1 ‡ þ2 (s ) 26 ± 0.8* þ5 (s ) 3.9 ± 0.06 0 K 0k 0 (μM−1 s−1) 0.26 ± 0.04† k 0 (μM−1 s−1)18±4† -30 -20 -10 0 10 20 30 40 50 1 þ2 −5 Distance (nm)

Nucleotide-free isomerization step Actin binding and release Fig. 1. Optical-trapping experiments performed in the absence of an ap- ‡ 0 −1 ‡ plied load (369 binding events). (A) Sample data trace of single myosin inter- Kα 0.33 ± 0.03 kþ6 (s ) 0.0011 −1 ‡ −1 −1 ‡ kþα (s ) 4.0 ± 0.03 k−6 (μM s ) 3.4 ± 0.2 actions with actin acquired in the presence of 5 mM ATP. (B) The distribution −1 † † k−α 12 1 K6 ¼ KA of attachment durations in the presence of 5 mM ATP. A single exponential (s ) 0.29 nM −1 0 −1 ‡ function was fit to the data, giving a detachment rate (k )of3.6 0.2 s . kþ10 (s ) 0.011 det (C) Ensemble averages of single-molecule interactions were constructed as −1 −1 ‡ Steady-state actin-activated ATPase k−10 (μM s ) 6.2 ± 0.2 described in Materials and Methods. Fitting a single exponential function −1 −1 −1 § † to the time-forward average yields a rate (k )of4.3 s . Two exponen- kss (μM s ) 0.0046 ± 0.0005 K10 ¼ KDA 1.6 nM forward −1 § † tial functions were summed to fit the time-reverse average, with the major V (s ) >0.8 KDA∕KA 5.5 max 67 4% k amplitude component ( of the total amplitude) having a rate ( reverse-fast ) 27 3 −1 k 3 8 of s and the minor component having a rate ( reverse-slow )of . ATP hydrolysis 0.2 s−1. The size of the working stroke is equal to 7.8 nm, comprised of a first

app −1 ¶ substep of 5.8 nm followed by a 2.0-nm substep. (Inset) A cartoon of the displace- k3 (s )33±2 ment generated by the myo1c3IQ working stroke where the myosin sequentially *Light scattering. undergoes a substep from state 1 to 2 before detaching. (D) Distribution of the † Calculated. sizes of the total working stroke for myo1c3IQ. A single Gaussian function was fit ‡ Pyrene-actin fluorescence. to the data, and the reported error (σ) is the standard deviation of the Gaussian §NADH-linked assay. function. The total step has a size of 7.8 13 nm. Note that the large variance is ¶Intrinsic tryptophan fluorescence. caused by Brownian motion–driven fluctuations of the actin filament position.

2of8 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1207811109 Greenberg et al. Downloaded by guest on September 27, 2021 The myo1c working stroke has been shown to be composed of force, whereas at forces >1 pN, there is a transition to force-sen- PNAS PLUS k two substeps (12). We determined the substep sizes and kinetics in sitive detachment where det slows with increasing force. Force the presence of 5 mM ATP by ensemble averaging the time courses independence of detachment at forces <1 pN was verified by of individual actomyo1c3IQ interactions that were synchronized at comparing the distribution of attachment durations between the time when the interactions started (i.e., upon actomyosin at- −1 and 0 pN to those between 0 and 1 pN using a two-sample tachment) or ended (i.e., upon actomyosin detachment) (20, 24, Kolmogorov–Smirnov test (37), which showed no statistical dif- 32). Ensemble averages that were synchronized at actomyosin at- ference between these conditions (P ¼ 0.99). tachment were averaged forwards in time (time-forward averages) The force independence of actomyosin detachment at forces whereas the interactions that were synchronized at actomyosin <1 pN can be further tested utilizing the Bell equation (38), detachment were averaged backwards in time (time-reverse which describes the force dependence of a mechanochemical averages). Time-forward averages have a rapid initial substep to transition by: state 1 (5.8 nm displacement) followed by a slower increase to state − F·d 2, which has a final displacement of 7.8 nm (Fig. 1C and Table 2). k T kf ðFÞ¼kf 0 · e B· ; [1] The total step sizes measured from both the time-forward and time-reverse averages differ by only 5.6%, with the small difference where kf0 is the rate in the absence of load, d is the distance to the arising from limitations in selecting extension points for ensemble 3 transition state (a measure of force sensitivity, also known as averaging. The distribution of individual myo1c IQ displacements the distance parameter), kB is Boltzmann's constant, and T is is well described by a single Gaussian distribution centered at the temperature. Fitting the Bell equation to the data at forces 7.8 nm (Fig. 1D), agreeing with the ensemble averages. <0.5 pN yields a very small distance parameter for this limited The rate of exit from state 1 was determined by fitting the time dataset (d0 5 ¼ 0.12 0.49 nm), reinforcing the force inde- course of the time-forward average (Fig. 1C) with a single expo- . pN k ¼ 4 3 0 1 −1 pendence of actomyosin detachment at low forces. nential function ( forward . . s ). This rate is similar to 0 −1 We modeled the observed detachment rate for the entire range the measured ADP-release rate (kþ5 ¼ 3.9 0.06 s ), and it is of forces as two sequential steps, one force-independent (ki) and consistent with the second substep of the working stroke being one force-dependent [kf ðFÞ]: associated with ADP release as shown previously for myo1c (12) and other myosins (20, 24, 25, 27, 33). The rate of exit from ki kf ðFÞ kf ðFÞ ki state 2 was determined by fitting the time course of the time-re- A→B → C or A → B→C [2]

