Actin Translocation Research Articles

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays.

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic properties are adapted for the particular function they execute in cells. Myosin 5a processively walks on F-actin to transport melanosomes and vesicles in cells. Conversely, nonmuscle myosin 2b operates as a bipolar filament containing approximately 30 molecules. It moves F-actin of opposite polarity toward the center of the filament, where the myosin molecules work asynchronously to bind actin, impart a power stroke, and dissociate before repeating the cycle. Nonmuscle myosin 2b, along with its other nonmuscle myosin 2 isoforms, has roles that includecell adhesion, cytokinesis, and tension maintenance. The mechanochemistry of myosins can be studied by performing in vitro motility assays using purified proteins. In the gliding actin filament assay, the myosins are bound to a microscope coverslip surface and translocate fluorescently labeled F-actin, which can be tracked. In the single molecule/ensemble motility assay, however, F-actin is bound to a coverslip and the movement of fluorescently labeled myosin molecules on the F-actin is observed. In this report, the purification of recombinant myosin 5a from Sf9 cells using affinity chromatography is outlined. Following this, we outline two fluorescence microscopy-based assays: the gliding actin filament assay and the inverted motility assay. From these assays, parameters such as actin translocation velocities and single molecule run lengths and velocities can be extracted using the image analysis software. These techniques can also be applied to study the movement of single filaments of the nonmuscle myosin 2 isoforms, discussed herein in the context of nonmuscle myosin 2b. This workflow represents a protocol and a set of quantitative tools that can be used to study the single molecule and ensemble dynamics of nonmuscle myosins.

Cellular fate of deformable needle-shaped PLGA-PEG fibers

Deformability of micro/nanometer sized particles plays an important role in particle-cell interactions and thus becomes a key parameter in carrier design in biomedicine application such as drug delivery and vaccinology. Yet the influence of material's deformability on the cellular fate of the particles as well as physiology response of live cells are to be understood. Here we show the cellular fate of needle shaped (high aspect ratio ~25) PLGA-PEG copolymer fibers depending on their deformability. We found that all the fibers entered murine macrophage cells (RAW 264.7) via phagocytosis. While the fibers of high apparent Young's modulus (average value = 872 kPa) maintained their original shape upon phagocytosis, their counterparts of low apparent Young's modulus (average value = 56 kPa) curled in cells. The observed deformation of fibers of low apparent Young's modulus in cells coincided with abnormal intracellular actin translocation and absence of lysosome/phagosome fusion in macrophages, suggesting the important role of material mechanical properties and mechano-related cellular pathway in affecting cell physiology. Statement of SignificanceParticles are increasingly important in the field of biomedicine, especially when they are serving as drug carriers. Physical cues, such as mechanical properties, were shown to provide insight into their stability and influence on physiology inside the cell. In the current study, we managed to fabricate 5 types of needle shaped PLGA-PEG fibers with controlled Young's modulus. We found that hard fibers maintained their original shape upon phagocytosis, while soft fibers were curled by actin compressive force inside the cell, causing abnormal actin translocation and impediment of lysosome/phagosome fusion, suggesting the important role of material mechanical properties and mechano-related cellular pathway in affecting cell physiology.

Functional Differences in MYH7b That Contribute to Distinct Biological Roles across Species and in Health and Disease

