Professor Michael Geeves
Mike Geeves joined the School of Biosciences in January 1999. He studied biochemistry as an undergraduate at the University of Birmingham then went on to the University of Bristol to work on a PhD with David Trentham. It was here that he first came to work on the myosin motor which has been the focus of his work ever since. In those early days it was muscle myosin - the only known form of myosin. After completing his PhD he spent 2 years at the University of California, Santa Cruz studying enzymology at sub-zero temperatures with Anthony Fink. He then return to spend 14 years at the University of Bristol working alongside Freddie Gutfreund, first as an SERC Junior Fellow then as a Royal Society University Fellow. At the end of the Fellowship he moved to become a Group Leader in the new Max Planck Institute of Molecular Physiology that was being established in Dortmund by Roger Goody. He left there to take up the current position as Professor of Physical Biochemistry. He was Head of School between 2006 - 2010.
Mike is a member of the Mechanobiology group also known as MaDCaP
ORCID ID: 0000-0002-9364-8898
Molecular motors particularly the motors of the myosin family including the myosin IIs responsible for muscle contraction.
Regulation of muscle contraction via calcium.
Inherited diseases of skeletal and cardiac muscle.
Novel fast reaction methods.
MSc-R project available for 2019/20
Evolution of the muscle sarcomere. A bioinformatics approach to the interaction between myosin and myosin binding protein-C
(joint supervision with Dr Mark Wass)
Following on from a study of how muscle-type myosins have adapted, over evolutionary timescales, for different types of muscle contraction, we will explore the co-evolution of myosin and the myosin binding proteins C. MyBP-C is well known to carry mutations linked to inherited heart disease.
Additional research costs: £1200
Protein based temperature sensors for use inside cells and organelles (project jointly supervised with Dr Dan Mulvihill)
We have a created a series of prototype protein based sensors to allow variation and changes in temperature to be monitored within living cells. Additional research costs: £1200
This project will;
- modify the protein to optimise the temperature profile for different cell types.
- express the protein in a variety of prokaryote and eukaryote cells and organisms.
- use signal peptides to target the sensor to specific cellular organelles.
Live Cell Imaging under Pressure
(project jointly supervised with Dr Dan Mulvihill)
We have a built a novel live cell imaging system that allows protein dynamics to be followed at 100 atmospheres, a pressure which specifically and reversibly disrupts protein dynamics.
This project will use this system to follow actin and microtubule dynamics in a variety of cell types to study how perturbations of cytoskeleton dynamics affect growth and organisation of cells and development.
Additional research costs: £1200
Showing 50 of 130 total publications in the Kent Academic Repository. View all publications.
Mitrou, G. et al. (2019). Evidence of functional deficits at the single muscle fiber level in experimentally-induced renal insufficiency. Journal of Biomechanics [Online] 82:259-265. Available at: https://doi.org/10.1016/j.jbiomech.2018.10.035.Chronic kidney disease patients present with metabolic and functional muscle
abnormalities, called uremic myopathy, whose mechanisms have not yet been fully
elucidated. We investigated whether chronic renal insufficiency (CRI) affects skeletal
muscle contractile properties at the cellular level. CRI was induced surgically in New
Zealand rabbits (UREM), with sham-operation for controls (CON), and samples were
collected at 3 months post-surgery, following euthanasia. All protocols had University
Ethics approval following national and European guidelines. Sample treatments and
evaluations were blinded. Maximal isometric force was assessed in 382 permeabilized
psoas fibers (CON, n=142, UREM, n=240) initially at pH7, 10oC (‘standard’
conditions), in subsets of fibers in acidic conditions (pH6.2, 10oC) but also at near
physiological temperature (pH7, 30oC and pH6.2, 30oC). CRI resulted in significant
smaller average CSA (~11%) for UREM muscle fibers (vs CON, P<0.01). At
standard conditions, UREM fibers produced lower absolute and specific forces (i.e.
normalized force per fiber CSA) (vs CON, P<0.01); force increased in 30oC for both
groups (P<0.01), but the disparity between UREM and CON remained significant.