verse average by exponential functions. We found that the data BIOPHYSICS AND

were best fit by the sum of two exponentials, with the major com- This modeling alone cannot distinguish the order of the steps COMPUTATIONAL BIOLOGY k ¼ 27 3 −1 67 4% ponent having a faster rate ( reverse-fast s ; of because the two models in Eq. 2 have the same force dependence k ¼ the total amplitude) than the minor component ( reverse-slow of the actomyosin-detachment rate, and thus the probability den- 3 8 0 2 −1 k . . s ). The reverse fast rate is similar to the rate of the bio- sity functions used for maximum-likelihood estimation are identi- - 3 chemical step that limits actomyo1c IQ detachment from state 2 cal. The force-dependent rate was modeled using the Bell equation k 0 ¼ 26 0 8 −1 1 k ( þ2 . s ; Scheme 1 and Table 1). (Eq. ), and thus the detachment rate ( det) is given by:

Force Dependence of Actomyo1c3IQ Detachment. The force sensitiv- 1 1 1 1 1 3 ¼ þ ¼ þ [3] 1 IQ F d ity of the rate of myo c detachment from actin was measured k ðFÞ k k ðFÞ k − · det det i f i k ·T using a feedback system that keeps the actin near its isometric kf0 · e B position while myosin undergoes its working stroke (20, 30, 34, 35). Forces applied to the actomyo1c3IQ are determined by the Maximum-likelihood estimation was used to fit the model to stiffness of the actomyo1c3IQ (and its linkage to the substrate), distribution of attachment durations (Fig. 2A), and bootstrapping the position at which myosin binds to the actin filament (which was used to determine confidence limits (20). We determined va- −1 is stochastic) (36), and the size of the working stroke (Materials lues for the force-independent rate [ki ¼ 5.6ðþ1.6∕−0.8Þ s ], and Methods). the force-dependent rate in the absence of force [kf0 ¼ At forces less than 1 pN, the durations of actin attachments 29ðþ9∕−6Þ s−1], and the distance parameter for detachment d ¼ 5 2ðþ0 5∕−0 6Þ k appear to be force-independent (Fig. 2A). Only at forces that ex- [ det . . . nm]. The rate i is similar to the acto- ceed 1 pN does there appear to be an increase in the mean dura- myosin-detachment rate determined in the absence of force k ¼ 3 6 −1 B tion of attachments. The force dependence of the duration of ( det . s ;Fig.1), the rate of the exponential increase actin binding is better visualized by plotting the inverse average resolved in the time-forward ensemble average recording k ¼ 4 3 −1 C of attachment durations binned by force (Fig. 2B) to yield detach- ( forward . s ;Fig.1 ), and the rate of ADP release mea- k 1 k k 0 ¼ 3 9 −1 ment rates ( det). At low forces (< pN), det is independent of sured by stopped-flow kinetics ( þ5 . s ; Table 1). Surpris-

Table 2. Single molecule optical trapping values Working stroke displacement State 1 5.8 nm State 2 7.8 nm

Attachment duration (5 mM MgATP) k 3 6 0 2 −1 det . . s

Force dependence of the attachment lifetime 5 mM MgATP 1 mM MgATP, 50 μM ADP 1 mM MgATP (FPLC purified)* 5 mM MgATP (high gain) −1 ki (s ) 5.6ðþ1.6∕−0.8Þ 4.0ð0.5Þ 7.4ðþ3.9∕−0.3Þ 6.7ðþ3.3∕−1.2Þ −1 kf0 (s ) 29ðþ9∕−6Þ 25ðþ5∕−2Þ 24ðþ10∕−13Þ 33ðþ12∕−9Þ d 5 2ðþ0 5∕−0 6Þ 6 3ðþ0 6∕−0 5Þ 4 3ðþ1 5∕−0 4Þ 6 2ðþ0 9∕−1 1Þ det (nm) ...... *Experiment was conducted at a higher ionic strength due to the salt necessary to elute the ATP from the column.

Greenberg et al. PNAS Early Edition ∣ 3of8 Downloaded by guest on September 27, 2021 A 10 transition in myo1c3IQ is not ADP release, as it is in many other slow myosins; rather, the step that follows ATP binding is force sen- sitive and limits the rate of detachment at high forces (see below). The rate at which the feedback loop applies force to keep the 1 actin near its isometric position is set by the response time of the feedback loop, 50 ms (20). To determine if the actomyo1c3IQ-at- tachment durations were sensitive to the rate of the feedback re- sponse, we acquired interactions with an approximately 10-fold 0.1 faster feedback–response time (5–7 ms; Table 2 and Fig. S4) and found no change in the force dependence of the actomyosin- Attachment Duration (s) Duration Attachment -1 0 1 2 3 4 5 detachment rate. Force (pN) 100 The Force-Dependent Transition Occurs After ADP Release. Because B the rate of the biochemical step that limits actomyo1c3IQ detach- ment from state 2 appears to be the primary force-sensitive tran- 10 sition, we examined the force dependence of the lifetime of state )

