Myosin 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 (myo1c), a widely expressed motor protein that links the fine the myo1c actin-activated ATPase pathway in solution are actin cytoskeleton to cell membranes, has been associated with very similar to myo1b (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 transcription 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 myosins. 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 cell membrane; 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 genes 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 Æ 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 Æ 2 s (Fig. S1B). 3 the actin-binding domain of α-actinin fused to the HaloTag gene 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 Æ 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 Æ 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 Æ 0.03) (Table 1).