Acidosis significantly reduced force (vs pH7, 10oC P<0.01), similarly in both groups
(in UREM by -48% and in CON by -43%, P>0.05). For the first time, we give
evidence that CRI can induce significant impairments in single psoas muscle fibers
force generation, only partially explained by fiber atrophy, thus affecting muscle
mechanics at the cellular level.
Sparrow, A. et al. (2019). Measurement of Myofilament-Localised Calcium Dynamics in Adult Cardiomyocytes and the Effect of Hypertrophic Cardiomyopathy Mutations. Circulation Research [Online]. Available at: https://doi.org/10.1161/CIRCRESAHA.118.314600.Rationale: Subcellular Ca2+ indicators have yet to be developed for the myofilament where disease mutation, or small molecules may alter contractility through myofilament Ca2+ sensitivity. Here we develop and characterise genetically encoded Ca2+ indicators restricted to the myofilament to directly visualise Ca2 changes in the sarcomere.
Objective: To produce and validate myofilament restricted Ca2+ imaging probes in an adenoviral transduction adult cardiomyocyte model using drugs that alter myofilament function (MYK-461, omecamtiv mecarbil and levosimendan) or following co-transduction of two established hypertrophic cardiomyopathy (HCM) disease causing mutants (cTnT R92Q and cTnI R145G) that alter myofilament Ca2+ handling.
Methods and Results: When expressed in adult ventricular cardiomyocytes RGECO-TnT/TnI sensors localise correctly to the sarcomere without contractile impairment. Both sensors report cyclical changes in fluorescence in paced cardiomyocytes with reduced Ca2+ on and increased Ca2+ off rates compared with unconjugated RGECO. RGECO-TnT/TnI revealed changes to localised Ca2+ handling conferred by MYK-461 and levosimendan, including an increase in Ca2+ binding rates with both levosimendan and MYK-461 not detected by an unrestricted protein sensor. Co-adenoviral transduction of RGECO-TnT/TnI with HCM causing thin filament mutants showed that the mutations increase myofilament [Ca2+] in systole, lengthen time to peak systolic [Ca2+], and delay [Ca2+] release. This contrasts with the effect of the same mutations on cytoplasmic Ca2+, when measured using unrestricted RGECO where changes to peak systolic Ca2+ are inconsistent between the two mutations. These data contrast with previous findings using chemical dyes that show no alteration of [Ca2+] transient amplitude or time to peak Ca2+.
Conclusions: RGECO-TnT/TnI are functionally equivalent. They visualise Ca2+ within the myofilament and reveal unrecognised aspects of small molecule and disease associated mutations in living cells.
Behrens, V. et al. (2019). Mg2+ -free ATP regulates the processivity of native cytoplasmic dynein. FEBS Letters [Online] 593:296-307. Available at: https://doi.org/10.1002/1873-3468.13319.Cytoplasmic dynein, a microtubule?based motor protein, is responsible for many cellular functions ranging from cargo transport to cell division. The various functions are carried out by a single isoform of cytoplasmic dynein, thus requiring different forms of motor regulation. A possible pathway to regulate motor function was revealed in optical trap experiments. Switching motor function from single steps to processive runs could be achieved by changing Mg2+ and ATP concentrations. Here, we confirm by single molecule total internal reflection fluorescence microscopy that a native cytoplasmic dynein dimer is able to switch to processive runs of more than 680 consecutive steps or 5.5 ?m. We also identified the ratio of Mg2+?free ATP to Mg.ATP as the regulating factor and propose a model for dynein processive stepping.
