William C. Unrath

Myosin IIIB Uses an Actin-Binding Motif in Its Espin-1 Cargo to Reach the Tips of Actin Protrusions

Myosin IIIA (MYO3A) targets actin protrusion tips using a motility mechanism dependent on both motor and tail actin-binding activity [1]. We show that myosin IIIB (MYO3B) lacks tail actin-binding activity and is unable to target COS7 cell filopodia tips, yet is somehow able to target stereocilia tips. Strikingly, when MYO3B is coexpressed with espin-1 (ESPN1), a MYO3A cargo protein endogenously expressed in stereocilia [2], MYO3B targets and carries ESPN1 to COS7 filopodia tips. We show that this tip localization is lost when we remove the ESPN1 C terminus actin-binding site. We also demonstrate that, like MYO3A [2], MYO3B can elongate filopodia by transporting ESPN1 to the polymerizing end of actin filaments. The mutual dependence of MYO3B and ESPN1 for tip localization reveals a novel mechanism for the cell to regulate myosin tip localization via a reciprocal relationship with cargo that directly participates in actin binding for motility. Our results are consistent with a novel form of motility for class III myosins that requires both motor and tail domain actin-binding activity and show that the actin-binding tail can be replaced by actin-binding cargo. This study also provides a framework to better understand the late-onset hearing loss phenotype in patients with MYO3A mutations.

Intermolecular Autophosphorylation Regulates Myosin IIIa Activity and Localization in Parallel Actin Bundles

Myosin IIIa (Myo3A) transports cargo to the distal end of actin protrusions and contains a kinase domain that is thought to autoregulate its activity. Because Myo3A tends to cluster at the tips of actin protrusions, we investigated whether intermolecular phosphorylation could regulate Myo3A biochemical activity, cellular localization, and cellular function. Inactivation of Myo3A 2IQ kinase domain with the point mutation K50R did not alter maximal ATPase activity, whereas phosphorylation of Myo3A 2IQ resulted in reduced maximal ATPase activity and actin affinity. The rate and degree of Myo3A 2IQ autophosphorylation was unchanged by the presence of actin but was found to be dependent upon Myo3A 2IQ concentration within the range of 0.1 to 1.2 μm, indicating intermolecular autophosphorylation. In cultured cells, we observed that the filopodial tip localization of Myo3A lacking the kinase domain decreased when co-expressed with kinase-active, full-length Myo3A. The cellular consequence of reduced Myo3A tip localization was decreased filopodial density along the cell periphery, identifying a novel cellular function for Myo3A in mediating the formation and stability of actin-based protrusions. Our results suggest that Myo3A motor activity is regulated through a mechanism involving concentration-dependent autophosphorylation. We suggest that this regulatory mechanism plays an essential role in mediating the transport and actin bundle formation/stability functions of Myo3A.

Omecamtiv Mecarbil Enhances the Duty Ratio of Human β-Cardiac Myosin Resulting in Increased Calcium Sensitivity and Slowed Force Development in Cardiac Muscle

The small molecule drug omecamtiv mecarbil (OM) specifically targets cardiac muscle myosin and is known to enhance cardiac muscle performance, yet its impact on human cardiac myosin motor function is unclear. We expressed and purified human β-cardiac myosin subfragment 1 (M2β-S1) containing a C-terminal Avi tag. We demonstrate that the maximum actin-activated ATPase activity of M2β-S1 is slowed more than 4-fold in the presence of OM, whereas the actin concentration required for half-maximal ATPase was reduced dramatically (30-fold). We find OM does not change the overall actin affinity. Transient kinetic experiments suggest that there are two kinetic pathways in the presence of OM. The dominant pathway results in a slow transition between actomyosin·ADP states and increases the time myosin is strongly bound to actin. However, OM also traps a population of myosin heads in a weak actin affinity state with slow product release. We demonstrate that OM can reduce the actin sliding velocity more than 100-fold in the in vitro motility assay. The ionic strength dependence of in vitro motility suggests the inhibition may be at least partially due to drag forces from weakly attached myosin heads. OM causes an increase in duty ratio examined in the motility assay. Experiments with permeabilized human myocardium demonstrate that OM increases calcium sensitivity and slows force development (ktr) in a concentration-dependent manner, whereas the maximally activated force is unchanged. We propose that OM increases the myosin duty ratio, which results in enhanced calcium sensitivity but slower force development in human myocardium.

Myosin 3A kinase activity is regulated by phosphorylation of the kinase domain activation loop

Background: Class III myosins contain both a motor and kinase domain. Results: Phosphorylation of the kinase activation loop enhances MYO3A kinase activity, augmenting autophosphorylation-induced attenuation of motor and cellular activity. Conclusion: MYO3A kinase activity mediates localization and function within actin protrusions. Significance: Characterizing MYO3A kinase regulation enhances our understanding of the role of MYO3A in the maintenance of actin protrusions found in sensory epithelia. Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells.

