Study of actin mutations linked to muscle disease

Imperial Users onl

The purification and in vitro motility analysis of Drosophila melanogaster ACT88F mutants.

The indirect flight muscle (IFM) specific ACT88F actin isoform o f Drosophila melanogaster was purified from flies on both a large and small scale. The development o f a mini-actin purification protocol allowed the isolation o f pure ACT88F actin from ten pairs o f dissected IFMs in sufficient quantities, (approximately 5pg), for many in vitro motility assays. An actin preparation, starting with 10,000 flies, (10g), was also developed, using anion exchange chromatography to isolate the ACT88F isoform from the five other Drosophila isoforms. Milligram quantités of ACT88F, containing a 10% “contamination” of an unknown type III actin isoform, provide sufficient material for the future in vitro biochemical and kinetic characterisation of ACT88F mutants. The in vitro motility o f four ACT88F mutants, G368E, E316K, E334K and E93K was investigated using a rabbit skeletal muscle HMM. A significant 35% reduction in G368E filament velocity under standard assay conditions (SAC) was also seen when under various ionic and ATP concentrations. E316K only showed a significant 36% reduction in its filament velocity at limiting ATP concentrations. Under all conditions in the motility assay, E334K mutant filaments displayed no in vitro movement. Where wild-type (WT) actin washed off the surface at 50mM KC1 in the motility assay, E334K actin dissociated at 30mM KC1. Three copolymers o f E334K and WT actin, each representing a different proportion and distribution o f mutant monomers in the cofilament, were all able to move under SAC. As the percentage fraction of E334K actin in the cofilament was increased, filament velocity decreased. Although E93K actin filaments washed off the surface under standard assay conditions, binding to, and movement o f this mutant over the surface was seen at lower ionic strengths, albeit with a significant 50% reduction in filament velocity. These results are discussed with respect to the atomic structure o f actin and the model o f the actin-Sl reconstruction. ACT88F was expressed in Saccharomyces cerevisiae. The expression o f WT and six ACT88F mutants was confirmed by two-dimensional gel electrophoresis and/or Western blotting with actin specific antibodies. However, the levels o f recombinant protein were not great enough to support in vitro biochemical studies

Investigating Myosin Ensemble Force Generation Using Optical Tweezers

Myosin is a motor protein that facilitates muscle contraction and movement by stepping along actin filaments using energy from ATP hydrolysis. If myosin or other motors are disrupted throughout the body, it can have many harmful effects. Hypertrophic cardiomyopathy is a disease caused by gene mutations that affect myosin heavy chains in the heart. The tissue of the heart becomes abnormally thick, which can make it more difficult to pump blood or block blood flow out of the heart. Our goal is to discern the mechanistic difference function of a healthy and diseased heart. To accomplish this, we construct model actomyosin environments and measure their force generation using optical tweezers. Most studies completed on these subjects in the past have been done on individual motor proteins, which is not the best representation of how they function in the human body. We have worked to make models of myosin and actin bundles that function as similarly to human muscle as possible. These models produce displacement and force profiles that can be analyzed to find the average cycles and work done for each bundle. These tests have resulted in three main force profiles, each showing the distinct signs of myosin stepping even with different optimized force production from the proteins

Biomolecular Shuttles under Dielectrophoretic Forces

Motor proteins and filaments are essential elements in living cells. They are employed in skeletal muscles to generate forces, they transport cargos such as organelles to specific locations in the cells or they reorganize themselves to change a cell\u27s structure. Moreover, motor proteins and filaments use hydrolysis of adenosine triphosphate (ATP) as chemical fuel to generate mechanical movement in their interaction. Understanding the behavior of these enticed nano-sized machines and their properties, yet to be mimicked and synthesized by humans is very important to the future development of transport in nanoscale. Thus far, researchers succeeded in demonstrating the interaction of motor proteins and filaments in in vitro environment and controlling their random movement by various methods such as with the influence of DC electric field, driven flow field and engineered tracks by photolithographic method. In this thesis, dielectrophoretic forces, which are generated under nonuniform electric field by AC, are explored as a candidate to control the direction of biomolecular shuttles, actin filaments which glide on heavy meromyosin coated surface. Under dielectrophoretic forces, actin filaments showed bidirectional movement between embedded electrodes. The orientation and velocity of actin filaments were measured under various AC voltages, frequencies and distances between electrodes. Additionally, the effect of temperature on myosin-actin motility was further investigated and loading cargo on actin filaments was demonstrated by using a streptavidin-biotin binding system

Tropomyosin-Based Effects of Acidosis on Thin-Filament Regulation During Muscle Fatigue