-1 2 using the isometric optical clamp. Individual attachment events

(s 1 were binned based on the average interaction force and then

det time-reverse ensemble averages were constructed as described k 0.1 earlier. Fitting single exponential functions to the time-reverse Assisting Resisting ensemble averages shows that the lifetime of state 2 is strongly force-dependent (Fig. 2C). At forces below 1 pN, the rate of exit 0.01 3IQ -1012345 from state 2 is faster than the rate of actomyo1c detachment, >1 Force (pN) but at forces pN, the rate of exit from state 2 is equal to the force-dependent detachment rate (Fig. 2B). Fitting the Bell equa- C 6 tion (Eq. 1) to the rates from the loaded time-reverse ensemble 5 averages (Fig. 2B, dashed line) yields a rate at zero force −1 (kf0 )of26 2 s , which is similar to the rate of the fast- 4 -ens avg exponential increase in the time-reverse averages in the absence k ¼ 27 3 −1 C 3 of load ( reverse-fast s ; Fig. 1 ) and the rate of the biochemical transition that limits actomyo1c3IQ detachment from 2 0 −1 state 2 (kþ2 ¼ 26 0.8 s ; Scheme 1 and Table 1). Further- Force (pN) Force 1 d more, the distance to the transition state ( ens avg) of this step is 0 4.6 0.8 nm, similar to the distance parameter of the transition d ¼ 5 2ðþ0 5∕ -1 that limits overall actomyosin detachment [ det . . -7 -6 -5 -4 -3 -2 -1 0 1 −0.6Þ nm]. Thus, the force-dependent transition that limits de- Time (s) tachment at loads greater than 1 pN occurs after the second step Fig. 2. Force sensitivity of myo1c3IQ in the presence of 5 mM ATP. (A) Using of the working stroke and before detachment. the isometric optical clamp to apply a load to the myosin, the force on the 3IQ myosin and the actomyosin-attachment duration were measured for each Added ADP Has Little Effect on the Force Dependence of Actomyo1c single-molecule interaction (n ¼ 670). The 90% confidence intervals were cal- Detachment. The force-dependent behavior of some myosins has culated by maximum-likelihood estimation (MLE) of 1,000 bootstrap simula- been shown to be highly sensitive to the presence of ADP (39, tions of the data (see Materials and Methods). The overall detachment rate as 40). Therefore, we performed experiments with the isometric op- a function of force was modeled as the sum of two rates, one force-depen- tical clamp engaged in the presence of 50 μM ADP and 1 mM dent and one force-independent (Eq. 3). The rate of the force-independent −1 ATP (Fig. 3). These conditions resulted in force-dependent de- transition (ki ) is equal to 5.6ðþ1.6∕−0.8Þ s . The rate of the force-depen- −1 tachment rates similar to those acquired in the presence of 5 mM dent transition in the absence of force (kf0) is equal to 29ðþ9∕−6Þ s with d 5 2ðþ0 5∕−0 6Þ ATP. Fitting the attachment durations as a function of force a distance parameter ( det)of . . . nm. (B) For the sake of visua- lization, events were ordered by the average force on the myosin, and sets of (Fig. 3A) by Eq. 3 yields values that are equivalent to the 5 mM 10 points were binned and converted to detachment rates. The thick black ATP data (Fig. 2A and Table 2). Thus, the presence of ADP does 3 line shows the calculated detachment rate as a function of force based on the not substantially affect the load-dependent kinetics of myo1c IQ. MLE fitting of the unaveraged data (Fig. 2A). The shaded regions show the We also performed experiments using the isometric optical clamp 90% confidence interval. Colored diamonds show the rate of the time- with 1 mM FPLC-purified ATP to decrease ADP contamination to – reverse ensemble averages binned by force (C). The force bins are 0 1pN <1% of the ATP concentration (see Materials and Methods). We (red), 1–2 pN (orange), 2–3 pN (yellow), 3–4 pN (green), and >4 pN (purple). k observed the same biphasic force response, and the parameters ob- The black diamond is the rate ( reverse-fast) at zero force. The dotted line is a fit 3 of the Bell equation to the rates of the time-reverse ensemble averages as a tained from fitting Eq. to the data are similar to the values ob- k ¼ 26 2 −1 – function of force, giving a rate of f0-ens avg s with a distance para- tained using 5 mM non FPLC purified ATP (Table 2 and Fig. S4). d 4 6 0 8 meter ( ens avg)of . . nm. (C) Time-reverse ensemble averages binned by average interaction force. The force bin ranges and colors are as in (B). Single Discussion exponential functions were fit to the data, and these rates are plotted (B). To better understand the molecular role of myo1c in the cell, we utilized ensemble and single-molecule techniques to characterize 3IQ ingly, the force-dependent rate in the absence of force, kf0,issi- the mechanochemical cycle of myo1c . Utilizing a novel bead– milar to the rate of the fast exponential increase resolved in the actin–bead attachment strategy to maintain high actin pretension k ¼ 27 3 −1 C time-reverse average ( reverse-fast s ;Fig.1 ) and the in the presence of 5 mM ATP, we found that the actin-attachment rate of the biochemical step that limits actomyo1c3IQ detachment lifetime of myo1c3IQ increases only with forces >1 pN and that it 0 −1 from state 2 (kþ2 ¼ 26 0.8 s ; Scheme 1 and Table 1). Note is much less sensitive to force than the closely related myosin-I that decreasing the width of the smoothing window in the data ana- isoform, myo1b (20). Furthermore, the primary force-sensitive lysis to allow for detection of events as short as 15 ms did not transition in myo1c3IQ is not ADP release [as has been seen in change these values (Fig. S4). Thus, the primary force-sensitive other slow myosins (25, 27), including myo1b (20)], but is rather