Helassa, N. et al. (2018). Ultrafast glutamate sensors resolve high-frequency release at Schaffer collateral synapses. Proceedings of the National Academy of Sciences [Online]. Available at: https://doi.org/10.1073/pnas.1720648115.Glutamatergic synapses display a rich repertoire of plasticity mechanisms on many different time scales, involving dynamic changes in the efficacy of transmitter release as well as changes in the number and function of postsynaptic glutamate receptors. The genetically encoded glutamate sensor iGluSnFR enables visualization of glutamate release from presynaptic terminals at frequencies up to ?10 Hz. However, to resolve glutamate dynamics during high-frequency bursts, faster indicators are required. Here, we report the development of fast (iGluf) and ultrafast (iGluu) variants with comparable brightness but increased Kd for glutamate (137 ?M and 600 ?M, respectively). Compared with iGluSnFR, iGluu has a sixfold faster dissociation rate in vitro and fivefold faster kinetics in synapses. Fitting a three-state model to kinetic data, we identify the large conformational change after glutamate binding as the rate-limiting step. In rat hippocampal slice culture stimulated at 100 Hz, we find that iGluu is sufficiently fast to resolve individual glutamate release events, revealing that glutamate is rapidly cleared from the synaptic cleft. Depression of iGluu responses during 100-Hz trains correlates with depression of postsynaptic EPSPs, indicating that depression during high-frequency stimulation is purely presynaptic in origin. At individual boutons, the recovery from depression could be predicted from the amount of glutamate released on the second pulse (paired pulse facilitation/depression), demonstrating differential frequency-dependent filtering of spike trains at Schaffer collateral boutons.
Geeves, M. et al. (2018). Dilated cardiomyopathy myosin mutants have reduced force-generating capacity. Journal of Biological Chemistry [Online]. Available at: http://dx.doi.org/10.1074/jbc.RA118.001938.Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) can cause arrhythmias, heart failure, and cardiac death. Here, we functionally characterized the motor domains of five DCM-causing mutations in human ?-cardiac myosin. Kinetic analyses of the individual events in the ATPase cycle revealed that each mutation alters different steps in this cycle. For example, different mutations gave enhanced or reduced rate constants of ATP binding, ATP hydrolysis, or ADP release or exhibited altered ATP, ADP, or actin affinity. Local effects dominated, no common pattern accounted for the similar mutant phenotype, and there was no distinct set of changes that distinguished DCM mutations from previously analyzed HCM myosin mutations. That said, using our data to model the complete ATPase contraction cycle revealed additional critical insights. Four of the DCM mutations lowered the duty ratio (the ATPase cycle portion when myosin strongly binds actin) because of reduced occupancy of the force-holding A·M.D complex in the steady-state. Under load, the A·M·D state is predicted to increase owing to a reduced rate constant for ADP release, and this effect was blunted for all five DCM mutations. We observed the opposite effects for two HCM mutations, namely R403Q and R453C. Moreover, the analysis predicted more economical use of ATP by the DCM mutants than by WT and the HCM mutants. Our findings indicate that DCM mutants have a deficit in force generation and force holding capacity due to the reduced occupancy of the force-holding state.
Brooker, H. et al. (2018). A novel live cell imaging system reveals a reversible hydrostatic pressure impact on cell cycle progression. Journal of Cell Science [Online] 131:jcs212167. Available at: http://dx.doi.org/10.1242/jcs.212167.Life is dependent upon the ability of a cell to rapidly respond to changes in
environment. Small perturbations in local environments change the ability of
molecules to interact and hence communicate. Hydrostatic pressure provides
a rapid non-invasive, fully-reversible method for modulating affinities between
molecules both in vivo and in vitro. We have developed a simple fluorescence
imaging chamber that allows intracellular protein dynamics and molecular
events to be followed at pressures up to 200 bar in living cells. Using yeast we
investigate the impact of hydrostatic pressure upon cell growth and cell cycle
progression. While 100 bar has no affect upon viability, it induces a delay in
chromosome segregation, resulting in the accumulation of long-undividedbent
cells, consistent with disruption of the cytoskeletons. This delay is
independent of stress signalling and induces synchronisation of cell-cycle
progression. Equivalent affects were observed in Candida albicans, with
pressure inducing a reversible cell-cycle delay and hyphal growth. We present
a simple novel non-invasive fluorescence microscopy based approach to
transiently impact molecular dynamics to visualise, dissect and study signalling pathways and cellular processes in living cells.