Myosin heavy chain 7b (MYH7b) is a recently identified sarcomeric myosin gene family member that has a unique expression pattern in mammals. MYH7b protein is absent from the heart and skeletal muscle of mammals and is only expressed in specialized muscles and, surprisingly, in the inner ear and brain. Additionally, compound heterozygous mutations in MYH7b (one in the motor domain and one in the rod domain) cause hearing loss in humans by an unknown mechanism (Haraksingh et al., BMC Genomics, 2014) indicating that MYH7b has an important yet undefined role in mammalian non-muscle tissue. Intriguingly, in snakes, MYH7b protein is abundant in cardiac and skeletal muscles. Despite these divergent roles in mammals and reptiles, MYH7b shares high sequence identity between these vertebrate classes, so it is unclear how mammalian MYH7b function may deviate in non-muscle tissues from its expected sarcomeric role. To address this question, we have generated recombinant protein for human MYH7b, human MYH7b with the motor domain hearing loss mutation (D515N), and python MYH7b, and have measured their biochemical and biophysical properties. We have observed differences in the steady-state actin-activated ATPase activities and actin translocation as assessed by in vitro motility assays between these three recombinant proteins. Comparing the motor activity of human MYH7b and python MYH7b provides both an evolutionary perspective and functional analysis of highly conserved myosins that operate in different cell types. Further, comparing the function of human wild type MYH7b and D515N MYH7b provides greater insight into the etiology of human hearing loss.

Baculovirus AC102 Is a Nucleocapsid Protein That Is Crucial for Nuclear Actin Polymerization and Nucleocapsid Morphogenesis.

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type species of alphabaculoviruses, is an enveloped DNA virus that infects lepidopteran insects and is commonly known as a vector for protein expression and cell transduction. AcMNPV belongs to a diverse group of viral and bacterial pathogens that target the host cell actin cytoskeleton during infection. AcMNPV is unusual, however, in that it absolutely requires actin translocation into the nucleus early in infection and actin polymerization within the nucleus late in infection coincident with viral replication. Of the six viral factors that are sufficient, when coexpressed, to induce the nuclear localization of actin, only AC102 is essential for viral replication and the nuclear accumulation of actin. We therefore sought to better understand the role of AC102 in actin mobilization in the nucleus early and late in infection. Although AC102 was proposed to function early in infection, we found that AC102 is predominantly expressed as a late protein. In addition, we observed that AC102 is required for F-actin assembly in the nucleus during late infection, as well as for proper formation of viral replication structures and nucleocapsid morphogenesis. Finally, we found that AC102 is a nucleocapsid protein and a newly recognized member of a complex consisting of the viral proteins EC27, C42, and the actin polymerization protein P78/83. Taken together, our findings suggest that AC102 is necessary for nucleocapsid morphogenesis and actin assembly during late infection through its role as a component of the P78/83-C42-EC27-AC102 protein complex.IMPORTANCE The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an important biotechnological tool for protein expression and cell transduction, and related nucleopolyhedroviruses are also used as environmentally benign insecticides. One impact of our work is to better understand the fundamental mechanisms through which AcMNPV exploits the cellular machinery of the host for replication, which may aid in the development of improved baculovirus-based research and industrial tools. Moreover, AcMNPV's ability to mobilize the host actin cytoskeleton within the cell's nucleus during infection makes it a powerful cell biological tool. It is becoming increasingly clear that actin plays important roles in the cell's nucleus, and yet the regulation and function of nuclear actin is poorly understood. Our work to better understand how AcMNPV relocalizes and polymerizes actin within the nucleus may reveal fundamental mechanisms that govern nuclear actin regulation and function, even in the absence of viral infection.

The central domain of UNC-45 chaperone inhibits the myosin power stroke.

The multidomain UNC‐45B chaperone is crucial for the proper folding and function of sarcomeric myosin. We recently found that UNC‐45B inhibits the translocation of actin by myosin. The main functions of the UCS and TPR domains are known but the role of the central domain remains obscure. Here, we show—using in vitro myosin motility and ATPase assays—that the central domain alone acts as an inhibitor of the myosin power stroke through a mechanism that allows ATP turnover. Hence, UNC‐45B is a unique chaperone in which the TPR domain recruits Hsp90; the UCS domain possesses chaperone‐like activities; and the central domain interacts with myosin and inhibits the actin translocation function of myosin. We hypothesize that the inhibitory function plays a critical role during the assembly of myofibrils under stress and during the sarcomere development process.