Skeletal muscle fatigue is defined as a loss in the force/velocity generating capacity of a muscle. A portion of the loss in function is attributable to effects of acidosis (i.e. low pH) on the regulatory proteins, troponin and tropomyosin (Tm), which regulate the binding of myosin and actin in a calcium (Ca++) dependent manner. However, the relative role of troponin and Tm on myosin-actin function during acidosis is not clear, nor are the mechanisms underlying these effects. PURPOSE: To determine the role of Tm in the acidosis-induced depression of muscle function using isolated muscle proteins in an in vitro motility assay. METHODS: Three mutant constructs of Tm were expressed by replacing the two amino acid (histidine) residues most likely affected by low pH with alanine residues (H153A, H276A, H153A/H276A). These mutant constructs were compared to wild-type Tm (wt-Tm) in order to test whether the acidosis-induced charge change of the histidine amino acid governs the pH-dependent alteration of tropomyosin and therefore the decrease in maximal RTF velocity and Ca++-sensitivity. The effect of acidosis on regulated thin filament (RTF) function was determined by assessing the impact of low pH (pH 6.8) versus neutral pH (pH 7.4) on myosin’s ability to move RTFs in the motility assay as a function of increasing levels of Ca++. This was done separately for the wt-Tm and each structural variant. RESULTS: A two-way ANOVA (pH x Tm construct) revealed that acidosis significantly (p\u3c0.05) depressed the maximal sliding velocity of the RTFs across all versions of Tm, but that the magnitude of the depression was similar among the wt and all of the Tm mutants. Acidosis did not significantly depress the sensitivity to Ca++ under the unloaded conditions of this assay (p\u3e0.05). CONCLUSIONS: These data suggest that the histidine residues in tropomyosin do not mediate the acidosis-induced depression in contraction velocity observed during muscle fatigue. However, since these residues may be more important in mediating the depression of force, we are currently testing the impact of the three mutant Tm constructs on the acidosis-induced depression in Ca++-sensitivity using a loaded in vitro motility assay

Effects of regulatory light chain phosphorylation on mutant and wild-type cardiac muscle myosin mechanochemistry

Cardiac muscle contraction is responsible for pumping blood throughout the body. The cyclical, ATP-hydrolysis dependent interaction of the myosin motor protein with filamentous actin drives muscle contraction. During this process the α-helical neck region of myosin acts as a lever arm, transmitting contractile force between thick and thin filaments by amplifying small conformational changes in the myosin motor domain. The resulting relative displacement of thick and thin filaments causes muscle shortening. The regulatory light chain (RLC) of myosin mechanically supports the lever arm by binding to the myosin heavy chain neck region; this is a crucial interaction in maintaining myosin's ability to produce force and motion. We investigated the role of N-terminal modifications of the RLC in modulating actomyosin contractility at the molecular level. Phosphorylation of the RLC is a naturally occurring post-translational modification of the RLC N-terminus that is important for cardiac function and has been shown to enhance contractility at the cellular level. In contrast, genetic mutations of the RLC that lead to familial hypertrophic cardiomyopathy (FHC) disrupt cardiac function and trigger remodeling of the cardiac muscle structure. We studied two FHC-linked mutations, N47K and R58Q, located in the N-terminus of the RLC in close proximity to the phosphorylation site. Using in vitro motility assays we examined how RLC modifications affect the mechanochemical properties of cardiac β-myosin. We found that the FHC mutations reduced myosin force and power generation, in contrast to RLC phosphorylation which increased myosin force and power for WT and mutant myosins. Phosphorylation of mutant RLC resulted in a restoration of the mutation-induced decreases in contractility to WT dephosphorylated levels. These results point to RLC phosphorylation as a general mechanism to increase force production of the individual myosin motor and as a potential target to ameliorate the fundamental contractile FHC-induced phenotype

Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism

Myosin-binding protein C (MyBP-C) is an accessory protein of striated muscle thick filaments and a modulator of cardiac muscle contraction. Defects in the cardiac isoform, cMyBP-C, cause heart disease. cMyBP-C includes 11 Ig- and fibronectin-like domains and a cMyBP-C-specific motif. In vitro studies show that in addition to binding to the thick filament via its C-terminal region, cMyBP-C can also interact with actin via its N-terminal domains, modulating thin filament motility. Structural observations of F-actin decorated with N-terminal fragments of cMyBP-C suggest that cMyBP-C binds to actin close to the low Ca(2+) binding site of tropomyosin. This suggests that cMyBP-C might modulate thin filament activity by interfering with tropomyosin regulatory movements on actin. To determine directly whether cMyBP-C binding affects tropomyosin position, we have used electron microscopy and in vitro motility assays to study the structural and functional effects of N-terminal fragments binding to thin filaments. 3D reconstructions suggest that under low Ca(2+) conditions, cMyBP-C displaces tropomyosin toward its high Ca(2+) position, and that this movement corresponds to thin filament activation in the motility assay. At high Ca(2+), cMyBP-C had little effect on tropomyosin position and caused slowing of thin filament sliding. Unexpectedly, a shorter N-terminal fragment did not displace tropomyosin or activate the thin filament at low Ca(2+) but slowed thin filament sliding as much as the larger fragments. These results suggest that cMyBP-C may both modulate thin filament activity, by physically displacing tropomyosin from its low Ca(2+) position on actin, and govern contractile speed by an independent molecular mechanism

