Tianming Lin

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

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 actin-attachment 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.

Biochemical and Motile Properties of Myo1b Splice Isoforms

Myo1b is a widely expressed myosin-I isoform that concentrates on endosomal and ruffling membranes and is thought to play roles in membrane trafficking and dynamics. Myo1b is alternatively spliced within the regulatory domain of the molecule, yielding isoforms with six (myo1ba), five (myo1bb), or four (myo1bc) non-identical IQ motifs. The calmodulin binding properties of the myo1b IQ motifs have not been investigated, and the mechanical and cell biological consequences of alternative splicing are not known. Therefore, we expressed the alternatively spliced myo1b isoforms truncated after the final IQ motif and included a sequence at their C termini that is a substrate for bacterial biotin ligase. Site-specific biotinylation allows us to specifically attach the myosin to motility surfaces via a biotin-streptavidin linkage. We measured the ATPase and motile properties of the recombinant myo1b splice isoforms, and we correlated these properties with calmodulin binding. We confirmed that calcium-dependent changes in the ATPase activity are due to calcium binding to the calmodulin closest to the motor. We found that calmodulin binds tightly to some of the IQ motifs (Kd < 0.2 μm) and very weakly to the others (Kd > 5 μm), suggesting that a subset of the IQ motifs are not calmodulin bound under physiological conditions. Finally, we found the in vitro motility rate to be dependent on the myo1b isoform and the calmodulin concentration and that the myo1b regulatory domain acts as a rigid lever arm upon calmodulin binding to the high affinity and low affinity IQ motifs.

A hearing loss-associated myo1c mutation (R156W) decreases the myosin duty ratio and force sensitivity.

myo1c is a member of the myosin superfamily that has been proposed to function as the adaptation motor in vestibular and auditory hair cells. A recent study identified a myo1c point mutation (R156W) in a person with bilateral sensorineural hearing loss. This mutated residue is located at the start of the highly conserved switch 1 region, which is a crucial element for the binding of nucleotide. We characterized the key steps on the ATPase pathway at 37 °C using recombinant wild-type (myo1c(3IQ)) and mutant myo1c (R156W-myo1c(3IQ)) constructs that consist of the motor domain and three IQ motifs. The R156W mutation only moderately affects the rates of ATP binding, ATP-induced actomyosin dissociation, and ADP release. The actin-activated ATPase rate of the mutant is inhibited >4-fold, which is likely due to a decrease in the rate of phosphate release. The rate of actin gliding, as measured by the in vitro motility assay, is unaffected by the mutation at high myosin surface densities, but the rate of actin gliding is substantially reduced at low surface densities of R156W-myo1c(3IQ). We used a frictional loading assay to measure the affect of resisting forces on the rate of actin gliding and found that R156W-myo1c(3IQ) is less force-sensitive than myo1c(3IQ). Taken together, these results indicate that myo1c with the R156W mutation has a lower duty ratio than the wild-type protein and motile properties that are less sensitive to resisting forces.

The myosin X motor is optimized for movement on actin bundles.

Myosin X has features not found in other myosins. Its structure must underlie its unique ability to generate filopodia, which are essential for neuritogenesis, wound healing, cancer metastasis and some pathogenic infections. By determining high-resolution structures of key components of this motor, and characterizing the in vitro behaviour of the native dimer, we identify the features that explain the myosin X dimer behaviour. Single-molecule studies demonstrate that a native myosin X dimer moves on actin bundles with higher velocities and takes larger steps than on single actin filaments. The largest steps on actin bundles are larger than previously reported for artificially dimerized myosin X constructs or any other myosin. Our model and kinetic data explain why these large steps and high velocities can only occur on bundled filaments. Thus, myosin X functions as an antiparallel dimer in cells with a unique geometry optimized for movement on actin bundles.

Mechanochemical tuning of myosin-I by the N-terminal region

Significance Myosin molecular motors generate forces in the cell and act as mechanosensors, adjusting their power outputs in response to mechanical loads. Little is known about the structural elements involved in myosin mechanosensing. Our results identify the N-terminal region (NTR) of the myosin-I protein as having an important role in tuning mechanochemistry. Appending the NTR from a highly tension-sensitive myosin (Myo1b) onto a less tension-sensitive motor (Myo1c) changes the identity of the primary force-sensitive transition of Myo1c, making it sensitive to forces <2 pN. Moreover, we show that the NTR stabilizes the post–power-stroke conformation. These results identify the NTR as an important structural element in myosin force sensing and suggest a mechanism for generating diversity of function among myosin isoforms. Myosins are molecular motors that generate force to power a wide array of motile cellular functions. Myosins have the inherent ability to change their ATPase kinetics and force-generating properties when they encounter mechanical loads; however, little is known about the structural elements in myosin responsible for force sensing. Recent structural and biophysical studies have shown that myosin-I isoforms, Myosin-Ib (Myo1b) and Myosin-Ic (Myo1c), have similar unloaded kinetics and sequences but substantially different responses to forces that resist their working strokes. Myo1b has the properties of a tension-sensing anchor, slowing its actin-detachment kinetics by two orders of magnitude with just 1 pN of resisting force, whereas Myo1c has the properties of a slow transporter, generating power without slowing under 1-pN loads that would stall Myo1b. To examine the structural elements that lead to differences in force sensing, we used single-molecule and ensemble kinetic techniques to show that the myosin-I N-terminal region (NTR) plays a critical role in tuning myosin-I mechanochemistry. We found that replacing the Myo1c NTR with the Myo1b NTR changes the identity of the primary force-sensitive transition of Myo1c, resulting in sensitivity to forces of <2 pN. Additionally, we found that the NTR plays an important role in stabilizing the post–power-stroke conformation. These results identify the NTR as an important structural element in myosin force sensing and suggest a mechanism for generating diversity of function among myosin isoforms.

A Hearing Loss-Associated myo1c Mutation (R156W) Decreases the Myosin Duty Ratio and Force Sensitivity

myo1c is a member of the myosin superfamily that has been proposed to function as the adaptation motor in vestibular and auditory hair cells. A recent study identified a myo1c point mutation (R156W) in a person with bilateral sensorineural hearing loss. This mutated residue is located at the start of the highly conserved switch 1 region, which is a crucial element for the binding of nucleotide. We characterized the key steps on the ATPase pathway at 37 °C using recombinant wild-type (myo1c3IQ) and mutant myo1c (R156W-myo1c3IQ) constructs that consist of the motor domain and three IQ motifs. The R156W mutation only moderately affects the rates of ATP binding, ATP-induced actomyosin dissociation, and ADP release. The actin-activated ATPase rate of the mutant is inhibited >4-fold, which is likely due to a decrease in the rate of phosphate release. The rate of actin gliding, as measured by the in vitro motility assay, is unaffected by the mutation at high myosin surface densities, but the rate of actin glidin...