4of8 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1207811109 Greenberg et al. Downloaded by guest on September 27, 2021 10 A was facilitated by the development of a unique bead–actin–bead PNAS PLUS attachment strategy (see Materials and Methods). k ¼ 3 8 0 2 −1 The origin of the minor slow phase ( reverse-slow . . s ) in the time-reverse average (Fig. 1C) is unclear. This slow rate is 1 similar to the rate of ADP release (3.9 0.06 s−1), so it is pos- sible that this component arises from rotation of the lever arm and population of state 2 before ADP release occurs (33% of the time), consistent with the finding that nucleotide and struc- 0.1 tural states are not necessarily tightly coupled (41). This rate is

Attachment Durations (s) Durations Attachment also similar to the rate of exit from the ATP-insensitive AM state −1 (kþα ¼ 4.0 0.03 s ; Table 1). However, the agreement of the 100 B biochemical kinetics (Table 1) with the actin-attachment lifetimes (Fig. 1B and Table2) suggests that the nucleotide-insensitive state 10 (AM; Scheme 1) is not substantially populated in the presence of )

-1 5 mM ATP. It is worth noting that this slow phase is not resolved (s 1 in the traces collected with the isometric optical clamp engaged, det

k likely because of noise and the fact that it becomes obscured by the slowing of the fast rate with force (see below). Further studies 0.1 are necessary to determine the origin of this slow phase.

0.01 3IQ -1.0 0.0 1.0 2.0 3.0 4.0 5.0 Myo1c Force-Dependent Detachment. The rate at which 1 3IQ Force (pN) myo c detaches from actin is largely independent of load at forces <1 pN, but is force-dependent at higher loads (Fig. 2). Fig. 3. Force sensitivity of myo1c3IQ in the presence of ADP. The isometric If we assume a sequential three-state model with a force-sensitive 3 optical clamp was used to examine the force sensitivity of myo1c IQ at 50 μM step that follows the Bell model (Eq. 3) (38), we conclude that the n ¼ 282 ADP and 1 mM ATP ( ). (A) Scatter plot of individual single-molecule rate of the force-sensitive transition is faster than the transition attachment durations as a function of force . The overall detachment rate as that limits detachment at low loads, yet it becomes rate-limiting a function of force was modeled as the sum of two rates, one force-depen- dent and one force-independent (Eq. 3). The best-fit parameters were calcu- for detachment when the force on the myosin exceeds 1 pN. Be- BIOPHYSICS AND

lated using MLE fitting and the 90% confidence intervals were calculated by cause ADP release limits the overall actomyosin-detachment rate COMPUTATIONAL BIOLOGY −1 bootstrapping. The force-independent rate (ki ) is equal to 4.0ð−0.5Þ s . The in the absence of force, ADP release is not the primary force-de- −1 force-dependent rate (kf0) is equal to 25ðþ5∕−2Þ s with a distance para- pendent transition as it is in some other myosins (20, 24, 25, 27). d 6 3ðþ0 6∕−0 5Þ meter ( det)of . . . nm. (B) For the sake of visualization, the de- This result is notable, because ADP release is associated with a tachment rates (the reciprocal of the attachment durations) were binned by mechanical step of 2.0 nm (Fig. 1). In fact, fitting Eq. 1 to the data force and averaged. The thick black line shows the best-fit curve and the acquired at forces <0.5 pN (i.e., where the rate of ADP release grey-shaded regions show the 90% confidence intervals. All MLE fitting limits actomyosin detachment) yields a very small distance para- and bootstrapping was performed on the unaveraged data (Fig. 3A). d ¼ 0 12 0 49 meter ( 0.5 pN . . nm), which is far smaller than the size of the second mechanical step. Because the distance para- the isomerization following ATP binding (see below). These re- meter for this transition is small, the position of the transition sults help to shed light on the molecular role of myo1c in the cell state along the reaction coordinate must be closer to the and highlight the diversity of mechanochemical tuning within the AM.ADP state than the AM state (42). This feature contrasts myosin superfamily. with other characterized myosins, where the size of the distance

