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

Abstract

Myosin IC (myo1c), a widely expressed motor protein that links the actin cytoskeleton to cell membranes, has been associated with numerous cellular processes, including insulin-stimulated transport of GLUT4, mechanosensation in sensory hair cells, endocytosis, transcription of DNA in the nucleus, exocytosis, and membrane trafficking. The molecular role of myo1c in these processes has not been defined, so to better understand myo1c function, we utilized ensemble kinetic and single-molecule techniques to probe myo1c's biochemical and mechanical properties. Utilizing a myo1c construct containing the motor and regulatory domains, we found the force dependence of the actin-attachment lifetime to have two distinct regimes: a force-independent regime at forces <1 pN, and a highly force-dependent regime at higher loads. In this force-dependent regime, forces that resist the working stroke increase the actinattachment lifetime. Unexpectedly, the primary force-sensitive transition is the isomerization that follows ATP binding, not ADP release as in other slow myosins. This force-sensing behavior is unique amongst characterized myosins and clearly demonstrates mechanochemical diversity within the myosin family. Based on these results, we propose that myo1c functions as a slow transporter rather than a tension-sensitive anchor.is a widely expressed myosin-I isoform that has been associated with several important cellular processes, including endocytosis (1), exocytosis (2) (including insulin-stimulated GLUT4 translocation to the cell membrane; refs. 3-5), membrane ruffling (6), transcription of DNA in the nucleus (7,8), and mechanosensing in sensory hair cells (9-13). Although it is known that myo1c links cell membranes to the actin cytoskeleton (14, 15), its molecular role in these cellular processes has not been determined. For example, in its proposed role in exocytosis, it is not known if myo1c acts as a motor for transport, moving vesicles into position for plasma membrane fusion and/or as a tension-sensitive anchor that docks exocytic vesicles to the actin cytoskeleton and plasma membrane.Most members of the myosin family share the same kinetic pathway for ATP hydrolysis, in which force-generating structural changes are linked to release of inorganic phosphate and ADP, but different myosin isoforms have evolved different biochemical reaction rates and force-dependent kinetics to suit their cellular functions. For example, in myosins that are thought to act as tension-sensitive anchors, the kinetic steps that limit actomyosin detachment are highly sensitive to load. In contrast, myosins that are thought to act as transporters have actin-detachment kinetics that are less sensitive to load, allowing work to be performed over a range of forces. Thus, insight into the molecular role of myosin in the cell can be gained from evaluating the kinetic and mechanical properties of the motor.Previous biochemical analyses have shown that myo1c is a lowduty ratio motor (i.e., it spends most of its biochemical cycle detached from act...

Myosin-10 produces its power-stroke in two phases and moves processively along a single actin filament under low load

“…Ensemble averaging of acto-M10HMM interactions measured using optical tweezers shows the power stroke occurs in two discrete phases, as found for several other myosin types (33,35,36,38,41). A large amplitude (15 nm) initial movement occurred within the first 5 ms of myosin binding to actin, presumably while ADP is still bound at the catalytic site.…”

“…S3A). Initial experiments conducted under our standard conditions using an optical trap stiffness of ∼0.015-0.045 pN/nm showed mostly unitary acto-M10HMM interactions or events, reminiscent of the nonprocessive behavior of myosins such as muscle myosin-2 and nonmuscle myosin-1 (33)(34)(35)(36). The experiments were then repeated in the presence of sinusoidal oscillations (200 Hz; ∼200 nm peak-to-peak) that were applied to one of the optical tweezers, with the aim being twofold: (i) to improve our ability to discriminate between detached and attached states and (ii) to determine the stiffness of a single actomyosin-10 attachment (32,34,37,38).…”

“…Previous studies have shown that the power strokes of some myosins are composed of more than one discrete motion of the lever arm (33,35,36,38,(41)(42)(43)(44). To test whether myosin-10 exhibits this behavior, individual binding events (recorded at 250 nM ATP) were identified in the raw data traces and synchronized (n = 220 interactions) to their starting and ending point based on changes in signal variance.…”

High-resolution helix orientation in actin-bound myosin determined with a bifunctional spin label

“…The physiological significance of the myosin ADP release step has become increasingly apparent. Actin decreases myosin's ADP affinity by a factor of roughly 100 (37,38), implying crucial allosteric coupling between the nucleotide-binding pocket (ATPase) and the actin-binding interface (cross-bridge dynamics). Strain on the lever arm affects the rate of ADP release in several myosins (39)(40)(41)(42), and single-molecule studies have correlated this rate with lever arm movement (39,43).…”

Local Turgor Pressure Reduction via Channel Clustering

“…An alternate possibility is one analogous to tubulation of giant unilamellar vesicles driven by type-1 myosin (45), where the driving force for tubulation came from myosin motion along a preexisting actin scaffold. We are not aware of measurements of the force-generating capability of yeast type-1 myosins, but mammalian type-1 myosins are able to generate up to 5 pN of force per molecule (46). It is also possible that there are growing actin filaments that were not detected in the previous filament counts based on Arp2/3 complex-mediated branching (20,23).…”