Myosin I Can Act As a Molecular Force Sensor

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Science  04 Jul 2008:
Vol. 321, Issue 5885, pp. 133-136
DOI: 10.1126/science.1159419


The ability to sense molecular tension is crucial for a wide array of cellular processes, including the detection of auditory stimuli, control of cell shape, and internalization and transport of membranes. We show that myosin I, a motor protein that has been implicated in powering key steps in these processes, dramatically alters its motile properties in response to tension. We measured the displacement generated by single myosin I molecules, and we determined the actin-attachment kinetics with varying tensions using an optical trap. The rate of myosin I detachment from actin decreases >75-fold under tension of 2 piconewtons or less, resulting in myosin I transitioning from a low (<0.2) to a high (>0.9) duty-ratio motor. This impressive tension sensitivity supports a role for myosin I as a molecular force sensor.

Myosin I's are the widely expressed, single-headed, and membrane-associated members of the myosin superfamily that participate in regulating membrane dynamics and structure in nearly all eukaryotic cells. Eight myosin I isoforms are expressed in humans, making it the largest “unconventional” myosin family (1). One specific and well-characterized molecular function of a myosin I isoform (myo1c) is to dynamically provide tension to sensitize mechanosensitive ion channels responsible for hearing (24). Myosin I's also power the transport and deformation of membranes in the cell cortex and in apical cell projections (58). To perform these roles, myosin I's have been proposed to act as tension-sensing proteins that alter their adenosine triphosphatase (ATPase) and mechanical properties in response to changes in loads imparted by their cellular cargos (3, 9).

Biochemical, structural, and single-molecule experiments suggest that some myosin I isoforms (myo1a, myo1b, and myo1c) are adapted to sense tension. Specifically, it has been shown that myosin I produces its working stroke displacement in two substeps (10). An initial displacement of the lever arm is followed by an additional ∼32° rotation that accompanies adenosine diphosphate (ADP) release (11). Because ADP release kinetically limits the rate of the detachment of myosin from actin (12, 13), the extra lever-arm rotation has been proposed to be a force-sensing substep, with the presence of resisting loads preventing this lever-arm rotation and thus inhibiting ADP release and actin detachment. A similar model has been proposed for gating of myosin V motor activity during processive motility (14).

We characterized the motor activity of myosin I by measuring single-molecule force-generating events using the three-bead configuration, 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 myosin (15). A recombinant myo1b splice isoform containing five calmodulin-binding IQ motifs and a C-terminal biotinylation tag (16) was attached to streptavidin-coated pedestal beads (17). Single-molecule actomyosin interactions at low loads were acquired using low trap stiffness (∼0.022 pN/nm, Fig. 1A) in the presence of 1 to 50 μM ATP. Because the myo1b working stroke is the sum of two substeps, we examined the substep sizes and kinetics of the actomyosin interactions by ensemble-averaging the time courses of individual actomyo1b interactions that were synchronized at the times when the interactions started or ended (Fig. 1B). The time courses of the start-time averages reveal the lifetimes of the first substep, and the time courses of the end-time averages reveal the lifetimes of the second sub-step (18).

Fig. 1.

Single and ensemble-averaged actomyo1b interactions. (A) Representative time traces showing force during unitary actomyo1b attachments acquired with or without engagement of the isometric clamp in the presence of 50 μM ATP. There is a strong increase in attachment durations during high-force attachments as a result of engagement of the isometric clamp. The insets in blue boxes show 4 s of three different attachment events acquired without the isometric clamp. Arrows indicate attached and detached positions. (B) Ensemble-averaged interactions at five different ATP concentrations. Single attachments were synchronized at the times when the attachments started or ended and were averaged as described (17). The number of unitary interactions in each average is indicated. The time scale for all start-time averages is the same, and the time scales for the end-time averages vary as indicated. The red lines are fits of the start-time (kstart) and end-time (kend) averages to a single exponential rate function. (C) ATP concentration dependence of kstart (open circles) and kend (solid circles) rates. The red line is a linear fit of the kend rates, yielding a slope of 0.48 μM–1 s–1 (correlation coefficient = 0.985).

The time courses of the start-time averages have rapid initial 5.1 ± 0.43–nm substeps of actin displacement followed by slower 3.3 ± 0.35–nm increases to the final displacement. If the 3.3-nm substep is the result of a 32° lever-arm rotation (11), we calculate the effective myo1b lever-arm length to be 6.0 ± 0.63 nm. This length is shorter than expected given that the regulatory domain (the lever-arm region) contains five calmodulin-binding IQ motifs, which would have a length of ∼20 nm if the lever arm were rigid (19). The short effective lever-arm of myo1b is probably due to weak calmodulin binding to a subset of the IQ motifs, resulting in a flexible regulatory domain (16). The regulatory domain is alternatively spliced in vivo (20), so it is possible that the compliance of this region is transcriptionally regulated so as to modulate the mechanical properties of this motor (16).