3IQ parameter is similar to the size of the mechanical step (27, 33), The Myo1c Working Stroke. We resolved two actin-bound me- suggesting the position of the transition state is closer to the AM 1 3IQ chanical states populated during the myo c working stroke state in these myosins. C (Fig. 1 ), as described previously (12). State 1 has a displacement A fit of Eq. 3 to the attachment durations indicates that the −1 of 5.8 nm and a lifetime equivalent to the AM.ADP biochemical force-dependent rate in the absence of force (kf0 ¼ 29 s ) is si- state (Scheme 1 and Tables 1 and 2). Thus, exit from state 1 is milar to the rate of the biochemical step that follows ATP binding 1 3IQ k 0 0 1 associated with the release of ADP from actomyo c ( þ5 ). (kþ2 ¼ 26 0.8 s ; Scheme 1 and Table 1) and the rate of the Furthermore, this transition limits the overall actin-detachment time-reverse ensemble average in the absence of applied force k ¼ 27 s−1 1 rate at low loads and saturating ATP concentrations. ( reverse-fast ). Fitting Eq. to the force dependence of State 2 has a displacement of 7.8 nm (2.0 nm larger than state the rates of the time-reverse ensemble averages in the presence 1). At subsaturating ATP concentrations, the rate of exit from of load yields a similar rate for the force-sensitive transition in the k ¼ 26 −1 state 2 has been shown to correlate with the second-order rate absence of force ( f0-ens avg s ) with a distance parameter d ¼ 4 6 constant for ATP binding in other myosins (20, 24, 27). Thus, at ( ens avg . nm) that is similar to the distance parameter for low ATP concentrations where ATP binding is rate-limiting, state the transition that limits force-dependent detachment at forces >1 d ¼ 5 2 2 is populated by the nucleotide-free AM’ biochemical state. Our pN ( det . nm). It is unlikely that the formation of the K 0 experiments were performed at a saturating ATP concentration AM(ATP) collision complex ( 1 ) is the primary force-depen- dent step because this would result in a decrease in the second- (5 mM), and so, based on the transient-kinetics experiments, exit k k 0 order rate constant for ATP binding, which would decrease f0 at from state 2 is expected to be limited by the rate of þ2 , the iso- 3 C lower ATP concentrations (Eq. ). However, experiments per- merization following ATP binding (Fig. 1 and Scheme 1). This formed at 1 mM ATP (Fig. 3 and Fig. S4) and 5 mM ATP (Fig. 2B) conclusion is supported by the finding that the predominant com- show the same relationship between the detachment rate and k ¼ ponent of the time-reverse averages has a rate ( reverse-fast force (Table 2). Therefore, our data support the primary force- 27 3 −1 s ) similar to the rate of the biochemical transition that dependent transition as the isomerization between ATP binding k 0 ¼ 26 0 8 −1 0 limits actomyosin detachment from state 2 ( þ2 . s ; and actomyosin detachment (kþ2 ) (Fig. 4A). It is important to Table 1). Thus, AM(ATP) is the predominant biochemical inter- note that other transitions may be force-sensitive; however, these mediate of state 2 under our experimental conditions (Scheme 1). force sensitivities are likely of secondary importance because they Probing the myosin mechanics at saturating ATP concentrations do not limit detachment under load.

Greenberg et al. PNAS Early Edition ∣ 5of8 Downloaded by guest on September 27, 2021 A Force-Insensitive Force-Sensitive 25, 27). For example, myo1b is a closely related myosin-I isoform Transition Transition with ATPase kinetics similar to myo1c, but the primary force- (Limits rate of detachment at (Limits rate of detachment at forces <1 pN) forces >1 pN) dependent transition is associated with ADP release (20). More- Pi ADP over, the myo1b-attachment lifetime is extremely sensitive to ATP applied loads (20, 30). The distance parameter for myo1ba (a ATP ADP.Pi myo1b splice isoform with six IQ motifs) is 18 nm, more than threefold greater than the distance parameter for myo1c3IQ (30). This dramatic force sensitivity enables myo1b to function B 1 C 25 as a tension-sensitive anchor, changing from a low- to a high-duty ) 80 0.8 -1 20 myo5 ratio motor with forces of <2 pN. Although it has been proposed SM myo2 40 that myo1c also acts as a tension sensor (10, 12, 45), the data pre- 0.6 myo1b 15 3 0 1 IQ 02.55 sented here show that myo c is substantially less force-sensi- 0.4 10 Force (pN) myo1c B myo1c tive than myo1b (Fig. 2 ). The structural basis for these Duty Ratio Duty 0.2 5 differences is unclear without high-resolution information.