The Unique Roles of UNC-45 Domains in Chaperoning Myosin Folding and Modulating Myosin Powerstroke

Molecular chaperones increase de novo protein folding efficiency in the crowded environment of the cell. They typically function by shielding hydrophobic regions of unfolded and partially folded proteins, preventing misfolding and aggregation. Also, certain chaperones interact with proteins further along their folding process, including the native state, but the relevance of this role remains unclear. Investigation of the mechanisms used by the chaperones UNC-45 and Hsp90 to assist in folding the myosin motor domain offers an ideal opportunity to investigate these gaps in knowledge.The main driver of current research is our unexpected finding that the myosin chaperone UNC-45 inhibits myosin motor domain powerstroke until its partner chaperone Hsp90 reverses the inhibition. We have found that the distinct domains comprising UNC-45 participate in discretely different transactions during myosin folding and assembly: 1) the UCS domain shields misfolding of folding intermediates as they occur heat shock conditions (and hence is responsible for the ‘classic chaperone’ function); 2) Our binding studies indicate that the Central domain also interacts with myosin but does not prevent myosin aggregation thus it does not possess chaperone-like activities. We found that the Central domain alone acts as an inhibitor of myosin power stroke. This different behavior and the fact that the Central domain induces significant conformational changes within the myosin--UNC-45 complex through allosteric interactions, suggest that the Central domain performs a different function than the UCS domain; 3) the TPR domain recruits Hsp90 to allow the myosin powerstroke to resume actin translocation(‘unblocking’ function).Our studies should provide novel mechanistic insights into the pathogenesis of increased or reduced myosin activity (such as in certain hypertrophic and dilated cardiomyopathies) and its modulation by chaperones.This work was supported by the American Heart Foundation (AHA 13GRNT17290006).

Chaperone-mediated reversible inhibition of the sarcomeric myosin power stroke

Molecular chaperones are required for successful folding and assembly of sarcomeric myosin in skeletal and cardiac muscle. Here, we show that the chaperone UNC-45B inhibits the actin translocation function of myosin. Further, we show that Hsp90, another chaperone involved in sarcomere development, allows the myosin to resume actin translocation. These previously unknown activities may play a key role in sarcomere development, preventing untimely myosin powerstrokes from disrupting the precise alignment of the sarcomere until it has formed completely.

Global Fit Analysis of Myosin-5b Motility Reveals Thermodynamics of Mg2+-Sensitive Acto-Myosin-ADP States

Kinetic and thermodynamic studies of the mechanochemical cycle of myosin motors are essential for understanding the mechanism of energy conversion. Here, we report our investigation of temperature and free Mg2+-ion dependencies of sliding velocities of a high duty ratio class-5 myosin motor, myosin-5b from D. discoideum using in vitro motility assays. Previous studies have shown that the sliding velocity of class-5 myosins obeys modulation by free Mg2+-ions. Free Mg2+-ions affect ADP release kinetics and the dwell time of actin-attached states. The latter determines the maximal velocity of actin translocation in the sliding filament assay. We measured the temperature dependence of sliding velocity in the range from 5 to 55°C at two limiting free Mg2+-ion concentrations. Arrhenius plots demonstrated non-linear behavior. Based on this observation we propose a kinetic model, which explains both sensitivity towards free Mg2+-ions and non-linearity of the temperature dependence of sliding velocity. According to this model, velocity is represented as a simple analytical function of temperature and free Mg2+-ion concentrations. This function has been applied to global non-linear fit analysis of three data sets including temperature and magnesium (at 20°C) dependence of sliding velocity. As a result we obtain thermodynamic parameters (ΔHMg and ΔSMg) of a fast equilibrium between magnesium free (AM·D) and magnesium bound acto-myosin-ADP (AM· Mg2+D) states and the corresponding enthalpic barriers associated with ADP release (ΔH1 ‡ and ΔH2 ‡). The herein presented integrative approach of data analysis based on global fitting can be applied to the remaining steps of the acto-myosin ATPase cycle facilitating the determination of energetic parameters and thermodynamics of acto-myosin interactions.