The initial 5.1-nm working-stroke substep occurred within the experimental response time and most likely corresponds to the displacement that accompanies phosphate (Pi) release (10). The time courses of the 3.3-nm increase in the start-time averages reveal the lifetimes of the 5.1-nm substep at the different ATP concentrations. These time courses, at all ATP concentrations, were well fit by a single exponential function with rates (kstart = 0.37 to 0.77 s–1) slower than reported for ADP release from actomyo1b in solution [1.8 s–1 (13)]. Small loads on the actomyo1b interactions probably lead to these slower rates.

The time courses of end-time averages have the same initial and total displacements observed in the start-time averages (Fig. 1B), and they reveal the lifetimes of attachment after the 3.3-nm substep. The time courses were well fit by single exponential functions with rates (kend) that are linearly related to the ATP concentration with a slope of 0.48 μM–1 s–1 (Fig. 1C), which is similar to solution measurements of the apparent second-order rate constant for ATP binding [0.22 μM–1 s–1 (13)]. A two-step model for myo1b detachment from actin with the rates obtained from the ensemble-averaged interactions describes the distributions of actomyo1b attachment lifetimes (fig. S2). These data confirm that the myo1b working stroke occurs in two substeps (10) with lifetimes consistent with solution kinetic measurements (13) of the rates of ADP release (lifetime of the 5.1-nm substep) and ATP binding (lifetime of the 3.3-nm substep).

The force dependence of actomyo1b attachment lifetimes was measured with a feedback system that applies a dynamic load to the actomyo1b to keep the actin near its isometric position while myosin undergoes its working stroke (21). With the feedback system, the resisting force applied to the actomyo1b attachment was determined primarily by the size of the myo1b powerstroke, the stiffness of the myo1b lever arm, and the position at which myosin binds to the actin filament.

We observed dramatic increases in actomyo1b attachment durations in the presence of 50 μM ATP upon engagement of the isometric clamp, with many durations exceeding 1 min (Fig. 2A). The attachment durations increased with increasing force until ∼1.5 pN, after which they appeared to be force-independent. We assumed a model for the rate of actomyo1b detachment that includes force-dependent and force-independent pathways Embedded Image(1) Embedded Image Embedded Image where kg is a force-dependent rate constant and ki is a force-independent rate constant for actomyo1b dissociation. The force dependence of the detachment rate (kdet) can be calculated as (22) Embedded Image(2) where kg0 is therateof kg in the absence of force, ddet is the distance parameter (the distance to the transition state), F is force, k is the Boltzmann constant, and T is the temperature. Because the attachment durations at each force are expected to be exponentially distributed, we used bootstrap Monte Carlo simulations to simulate data to use in maximum likelihood estimations (MLEs) (17). From the MLEs, we determined the values and confidence limits of ddet, kg0, and ki that best describe the distribution of attachment durations. The best-fit value of kg0 = 1.6 s–1 (+0.5/–0.35 s–1) is consistent with the rate of ADP release measured via biochemical methods [1.8 s–1 (13)], which limits kdet at 50 μM ATP in the absence of force. The distance parameter ddet = 12 nm (+1.6/–3.0 nm), which is a measure of strain sensitivity, is very large and distinguishes myo1b as an extraordinarily strain-dependent motor at loads <2 pN. This sensitivity is very different from that of other characterized myosins, including strain-sensitive myosin VI (23). Unlike myo1b, the strain sensitivity of myosin VI is seen only at low forces (<2 pN) in the presence of ADP.

Fig. 2.

Force dependence of actomyo1b attachment durations. (A) Attachment durations (n = 638 from 12 experiments) plotted as a function of the average force during the attachment in the presence of 50 μM ATP. (B) Force dependence of the actin-detachment rate (kdet) as calculated using the parameters from the fit of the data in (A) to Eq. 2 as determined by MLE. The best-fit parameters are kg0 = 1.6 s–1 (+0.5/–0.35 s–1), ddet = 12 nm (+1.6/–3.0 nm), and ki = 0.021 s–1 (+0.007/–0.004 s–1). Points are the inverse averages of 20 consecutive attachment durations from the plot in (A). (Inset) Predicted myo1b duty ratio as a function of force. The shaded areas show the 97% confidence limit based on the uncertainties listed above. Arrows show the force at which myo1b becomes a high–duty-ratio motor (∼0.5 pN).