Mutations in repeating structural motifs of tropomyosin cause gain of function in skeletal muscle myopathy patients

The congenital myopathies include a wide spectrum of clinically, histologically and genetically variable neuromuscular disorders many of which are caused by mutations in genes for sarcomeric proteins. Some congenital myopathy patients have a hypercontractile phenotype. Recent functional studies demonstrated that ACTA1 K326N and TPM2 ΔK7 mutations were associated with hypercontractility that could be explained by increased myofibrillar Ca(2+) sensitivity. A recent structure of the complex of actin and tropomyosin in the relaxed state showed that both these mutations are located in the actin–tropomyosin interface. Tropomyosin is an elongated molecule with a 7-fold repeated motif of around 40 amino acids corresponding to the 7 actin monomers it interacts with. Actin binds to tropomyosin electrostatically at two points, through Asp25 and through a cluster of amino acids that includes Lys326, mutated in the gain-of-function mutation. Asp25 interacts with tropomyosin K6, next to K7 that was mutated in the other gain-of-function mutation. We identified four tropomyosin motifs interacting with Asp25 (K6-K7, K48-K49, R90-R91 and R167-K168) and three E-E/D-K/R motifs interacting with Lys326 (E139, E181 and E218), and we predicted that the known skeletal myopathy mutations ΔK7, ΔK49, R91G, ΔE139, K168E and E181K would cause a gain of function. Tests by an in vitro motility assay confirmed that these mutations increased Ca(2+) sensitivity, while mutations not in these motifs (R167H, R244G) decreased Ca(2+) sensitivity. The work reported here explains the molecular mechanism for 6 out of 49 known disease-causing mutations in the TPM2 and TPM3 genes, derived from structural data of the actin–tropomyosin interface

Observing the Molecular Basis of Thin Filament Activation with a Three Bead Laser Trap Assay

Muscle contracts after calcium (Ca++) is released into the muscle cell, resulting from a cascade of events which result in myosin, the molecular motor of muscle, to produce force and motion. Myosin cyclically binds to a regulated thin filament, using the chemical energy of ATP to produce force and motion. Perturbations in muscle, such as a build-up of metabolic by-products or point mutations in key contractile proteins, can inhibit these functions in both skeletal and cardiac muscle either acutely or chronically. Despite the many years we have studied skeletal and cardiac muscle, we still do not have a clear picture of the effect of these perturbations at the molecular level. Indeed, we do not even have a clear picture of how muscle is activated at the molecular level. Such an understanding would provide a foundation for future work, and aid our understanding of perturbations such as muscle fatigue or point mutations. Recent advances in biophysical techniques have allowed us to directly observe both single myosin and small ensembles of myosin interacting with an actin filament. We build off this previous work by examining 1) a single myosin molecule interacting with a regulated actin thin filament, and 2) small ensembles of myosin working together to pull on a regulated thin filament. We set out to directly determine how Ca++ and myosin activate the thin filament and thus muscle. Our approach to directly observe these molecular phenomena across physiological Ca++ levels as well as high ATP concentrations is novel. The first goal of this work was to examine how 1) a single myosin interacts with a regulated thin filament and 2) how a small ensemble of myosins work together to generate force and motion in a Ca++ dependent manner as they interact with a regulated thin filament. The findings build on previous work using similar techniques, but are rich in data and provide a more physiological viewpoint. The second goal of this work was to examine a particular mutation that leads to hypertrophic cardiomyopathy. The current evidence suggests that this mutation alters both thin filament dynamics and myosin kinetics. This mutation was thus chosen not only to add to our understanding of this particular mutation in hopes of developing a therapeutic, but also to provide insight into the role of thin filament activation and myosin dynamics in hopes of better understanding muscle contraction overall. With our experiments, supplemented with a mathematical model, we have de-convolved the role of Ca++ and myosin in thin filament activation, and developed a model of muscle activation from the single molecule up to the scale of a cell