Nuclear localization of actin requires AC102 in Autographa californica multiple nucleopolyhedrovirus-infected cells

Autographa californica multiple nucleopolyhedrovirus requires nuclear actin for progeny virus production and thereby encodes viral products that ensure actin's translocation to and retention within the nucleus. Current evidence suggests that the ie0-ie1 gene complex along with five nuclear localization of actin (NLA) genes are sufficient for NLA in transient transfection experiments. Here we report that, during infection, only one of the five NLA genes, Ac102, was essential for NLA, and that AC102 had at least one other activity critical for budded virus (BV) production. Viral deletion mutants in the other four NLA genes were viable, with only two having replication phenotypes different from that of the wild type. Infection with AcΔpe38 revealed a delay in both BV production and NLA. Infection with AcΔ152 revealed a delay in BV production, but no corresponding delay in NLA. Infection with either AcΔpe38 or AcΔ152 resulted in slightly reduced BV titres. Deletion of Ac004 or he65 had no impact on actin translocation kinetics, timing of BV production or BV titres. These results implicate AC102 as a key player in baculovirus manipulation of actin.

Light‐Triggered Myosin Activation for Probing Dynamic Cellular Processes

Myosin II is an ATPase motor protein essential for many cellular functions including cell migration[1] and division.[2] In nonmuscle cells, myosin modulates protrusions at the leading edge and promotes retraction at the trailing edge during migration,[3] while during cytokinesis, myosin is required for contraction of the cleavage furrow.[4] For nonmuscle myosin, these varied functions are regulated by phosphorylation of the associated myosin regulatory light chain (mRLC) protein at Ser19, which activates the myosin complex to promote myosin assembly, contractility, and stress fiber formation.[5] Upon phosphorylation of the mRLC at both Thr18 and Ser19, these activities are further enhanced.[6 ] The dramatic effects of phosphorylation can also be recapitulated in vitro. Specifically, myosin and the proteolytic derivative heavy meromyosin (HMM),[7] which contains only one-third of the C-terminal myosin tail, exhibit low in vitro activities when associated with the nonphosphorylated mRLC. Phosphorylation of Ser19 amplifies actin-activated ATPase activities 10 – 1000-fold[8] and leads to myosin-mediated actin translocation.[9]

Kinetic Characterization of Non-Muscle Myosin IIB Single-Headed Heavy Meromyosin on Single Molecule Level with Optical Tweezers

Non-muscle myosin IIB (NMIIB) is a cytoplasmic conventional myosin, which plays an important role in development of the brain and heart, and in directed growth cone motility by maintaining cortical tension in motile cells. It forms short bipolar filaments with ∼14 myosin molecules on each side of the bare zone. NMIIB is a very slow myosin both in terms of actin-activated ATPase activity and actin translocation capability. Our previous studies showed that the NMIIB is a moderately high duty ratio (at least 20-25%) motor. The ADP release step (∼0.35 s-1), of NMIIB is only ∼3 times faster than the rate-limiting phosphate release (0.13 ± 0.01 s-1). Because of its slow ADP off-rate, acto-NMIIB has the highest ADP-affinity reported so far for the myosin superfamily (<0.15 μM). To examine the mechanics and kinetics of NMIIB at the single-molecule level we used a dual-beam optical tweezer to perform the three-bead assay. The surface-immobilized bead was coated with recombinantly engineered single-headed heavy meromyosin-like (NMIIB-SH-HMM) molecules. We measured the lifetimes of unitary actomyosin interactions and determined the actin-detachment kinetics with varying ATP concentrations. Results showed that at physiological ATP concentration (1 mM), the rate of detachment of acto-NMIIB-SH-HMM interactions was ∼0.51 s-1, similar to the ADP release rate and steady-state ATPase rate reported from solution kinetic studies. Decreasing the ATP concentration to 1 μM did not alter this rate of detachment (∼0.47 s-1). Also, our results showed that the power-stroke of NMIIB-SH-HMM was ∼8 nm. We will discuss our single-molecule results from the perspective of the essential cellular functions of NMIIB in cell locomotion, tension generation and maintenance.