Biochemical and bioinformatic analysis of the MYO19 motor domain

Mitochondrial dynamics are dependent on both the microtubule and actin cytoskeletal systems. Evidence for the involvement of myosin motors has been described in many systems, and until recently a candidate mitochondrial myosin transport motor had not been described in vertebrates. Myosin‐XIX (MYO19) was predicted to represent a novel class of myosin and had previously been shown to bind to mitochondria and increase mitochondrial network dynamics when ectopically expressed. Our analyses comparing ∼40 MYO19 orthologs to ∼2000 other myosin motor domain sequences identified instances of homology well‐conserved within class XIX myosins that were not found in other myosin classes, suggesting MYO19‐specific mechanochemistry. Steady‐state biochemical analyses of the MYO19 motor domain indicate that Homo sapiens MYO19 is a functional motor. Insect cell‐expressed constructs bound calmodulin as a light chain at the predicted stoichiometry and displayed actin‐activated ATPase activity. MYO19 constructs demonstrated high actin affinity in the presence of ATP in actin‐co‐sedimentation assays, and translocated actin filaments in gliding assays. Expression of GFP‐MYO19 containing a mutation impairing ATPase activity did not enhance mitochondrial network dynamics, as occurs with wild‐type MYO19, indicating that myosin motor activity is required for mitochondrial motility. The measured biochemical properties of MYO19 suggest it is a high‐duty ratio motor that could serve to transport mitochondria or anchor mitochondria, depending upon the cellular microenvironment.

Biochemical and bioinformatic analysis of the myosin-XIX motor domain.

Mitochondrial dynamics are dependent on both the microtubule and actin cytoskeletal systems. Evidence for the involvement of myosin motors has been described in many systems, and until recently a candidate mitochondrial myosin transport motor had not been described in vertebrates. Myosin‐XIX (MYO19) was predicted to represent a novel class of myosin and had previously been shown to bind to mitochondria and increase mitochondrial network dynamics when ectopically expressed. Our analyses comparing ∼40 MYO19 orthologs to ∼2000 other myosin motor domain sequences identified instances of homology well‐conserved within class XIX myosins that were not found in other myosin classes, suggesting MYO19‐specific mechanochemistry. Steady‐state biochemical analyses of the MYO19 motor domain indicate that Homo sapiens MYO19 is a functional motor. Insect cell‐expressed constructs bound calmodulin as a light chain at the predicted stoichiometry and displayed actin‐activated ATPase activity. MYO19 constructs demonstrated high actin affinity in the presence of ATP in actin‐co‐sedimentation assays, and translocated actin filaments in gliding assays. Expression of GFP‐MYO19 containing a mutation impairing ATPase activity did not enhance mitochondrial network dynamics, as occurs with wild‐type MYO19, indicating that myosin motor activity is required for mitochondrial motility. The measured biochemical properties of MYO19 suggest it is a high‐duty ratio motor that could serve to transport mitochondria or anchor mitochondria, depending upon the cellular microenvironment.

Impact of the Motor and Tail Domains of Class III Myosins on Regulating the Formation and Elongation of Actin Protrusions

Class III myosins (MYO3A and MYO3B) are proposed to function as transporters as well as length and ultrastructure regulators within stable actin-based protrusions such as stereocilia and calycal processes. MYO3A differs from MYO3B in that it contains an extended tail domain with an additional actin-binding motif. We examined how the properties of the motor and tail domains of human class III myosins impact their ability to enhance the formation and elongation of actin protrusions. Direct examination of the motor and enzymatic properties of human MYO3A and MYO3B revealed that MYO3A is a 2-fold faster motor with enhanced ATPase activity and actin affinity. A chimera in which the MYO3A tail was fused to the MYO3B motor demonstrated that motor activity correlates with formation and elongation of actin protrusions. We demonstrate that removal of individual exons (30–34) in the MYO3A tail does not prevent filopodia tip localization but abolishes the ability to enhance actin protrusion formation and elongation in COS7 cells. Interestingly, our results demonstrate that MYO3A slows filopodia dynamics and enhances filopodia lifetime in COS7 cells. We also demonstrate that MYO3A is more efficient than MYO3B at increasing formation and elongation of stable microvilli on the surface of cultured epithelial cells. We propose that the unique features of MYO3A, enhanced motor activity, and an extended tail with tail actin-binding motif, allow it to play an important role in stable actin protrusion length and ultrastructure maintenance.

The HCM Loop Plays a Role in Actin-Activated Product Release in Myosin V

We examined the functional role of the upper 50 kDa hypertrophic cardiomyopathy (HCM) loop in myosin V. Hypertrophic cardiomyopathy is caused by missense mutations in highly conserved regions of myo2β and one deadly mutation occurs in the HCM loop (R403Q). Since the R403Q mutation has been shown to enhance or decrease the ATPase activity and in vitro motility of myosin II, it may be expected that the HCM loop plays a role in actin-activated product release. In our previous work we correlated the conformational change associated with ADP release and maximum ATPase rate in myosin V using FRET analysis. We engineered the R403Q mutation at an analogous site in myosin V 1IQ (R378Q) so that we would be able to investigate the impact of the mutation on actin-activated product release, maximum ATPase rate, in vitro motility, and FRET in myosin V. The R378Q mutation reduces the maximum ATPase rate two-fold while it slightly enhances sliding velocity compared to wild-type MV 1IQ. Our results suggest the duty ratio may be reduced as a result of the R378Q mutation. We will directly examine both ADP-release and phosphate-release to evaluate this possibility. To examine the impact of the point mutation on structural dynamics we will determine if conformational changes in the nucleotide-binding pocket and actin-binding cleft are disrupted using our established FRET probes. Our studies further establish a strategy for examining the mechanism of product release in myosin using the three assays: FRET, ATPase, and motility.