Johnson, C. et al. (2017). Temperature sensitive point mutations in fission yeast tropomyosin have long range effects on the stability and function of the actin- tropomyosin copolymer. Biochemical and Biophysical Research Communications [Online]. Available at: https://doi.org/10.1016/j.bbrc.2017.10.109.The actin cytoskeleton is modulated by regulatory actin-binding proteins which fine- tune the dynamic properties of the actin polymer to regulate function. One such actin-binding protein is tropomyosin (Tpm), a highly-conserved alpha-helical dimer which stabilises actin and regulates interactions with other proteins. Temperature sensitive mutants of Tpm are invaluable tools in the study of actin filament dependent processes, critical to the viability of a cell. Here we investigated the molecular basis of the temperature sensitivity of fission yeast Tpm mutants which fail to undergo cytokinesis at the restrictive temperatures. Comparison of Contractile Actomyosin Ring (CAR) constriction as well as cell shape and size revealed the cdc8.110 or cdc8.27 mutant alleles displayed significant differences in their temperature sensitivity and impact upon actin dependent functions during the cell cycle. In vitro analysis revealed the mutant proteins displayed a different reduction in thermostability, and unexpectedly yield two discrete unfolding domains when acetylated on their amino-termini. Our findings demonstrate how subtle changes in structure (point mutations or acetylation) alter the stability not simply of discrete regions of this conserved cytoskeletal protein but of the whole molecule. This differentially impacts the stability and cellular organisation of this essential cytoskeletal protein.
Geeves, M. (2017). More Can Mean Less, or: Simplifying Sometimes Requires Ideas To Be More Complicated. Biophysical Journal [Online] 112:2467-2468. Available at: http://dx.doi.org/10.1016/j.bpj.2017.05.004.
Mijailovich, S. et al. (2017). Modeling the Actin.myosin ATPase cross-bridge cycle for skeletal and cardiac muscle myosin isoforms. Biophysical Journal [Online] 112:984-996. Available at: http://dx.doi.org/10.1016/j.bpj.2017.01.021.Modeling the complete actin.myosin ATPase cycle has always been limited by the lack of experimental data concerning key steps of the cycle, because these steps can only be defined at very low ionic strength. Here, using human ?-cardiac myosin-S1, we combine published data from transient and steady-state kinetics to model a minimal eight-state ATPase cycle. The model illustrates the occupancy of each intermediate around the cycle and how the occupancy is altered by changes in actin concentration for [actin] = 1–20Km. The cycle can be used to predict the maximal velocity of contraction (by motility assay or sarcomeric shortening) at different actin concentrations (which is consistent with experimental velocity data) and predict the effect of a 5 pN load on a single motor. The same exercise was repeated for human ?-cardiac myosin S1 and rabbit fast skeletal muscle S1. The data illustrates how the motor domain properties can alter the ATPase cycle and hence the occupancy of the key states in the cycle. These in turn alter the predicted mechanical response of the myosin independent of other factors present in a sarcomere, such as filament stiffness and regulatory proteins. We also explore the potential of this modeling approach for the study of mutations in human ?-cardiac myosin using the hypertrophic myopathy mutation R453C. Our modeling, using the transient kinetic data, predicts mechanical properties of the motor that are compatible with the single-molecule study. The modeling approach may therefore be of wide use for predicting the properties of myosin mutations.