Myosin II Motors and F-Actin Dynamics Drive the Coordinated Movement of the Centrosome and Soma during CNS Glial-Guided Neuronal Migration

Lamination of cortical regions of the vertebrate brain depends on glial-guided neuronal migration. The conserved polarity protein Par6alpha localizes to the centrosome and coordinates forward movement of the centrosome and soma in migrating neurons. The cytoskeletal components that produce this unique form of cell polarity and their relationship to polarity signaling cascades are unknown. We show that F-actin and Myosin II motors are enriched in the neuronal leading process and that Myosin II activity is necessary for leading process actin dynamics. Inhibition of Myosin II decreased the speed of centrosome and somal movement, whereas Myosin II activation increased coordinated movement. Ectopic expression or silencing of Par6alpha inhibited Myosin II motors by decreasing Myosin light-chain phosphorylation. These findings suggest leading-process Myosin II may function to "pull" the centrosome and soma forward during glial-guided migration by a mechanism involving the conserved polarity protein Par6alpha.

Interaction of Processive Motor Proteins with ATP analogue Having Syn Conformation with respect to the Adenine-ribose bond

It is known that ATP analogues such as 8-Br-ATP with bulky substitution at the eight position of the adenine ring predominantly assume the syn conformation with respect to the adenine-ribose bond. Previously we have demonstrated that 8-Br-ATP induces intrinsic trp fluorescence enhancement of smooth muscle myosin that reflect the formation of the M∗∗·ADP·Pi state. Moreover, the phosphorylated smooth muscle myosin supported actin translocation using 8-Br-ATP. Contrary, for skeletal muscle myosin, 8-Br-ATP induced neither trp fluorescence enhancement nor actin translocation. Kinesin is also ATP driven motor protein that has strikingly similar structure of motor domain to myosin. In the present study, interaction of kinesin with 8-Br-ATP has been examined. Interestingly, conventional kinesin supported microtubules translocation using 8-Br-ATP. This suggests conventional kinesin adopts the 8-Br-ATP in the normal conformation. However, the sliding velocity was approximately one-fifth of regular ATP. Moreover single molecular measurment using optical tweezers revealed that for kinesin, 8-Br-ATP induced nearly similar force generation with that of ATP. Myosin V is also processive motor protein like kinesin. Interaction of unconventional myosin V with 8-Br-ATP was also analyzed. Myosin V supported actin translocation using 8-Br-ATP. Currently, we are examining single molecular measurment of myosin V in the presence of 8-Br-ATP.

A Viscous Drag Model of Frictional Loading Assays for Myosin Motors

The myosin superfamily is a diverse family of actin binding proteins that hydrolyze ATP and generate force. The in vitro motility assay, which combines the strengths of solution biochemistry with the ability to measure myosin force and motility, has allowed the molecular events that couple ATP hydrolysis to mechanical work to be probed. Several groups have used modifications of the in vitro motility assay to study the effects of load on actin filament velocity. One such method uses alpha-actinin to exert a frictional load on sliding actin filaments that are being propelled over a bed of a given myosin isoform. This method, which has been used to measure myosin isometric force, relies on the ability to determine the amount of alpha-actinin necessary to stop actin translocation over the bed of myosin. However, careful examination of frictional loading assay data shows that even in the presence of very high concentrations of alpha-actinin, actin filament sliding was never observed to completely stop, suggesting a non-linear relationship between the amount of alpha-actinin added and the frictional load acting on the myosin. In order to better characterize this behavior, we developed a model in which the alpha-actinin behaves as a viscoelastic rather than purely elastic element, giving a velocity dependence to the frictional load imposed by the alpha-actinin. This correction allows for the determination of the myosin isometric force in the absence of a clear zero velocity point. Furthermore, this model describes the absolute resisting force imposed by the alpha-actinin as function of velocity, allowing for the measurement of isotonic kinetics using the motility assay. Supported by NIH-HL077280, AHA-0435434T (J.R.M.), and AHA-0815704D (to M.J.G.).