Power (pN nm s myo1b 0 It is worth noting that ATP binding has been proposed to be 012345 0012345 Force (pN) Force (pN) the primary force-sensitive transition in myosin-VI (46), although this mechanism is still debated (39, 47, 48). The force-dependent Fig. 4. Comparison of myo1c3IQ and the closely related isoform, myo1b ATP-binding model proposes that myosin-VI’s primary force-de- (splice isoform “a”). (A) Model of myo1c (blue) interacting with actin (red) pendent transition is ATP binding (46) and not the isomerization in the presence of force. The transition corresponding to ADP release [the following ATP binding as seen here for myo1c3IQ. Moreover, the primary force-sensitive transition in myo1b (20), smooth muscle myosin-II force sensitivity of myo1c3IQ is different from myosin-VI in that it (27), and myosin-V (25)] is force-insensitive for myo1c3IQ and rate-limiting for actomyosin detachment at forces less than 1 pN. At forces in excess of has been shown that the addition of ADP causes an increase in 1 pN, the primary force-sensitive transition (the isomerization that follows the force sensitivity of myosin-VI (39, 47, 49), whereas ADP has 3IQ ATP binding) becomes rate-limiting for detachment. (B) The duty ratios of no appreciable effect on the force sensitivity of myo1c (Fig. 3). 3 myo1c3IQ (blue) and myo1b (red), calculated from the transient-kinetics and The basis for these differences between myo1c IQ and myosin-VI optical-trapping experiments (20), show that the duty ratio of myo1c3IQ is is not clear; however, the force-sensing mechanism of myo1c3IQ substantially less force-sensitive than myo1b. (C) The calculated average appears to be unique amongst all myosins that have been me- 3 power output (see Materials and Methods)ofmyo1c IQ (blue), myo1b chanochemically characterized. (red) (20), smooth muscle myosin-II (purple) (27), and myosin-V (Inset) (18, It has been suggested that the ADP coupling constant (i.e., 25) as a function of force. Note that myo1c3IQ is substantially different from myo1b. Myo1b has a very low power output that approaches zero with very the ratio of the dissociation constant of actomyosin in the pre- little force, consistent with myo1b acting as a tension-sensitive anchor. sence of ADP to the dissociation constant in the absence of ADP, Myo1c3IQ, on the other hand, has a higher power output and is able to gen- KDA∕KA) is an indicator for whether a myosin is a tension sensor erate power over a range of forces, more consistent with its functioning as a or a fast mover. A coupling constant of <10 is viewed as a sig- 3 transporter rather than as a tension-sensing anchor. The power output of nature for a tension sensor (22, 23), and myo1c IQ has a coupling myo1c3IQ is lower than smooth muscle myosin-II or myosin-V because of its constant of 5.5, placing it in the tension-sensor class. Myo1c3IQ slower overall kinetics; however, all three of these myosins are able to gen- (10) and myo1b (28, 29) have similar coupling constants and erate power over a range of forces. kinetic rate constants, and thus one would expect them to have similar mechanics and functions; however, this is clearly not the Interestingly, the distance parameters for ATP binding and the case (Figs. 3 and 4 B and C). Furthermore, myo1b and myo1c3IQ subsequent isomerization in myosin-V (25) (d ¼ 0.9 nm), smooth have different kinetic steps as their primary force-sensitive tran- muscle myosin-II (27) (d ¼ 1.3 nm), and myo1b (20) (d ¼ sitions, something not apparent from an analysis of the solution 2.5 nm) are much smaller that the distance parameter for the iso- kinetics alone. Previous assignments of the molecular roles of 3 merization following ATP binding in myo1c IQ (5.2 nm). How the other myosins based solely on kinetics should thus be revisited size of this distance parameter is related to conformational using mechanical measurements. changes in the nucleotide and actin-binding sites requires further investigation. The Molecular Role of Myo1c. The potential molecular roles for At low ATP concentrations (≤20 μM), the actomyosin-detach- myo1c can be broadly divided into two categories with distinct ment rate at low forces is limited by the apparent second-order kinetic and mechanical properties: transporter and tension-sen- 0 0 rate constant for ATP binding (K1 kþ2 ) and not ADP release. sitive anchor. A transporter, such as myosin-V,needs to be able to Because the force-independent regime at high [ATP] occurs over generate productive work in the presence of a load, and as such, the range of forces where the rate of ADP release is slower than its power output needs to be high and sustained over a range of ATP binding, a force-independent regime at forces <1 pN would forces. On the other hand, a tension-sensitive anchor, such as not be observed at low [ATP], and the actin-detachment rate myo1b, needs to be able to remain attached to actin in the pre- would appear to be sensitive to all forces > − 1 pN. This finding sence of load (i.e., its attachment duration is very force-sensitive), may explain a previous report of myo1c force dependence con- and as such, its power output should be low and sensitive to load. ducted at 20 μM ATP, which suggested that kinetics of actomyo1c The force sensitivity of myo1c3IQ (Figs. 2B and 4B) and its power detachment were sensitive over the range of all of the forces ex- output over a range of forces (Fig. 4C) are more consistent with amined (-1 to +1 pN) (43). Our results reinforce the importance myo1c acting as transporter, albeit a slow one, rather than as a of performing experiments at saturating ATP concentrations be- tension-sensitive anchor. The slower kinetics of myo1c may allow cause different force-dependent transitions may be observed at for the transport of cargos through highly cross-linked actin subsaturating ATP concentrations (44). It is worth pointing out meshworks without compromising their integrity because the 1 3IQ k that the myo c construct examined in these studies contained dissociation rate of cytoskeletal cross-linkers are similar to det the motor and regulatory domains. Future studies utilizing the (50, 51). In contrast, cargo transport by myosins with faster step- full-length myo1c molecule will show whether the tail domain ping kinetics, such as myosin-V, would require disassembly of the plays a role in modulating myo1c mechanochemistry. actin network because the motor’s stepping rate is faster than the dissociation rate of cytoskeletal cross-linkers. The maximum 3 Relationship to Other Myosins. ADP release is the primary force- power output of myo1c IQ is less than that of smooth muscle myo- sensitive transition for several characterized myosins (20, 24, sin-II and myosin-V because of its slower biochemical kinetics;