Karatzaferi, C., Adamek, N. and Geeves, M. (2017). Modulators of actin-myosin dissociation: basis for muscle type functional differences during fatigue. American Journal of Physiology - Cell Physiology [Online] 313:C644-C654. Available at: http://dx.doi.org/10.1152/ajpcell.00023.2017.The muscle types present with variable fatigue tolerance, in part due to the myosin isoform expressed. However, the critical steps that define 'fatigability' in vivo of fast vs slow myosin isoforms, at the molecular level, are not yet fully understood. We examined the modulation of the ATP-induced myosin sub-fragment 1 (S1) dissociation from pyrene-actin by inorganic phosphate (Pi), pH and temperature using a specially modified stopped-flow system that allowed fast kinetics measurements at physiological temperature. We contrasted the properties of rabbit psoas (fast) and bovine masseter (slow) myosins (obtained from samples collected from New Zealand rabbits and from a licensed abattoir, respectively, according to institutional and national ethics permits). To identify ATP cycling biochemical intermediates, we assessed ATP binding to a pre-equilibrated mixture of actomyosin and variable [ADP], pH (pH 7 vs pH 6.2) and Pi (zero, 15 or 30 added mM Pi) in a range of temperatures (5 to 45°C). Temperature and pH variations had little, if any, effect on the ADP dissociation constant (KADP) for fast S1 but for slow S1 KADP was weakened with increasing temperature or low pH. In the absence of ADP, the dissociation constant for phosphate (KPi) was weakened with increasing temperature for fast S1. In the presence of ADP, myosin type differences were revealed at the apparent phosphate affinity, depending on pH and temperature. Overall, the newly revealed kinetic differences between myosin types could help explain the in vivo observed muscle type functional differences at rest and during fatigue.
Brooker, H., Geeves, M. and Mulvihill, D. (2016). Analysis of biophysical and functional consequences of Tropomyosin - fluorescent protein fusions. FEBS letters [Online]:3111-3121. Available at: http://onlinelibrary.wiley.com/doi/10.1002/1873-3468.12346/full.The dynamic nature of actin polymers is modulated to facilitate a diverse
range of cellular processes. These dynamic properties are modulated by
different isoforms of Tropomyosin, which are recruited to distinct subpopulations
of actin polymers to differentially modulate their functional
properties. This makes them an attractive target for labelling discrete actin
populations. We have assessed the effect of different fluorescent labelling
strategies for this protein. Although tropomyosin fluorescent fusions
decorate actin in vivo, they are either non-functional or perturb regulation
of actin nucleation and cell cycle timings. Thus conclusions and
physiological relevance should be carefully evaluated when using
Geeves, M. (2016). The ATPase mechanism of myosin and actomyosin. Biopolymers [Online] 105:483-491. Available at: http://doi.org/10.1002/bip.22853.Myosins are a large family of molecular motors that use the common P-loop, Switch 1 and Switch 2 nucleotide binding motifs to recognize ATP, to create a catalytic site than can efficiently hydrolyze ATP and to communicate the state of the nucleotide pocket to other allosteric binding sites on myosin. The energy of ATP hydrolysis is used to do work against an external load. In this short review I will outline current thinking on the mechanism of ATP hydrolysis and how the energy of ATP hydrolysis is coupled to a series of protein conformational changes that allow a myosin, with the cytoskeleton track actin, to operate as a molecular motor of distinct types; fast movers, processive motors or strain sensors. This article is protected by copyright. All rights reserved.
Walklate, J. et al. (2016). The Most Prevalent Freeman-Sheldon Syndrome Mutations in the Embryonic Myosin Motor Share Functional Defects. Journal of Biological Chemistry [Online] 291:10318-10331. Available at: http://doi.org/10.1074/jbc.M115.707489.The embryonic myosin isoform is expressed during fetal development and rapidly down-regulated after birth. Freeman-Sheldon syndrome (FSS) is a disease associated with missense mutations in the motor domain of this myosin. It is the most severe form of distal arthrogryposis, leading to overcontraction of the hands, feet, and orofacial muscles and other joints of the body. Availability of human embryonic muscle tissue has been a limiting factor in investigating the properties of this isoform and its mutations. Using a recombinant expression system, we have studied homogeneous samples of human motors for the WT and three of the most common FSS mutants: R672H, R672C, and T178I. Our data suggest that the WT embryonic myosin motor is similar in contractile speed to the slow type I/? cardiac based on the rate constant for ADP release and ADP affinity for actin-myosin. All three FSS mutations show dramatic changes in kinetic properties, most notably the slowing of the apparent ATP hydrolysis step (reduced 5–9-fold), leading to a longer lived detached state and a slowed Vmax of the ATPase (2–35-fold), indicating a slower cycling time. These mutations therefore seriously disrupt myosin function.