The strain sensitivity of myo1b is most clearly illustrated in a plot of kdet versus force (Fig. 2B), where it can be seen that kdet decreases >75-fold with <2 pN of resisting force. At the low forces experienced by myo1b in the absence of the isometric clamp (<0.2 pN, Fig. 1B), a 1.5-fold decrease in kdet is predicted over the unloaded rate (kg0), which is consistent with the values of kstart measured from the start-time averages.

The fraction of the total ATPase cycle in which myo1b is bound to actin in a force-bearing state is termed the duty ratio, and the force dependence of the duty ratio can be calculated as Embedded Image(3) where katt is the rate of entry into the strong binding states. katt cannot be determined directly from the force time courses but can be estimated from the rate of phosphate release (katt = 0.38 s–1, fig. S3). Myo1b transforms from a low–duty-ratio motor (<0.2) to a high–duty-ratio motor (>0.5) when working against as little as 0.5 pN of force, and it approaches the duty ratio of processive myosins (>0.9) at forces as low as 1.5 pN (Fig. 2B, inset).

We investigated the effect of force on the lifetimes and force amplitudes of the working-stroke substeps. Interactions acquired in the presence of 50 μM ATP were sorted into bins based on the force immediately before detachment, and individual interactions were synchronized at their end times and ensemble-averaged. Transient increases in force due to substeps were observed in the 500 ms before detachment in all force bins (Fig. 3A). Single exponential fits of the time courses yielded rates that decreased with increasing force (Fig. 3B). The force dependence of the rates was fit to the equation Embedded Image(4) where kend0 is the rate of the time course in the absence of force and dend is the distance parameter for the substep (Fig. 3B). The best-fit rate of kend0 (22 ± 2.5 s –1) is consistent with the rate of 50 μM ATP binding in the absence of resisting loads (kend = 24 s–1, Fig. 1C), and the value dend = 2.5 ±0.83 nm is much smaller than ddet (Fig. 2B). Therefore, the ATP binding step is not the force-dependent step that limits the rate of actomyo1b detachment. ADP release is the most likely candidate for the force-dependent transition (Fig. 3C). ddet is substantially larger than the size of the substep that correlates with ADP release (3.3 ± 0.35 nm, Fig. 1B), indicating that the force-sensitive transition state is not on a coordinate that is in line with a rigid lever arm rotation (24).

Fig. 3.

Effect of force on working-stroke substep lifetimes. (A) Single interactions acquired with the isometric clamp in the presence of 50 μM ATP were sorted into bins based on the force immediately before detachment, synchronized to the interaction end-time, and averaged. From the bottom, the force bins (in piconewtons) are (0 to 0.125), (0.125 to 0.25), (0.25 to 0.50), (0.50 to 0.75), (0.74 to 1.0), (1.0 to 2.0), and (2.0 to 4.0). For clarity, the interaction averages in the three lowest force bins are shown on an expanded scale. The red lines are fits of the end-time averages to a single exponential rate function (kend). The rates of the end-time averages are faster than the feedback response time of the isometric clamp, resulting in the lower force amplitudes for the faster substep time courses. (B) Force dependence of kend rates obtained from the fits in (A). The red line is the best fit of the data to Eq. 4 with kend0 = 22 ± 2.5 s–1 and dend = 2.5 ± 0.83 nm. (C) Model for myo1b (blue) bound to actin (red) undergoing a working stroke. The 5.1-nm substep, 3.3-nm substep, and force-sensitive transition are identified. The rate of the ADP release step as a function of force is defined as kdet (Eq. 2), and the rate of ATP binding and subsequent actomyo1b detachment is defined as kend (Eq. 4). The extended spring signifies tension on the actomyo1b complex.

The presence of a substep in the end-time averages in all force bins indicates that actomyo1b detachment did not occur before ADP release, even at forces where the detachment rate is force-insensitive (> 1.5 pN; Fig. 2). The force-independent detachment rate (ki in Eq. 1) is probably the result of accelerated detachment due to force fluctuations in the system. Decreases in force before the ADP-release substep are observed in the force-binned start- and end-time averages (fig. S4). Thus, when force transiently drops, there is an exponentially higher probability of ADP release (Fig. 2B), which is followed by rapid ATP binding and detachment.

Our results show that myosin I responds to small resisting loads (<2 pN) by dramatically increasing the actin-attachment lifetime more than 75-fold. This impressive tension sensitivity supports models that identify myosin I as the adaptation motor in mechanosensory hair cells (2, 3). More generally, the load-dependent kinetics support a model in which myosin I's function to generate and sustain tension for extended time periods, rather than to rapidly transport cargos (fig. S5). This new understanding of myosin I mechanics allows a more rigorous assignment of this motor's molecular roles in controlling organelle morphology (8) and dynamics (5) in the wide variety of cells types in which it is expressed.

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Materials and Methods

Figs. S1 to S5


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