Smooth Muscle Myosin Phosphorylated at Single Head Shows Sustained Mechanical Activity

Smooth muscle contraction is regulated by the phosphorylation of myosin. It is well known that tonic smooth muscles can maintain force with low energy consumption (latch state); however, the molecular mechanism underlying this phenomenon is unresolved. Here we show that single-head phosphorylated smooth myosin (SHPMII) exhibits fast ( approximately 24 s(-1)) and slow prolonged ( approximately 1 s(-1)) actin interactions, whereas double-head phosphorylated myosin (DHPMII) predominantly exhibits the fast ( approximately 29 s(-1)) interaction, suggesting that the phosphorylated head of SHPMII is mechanically as active as that of DHPMII. Both the fast and the slow actin interactions of SHPMII support the positive net mechanical displacement of actin. The actin translocating velocity of SHPMII was much slower than that of DHPMII, which is consistent with the slow actin interaction of SHPMII. We propose that the "latch state" can be explained by the motor characteristics of SHPMII that is present during the sustained phase of contraction.

When fibres go slack and cross bridges are free to run: a brilliant method to study kinetic properties of acto‐myosin interaction

When fibres go slack and cross bridges are free to run: a brilliant method to study kinetic properties of acto‐myosin interaction

Protein kinase C-ζ regulation of GLUT4 translocation through actin remodeling in CHO cells

Actin remodeling plays a crucial role in insulin-induced translocation of glucose transporter 4 (GLUT4) from the cytoplasm to the plasma membrane and subsequent glucose transport. Protein kinase C (PKC) zeta has been implicated in this translocation process, although the exact mechanism remains unknown. In this study, we investigated the effect of PKCzeta on actin cytoskeleton and translocation of GLUT4 in CHO-K1 cells expressing myc-tagged GLUT4. Insulin stimulated the phosphorylation of PKCzeta at Thr410 with no apparent effect on its protein expression. Moreover, insulin promoted colocalization of PKCzeta and actin that could be abolished by Latrunculin B. The overexpression of PKCzeta mimicked the insulin-induced change in actin cytoskeleton and translocation of GLUT4. These effects were also completely abrogated by Latrunculin B treatment. Using cell-permeable pseudosubstrate (PS) inhibitor of PKCzeta, the response to insulin could be alleviated. Our results strongly suggest that PKCzeta mediates the stimulatory effect of insulin on GLUT4 translocation through its interaction with actin cytoskeleton.

A novel cyathane diterpene, cyrneine A, induces neurite outgrowth in a Rac1-dependent mechanism in PC12 cells

We examined the effects of cyrneine A, a novel diterpene isolated from the mushroom Sarcodon cyrneus, on morphology of rat pheochromocytoma cells (PC12). In response to cyrneine A, PC12 cells extended their neurites, an effect partially blocked by the extracellular signal-regulated kinase (ERK) kinase inhibitor PD98059, but not by the protein kinase C inhibitor GF109203X, nor the phosphatidylinositol-3-kinase inhibitor wortmannin. Cyrneine A did not activate ERK at any of the time points tested (5–120 min), indicating that only the basal activity of ERK is required for cyrneine A-induced neurite outgrowth. As transcriptional regulation is required for neurite extension, the activity of three major transcription factors was determined. Cyrneine A enhanced activation of the transcription factors activator protein-1 (AP-1) and nuclear factor-κB, but not CREB, and this was accompanied by enhanced c-fos expression. Moreover, we determined the role of Rac1, a small GTPase protein of the Rho family that regulates actin dynamics, in cyrneine A-induced neurite outgrowth. Treatment with cyrneine A led to actin translocation and subsequently, to accumulation of F-actin at the tip of neurites. Rac1 activity was increased by cyrneine A and expression of a dominant-negative Rac1 mutant significantly inhibited the cyrneine A-induced extension of neurites. These results suggest that cyrneine A induces neurite outgrowth in a Rac1-dependent mechanism.