6of8 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1207811109 Greenberg et al. Downloaded by guest on September 27, 2021 however, all three myosins are similar in that they are able to gen- 1 mM and allowed to incubate with the beads for 1 h at room temperature. PNAS PLUS erate power over a range of forces, enabling them to accomplish The beads were then washed three times in KMg25 buffer and resuspended work. It is interesting to note that, unlike myosin-VI (39), the with 100–200 μM HaloTag α-actinin in KMg25 buffer. This mixture was force-sensing behavior of myo1c3IQ is not highly ADP-sensitive, allowed to incubate for 1 h at 37 °C before being washed three times in KMg25 buffer. The beads were then resuspended in KMg25 buffer and either potentially enabling myo1c to generate power without stalling at used within 1.5 weeks or flash-frozen in liquid nitrogen and stored at −80 °C. cellular concentrations of ADP. HaloTagged α-actinin beads were able to bind to actin and maintain preten- Myo1c has been proposed to be the slow adaptation motor in sions in excess of 6 pN for greater than 15 min, even in the presence of sensory hair cells where it senses changes in the tension of the tip saturating (5 mM) ATP. link (9–13, 45). If this is myo1c’s role, then models need to incor- porate the motor’s unique tension-sensing properties. New mod- Motility Chamber Preparation. Nitrocellulose-coated motility chambers con- els of adaptation may require myo1c to work in ensembles to taining 1.9 μm silica beads as pedestals (Bangs Laboratories) were prepared maintain a dynamic stall where ATP would be continuously hy- as described (20). Solutions were added sequentially to the chamber as 0 1 ∕ 1 ∕ drolyzed to maintain tension, rather than acting as a myo1b-like follows: . mg mL streptavidin in water (5 min); mg mL bovine serum al- – 1 3IQ 25 þ tension-sensitive anchor. One of the assumptions of the current bumin in KMg25 (2 times 5 min); 2 10 nM biotinylated myo c in KMg 2 μM calmodulin (5 min); washed 2-times with KMg25 buffer; and 1 nM TRITC model of adaptation is that calcium will cause a decrease in the phalloidin–labeled F-actin in KMg25 with 1 mg∕mL glucose, 1 mg∕mL bovine force sensitivity of the adaptation motor, leading the myosin to serum albumin, the desired concentration of ATP, 2 μM calmodulin, detach from actin. Although our experiments have not examined 192 U∕mL glucose oxidase, and 48 μg∕mL catalase (Roche). Beads coated the effect of calcium on myo1c, our previous experiments with with HaloTagged α-actinin were added to one side of the chamber to replace myo1b showed that the distance parameter is proportional to approximately one-fourth the volume of the chamber. The chamber was the size of the working stroke (30) and that calcium causes a sealed with silicon vacuum grease (Dow Corning). reduction in both (52). In contrast, it has been suggested that calcium increases the size of the myo1c working stroke (12), and Optical Trapping. Single-molecule actomyosin interactions were recorded thus one would potentially expect calcium to increase the force using the three-bead assay geometry in a dual-beam optical-trap system as described (20, 31). For each bead–actin–bead assembly, the trap stiffness sensitivity, leading to prolonged attachment. Future studies are and the system-calibration factor were determined by fitting a Lorentzian warranted to examine the effect of calcium on myo1c mechano- function to the power spectrum. Trap stiffnesses were approximately 0.02– chemistry at saturating ATP concentrations. 0.03 pN∕nm. Bead–actin–bead assemblies were pre-tensioned to 3–6 pN and lowered onto the surface of a bead pedestal using a piezoelectric stage con-

Materials and Methods troller to scan for actomyosin interactions. Upon observation of interactions, BIOPHYSICS AND Reagents and Buffers. ADP and ATP concentrations were determined before