Packaging actomyosin-based biomolecular motor-driven devices for nanoactuator applications

Biomolecular motors such as the muscle protein myosin with its partner protein actin hold great promise for actuation in hybrid nanoscale biomicroelectromechanical systems devices (bio-MEMS), particularly for future biomedical applications that involve highly localized delivery of biomolecules over short distances (e.g., micrometers) to specific tissue or cellular locations. Two fundamental issues in the construction and packaging of actomyosin-based nanoactuators are the ability to electrically insulate microelectrical components while maintaining both biocompatibility and also compatibility with our functional assays for prototype development and identification of conditions for storage of assembled devices. Here, we show that sputter coating with SiO/sub 2/ provides a straightforward method for electrical insulation that can be readily integrated into existing assays of myosin function in a bio-MEMS setting. We also report using in vitro motility analysis that both rabbit skeletal muscle heavy meromyosin (HMM) and fish (Fundulus heteroclitus) myosin remained functional for at least six days in a bio-MEMS setting when hydrating conditions were maintained. The speed of actin sliding was faster after six days when rabbit HMM was stored in 10% DMSO than in its absence, but there was no effect of DMSO during storage on fish myosin. The speed of actin translocation by fish myosin was significantly increased when adenosine 5'-triphosphate (ATP), the chemical energy source for myosin function, was replaced by the analog 2' deoxy-ATP, as has been previously reported for rabbit HMM. Taken together, these results provide new direction for modulation and control of actomyosin-based nanoactuators and also for long-term storage of assembled nanoactuators.

Molecular and phenotypic effects of heterozygous, homozygous, and compound heterozygote myosin heavy-chain mutations

Autosomal dominant familial hypertrophic cardiomyopathy (FHC) has variable penetrance and phenotype. Heterozygous mutations in MYH7 encoding beta-myosin heavy chain are the most common causes of FHC, and we proposed that "enhanced" mutant actin-myosin function is the causative molecular abnormality. We have studied individuals from families in which members have two, one, or no mutant MYH7 alleles to examine for dose effects. In one family, a member homozygous for Lys207Gln had cardiomyopathy complicated by left ventricular dilatation, systolic impairment, atrial fibrillation, and defibrillator interventions. Only one of five heterozygous relatives had FHC. Leu908Val and Asp906Gly mutations were detected in a second family in which penetrance for Leu908Val heterozygotes was 46% (21/46) and 25% (3/12) for Asp906Gly. Despite the low penetrance, hypertrophy was severe in several heterozygotes. Two individuals with both mutations developed severe FHC. The velocities of actin translocation (V(actin)) by mutant and wild-type (WT) myosins were compared in the in vitro motility assay. Compared with WT/WT, V(actin) was 34% faster for WT/D906G and 21% for WT/L908V. Surprisingly V(actin) for Leu908Val/Asp906Gly and Lys207Gln/Lys207Gln mutants were similar to WT. The apparent enhancement of mechanical performance with mutant/WT myosin was not observed for mutant/mutant myosin. This suggests that V(actin) may be a poor predictor of disease penetrance or severity and that power production may be more appropriate, or that the limited availability of double mutant patients prohibits any definitive conclusions. Finally, severe FHC in heterozygous individuals can occur despite very low penetrance, suggesting these mutations alone are insufficient to cause FHC and that uncharacterized modifying mechanisms exert powerful influences.