data were filtered at 1 kHz and digitized with a 2-kHz sampling rate for up to COMPUTATIONAL BIOLOGY −1 −1 each transient-kinetic experiment by absorbance (ε259 ¼ 15;400 M cm ). 10 min. Single-molecule conditions were verified by (i) diluting the myosin The purity of ATP and ADP was assessed by running a 0–1 M salt gradient such that approximately one in three pedestals yielded actomyosin inter- over a FPLC MonoQ column with bound ATP or ADP. No differences in myo- actions, and (ii) examining the distributions of covariances for each single- sin-loaded or -unloaded kinetics in the optical trap were observed using FPLC- molecule trace (20) (see below). purified ATP, and thus, unless otherwise noted, non–FPLC purified (99% pure) The force dependence of actomyosin-attachment lifetimes was measured ATP was used. Unless stated otherwise, all experiments were performed in using an isometric optical clamp that applies a dynamic load to the actomyo- KMg25 buffer (10 mM MOPS, 25 mM KCl, 1 mM MgCl2, 1 mM EGTA, and sin to keep the actin filament near its isometric position during the myosin 1 mM DTT). ATP was always added as MgATP (e.g., solutions containing working stroke as described (20, 34, 35). Briefly, changes in the force on the 5 mM ATP contained 6 mM total MgCl2). All transient-kinetics experiments bead attached to the pointed end of the actin filament (transducer bead) were conducted in the presence of 1 μM free calmodulin (i.e., in excess of the were fed through an analog-integrating feedback amplifier to an acousto- calmodulin already bound to the myosin from the purification), and all optic deflector, which moved the position of the bead bound to the barbed optical-trapping experiments were conducted in the presence of at least end of the actin filament (motor bead) until the position of the transducer 2 μM free calmodulin. Apyrase VII was obtained from Sigma. bead was restored to its original position. The response time of the feedback loop in the absence of interactions was adjusted to 50 ms. A positive force is Protein Preparation. The mouse myo1c construct used in these studies defined as a force that opposes the myosin working stroke. consisted of the motor domain, complete regulatory domain with three IQ motifs, a C-terminal FLAG tag for affinity purification, and a C-terminal Event Selection and Ensemble Averaging. To determine the start and end AviTag for site-specific biotinylation (16). Actin was purified from rabbit points of actomyosin attachments, a covariance-threshold selection was used skeletal muscle as described (53). Actin concentrations were determined as described (20), using custom software written in LabView (National Instru- −1 −1 by absorbance at 290 nm (ε290 ¼ 26;600 M cm ). Calmodulin was pre- ments). The covariance of the transducer and motor beads was calculated pared as described (54). For single-molecule experiments, actin was polymer- with an 85-ms sliding window and the resulting signal was smoothed over ized and fluorescently labeled in the presence of 1∶1 molar equivalent TRITC a 50-ms sliding window to construct distributions of covariance values (20). phalloidin (Sigma). For transient-kinetics experiments utilizing N-(1-pyrenyl) The distribution of covariances was bimodal, with one peak corresponding to iodoacetamide–labeled actin (pyrene actin), the actin was labeled, gel fil- the detached state and the other corresponding to the actomyosin-attached tered (55), and stabilized with a 1∶1 molar ratio of unlabeled phalloidin. state. The beginning of the interaction is defined as the time when the mea- sured covariance became smaller than the covariance at the minimum value HaloTag α-Actinin–Coated Beads. Single-molecule optical-trapping experi- between the two covariance peaks. The end of the interaction was defined as ments conducted at saturating ATP require an ATP-insensitive linkage that the point where the covariance rose above that same threshold. To reduce does not interfere or compete with the biotin–streptavidin linkage used false-positive events caused by transient decreases in covariance during un- to attach the myosin specifically to the pedestal bead. To achieve this, the attached periods, only events longer than 50 ms (the width of the smoothing cDNA sequence for the actin-binding domain of human α-actinin 1 (ACTN1) window) were scored as events. Changing the width of the smoothing win- (residues 30–253) (56) containing a 5′ NdeI site and a 3′ BamH1 site was in- dow to 15 ms to allow for detection of events as short as 15 ms did not change serted into the pET28a vector. The pET28a vector also contains a N-terminal the force-dependent behavior (Fig. S4). Ensemble averages were constructed hexahistadine tag for protein purification. The HaloTag cDNA sequence was by synchronizing single-molecule interactions and then averaging the inter- cut from the pFC20A vector (Promega) between the BamH1 and HindIII sites actions forward and reverse in time as described (20, 24, 32). and inserted into the pET vector containing the α-actinin gene insert. This vector was inserted into BL21(DE3) cells and expressed via IPTG induction. Power-Output Calculations. The power output of the different myosin iso- The protein was purified by running the cell lysate over a NiNTA column fol- forms was calculated as the average power per myosin head. To do this, the – VðFÞ VðFÞ¼w k ðFÞ lowed by a FLPC MonoQ column (0 1-M KCl gradient). velocity as a function of force, , was calculated as: · det , α – w k ðFÞ HaloTag -actinin conjugated beads were prepared as follows. First, where is the size of the total working stroke and det is the force-de- 1.0-μm diameter amino-functionalized polystyrene beads (2.5% solids; Poly- pendent actomyosin-detachment rate. The power produced during actin at- sciences) were washed twice in distilled water and then resuspended in PBS, tachment is equal to the product of the force on the myosin and the velocity, pH 7.0. HaloTag succinimidyl ester O2 ligand (Promega) was then added to VðFÞ, at that force. Finally, the average power was calculated by multiplying

Greenberg et al. PNAS Early Edition ∣ 7of8 Downloaded by guest on September 27, 2021 the power produced by myosin during actin attachment by the myosin duty The force-dependent lifetimes are exponentially distributed at each force,

krls ratio, r, where the duty ratio is given by: rðFÞ¼k þk ðFÞ and k is the rate- and thus maximum-likelihood estimation was used to determine the para- rls det rls limiting step for the detached states [e.g., kss · ½actin for myo1c]. Thus, the meter values for these experiments (20). Confidence intervals for each un- average power per myosin head, PðFÞ, is given by the expression: iquely determined parameter were found by simulating the data with a bootstrap routine; 1,000 bootstrapped datasets were generated and fit inde- k pendently for all of the relevant parameters. The 90% confidence intervals PðFÞ¼F w k ðFÞ rls : [4] · · det · k þ k ðFÞ were determined by numerically ordering the values determined by boot- rls det strapping and then finding the bounds that contain 45% of the data from Statistics and Curve Fitting. Single or double exponential functions were the median value. fitted to stopped-flow fluorescence transients using the software supplied with the instrument. Fitting of the kinetic rates as a function of nucleotide ACKNOWLEDGMENTS. We thank John H. Lewis for his assistance with the was done in Kaleidagraph (Synergy Software). For all fitted curves, the error maximum-likelihood estimation and bootstrapping analysis, and Roberto bars represent the error in determination of the fit parameter. For all derived Dominguez for supplying us with the clone for the α-actinin actin-binding parameters, errors were propagated by calculating partial derivatives. Fitting domain. This work was supported by National Institute of General Medical of ensemble averages was performed with custom software written in Sciences Grant PO1 GM087253 (to E.M.O., H.S., and Y.E.G.), National Institute Matlab (Mathworks). Two-sample Kolmogorov–Smirnov tests were carried of Arthritis and Musculoskeletal and Skin Diseases Training Grant T32 out using Matlab. AR053461 (to M.J.G.), and F32 GM097889 (to M.J.G.).

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