Walklate, J., Ujfalusi, Z. and Geeves, M. (2016). Myosin isoforms and the mechanochemical cross-bridge cycle. Journal of Experimental Biology [Online] 219:168-174. Available at: http://doi.org/10.1242/jeb.124594.At the latest count the myosin family includes 35 distinct groups, all of which have the conserved myosin motor domain attached to a neck or lever arm, followed by a highly variable tail or cargo binding region. The motor domain has an ATPase activity that is activated by the presence of actin. One feature of the myosin ATPase cycle is that it involves an association/dissociation with actin for each ATP hydrolysed. The cycle has been described in detail for a large number of myosins from different classes. In each case the cycle is similar, but the balance between the different molecular events in the cycle has been altered to produce a range of very different mechanical activities. Myosin may spend most of the ATPase cycle attached to actin (high duty ratio), as in the processive myosin (e.g. myosin V) or the strain-sensing myosins (e.g. myosin 1c). In contrast, most muscle myosins spend 80% of their ATPase cycle detached from actin. Within the myosin IIs found in human muscle, there are 11 different sarcomeric myosin isoforms, two smooth muscle isoforms as well as three non-muscle isoforms. We have been exploring how the different myosin isoforms have adapted the cross-bridge cycle to generate different types of mechanical activity and how this goes wrong in inherited myopathies. The ideas are outlined here.
Mijailovich, S. et al. (2016). Three-dimensional stochastic model of actin–myosin binding in the sarcomere lattice. The Journal of General Physiology [Online] 148:459-488. Available at: http://doi.org/10.1085/jgp.201611608.
Nag, S. et al. (2015). Contractility parameters of human -cardiac myosin with the hypertrophic cardiomyopathy mutation R403Q show loss of motor function. Science Advances [Online] 1:e1500511-e1500511. Available at: http://doi.org/10.1126/sciadv.1500511.Hypertrophic cardiomyopathy (HCM) is the most frequently occurring inherited cardiovascular disease. It is caused by mutations in genes encoding the force-generating machinery of the cardiac sarcomere, including human ?-cardiac myosin. We present a detailed characterization of the most debated HCM-causing mutation in human ?-cardiac myosin, R403Q. Despite numerous studies, most performed with nonhuman or noncardiac myosin, there is no consensus about the mechanism of action of this mutation on the function of the enzyme. We use recombinant human ?-cardiac myosin and new methodologies to characterize in vitro contractility parameters of the R403Q myosin compared to wild type. We extend our studies beyond pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin. We find that, with pure actin, the intrinsic force generated by R403Q is ~15% lower than that generated by wild type. The unloaded velocity is, however, ~10% higher for R403Q myosin, resulting in a load-dependent velocity curve that has the characteristics of lower contractility at higher external loads compared to wild type. With regulated actin filaments, there is no increase in the unloaded velocity and the contractility of the R403Q myosin is lower than that of wild type at all loads. Unlike that with pure actin, the actin-activated adenosine triphosphatase activity for R403Q myosin with Ca2+-regulated actin filaments is ~30% lower than that for wild type, predicting a lower unloaded duty ratio of the motor. Overall, the contractility parameters studied fit with a loss of human ?-cardiac myosin contractility as a result of the R403Q mutation.
Bloemink, M. et al. (2015). The Relay/Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II. Journal of Biological Chemistry [Online] 291:1763-1773. Available at: http://doi.org/10.1074/jbc.M115.688002.The interface between relay and converter domain of muscle myosin is critical for optimal myosin performance. Using Drosophila melanogaster indirect flight muscle S1, we performed a kinetic analysis of the effect of mutations in the converter and relay domain. Introduction of a mutation (R759E) in the converter domain inhibits the steady-state ATPase of myosin S1, whereas an additional mutation in the relay domain (N509K) is able to restore the ATPase toward wild-type values. The R759E S1 construct showed little effect on most steps of the actomyosin ATPase cycle. The exception was a 25–30% reduction in the rate constant of the hydrolysis step, the step coupled to the cross-bridge recovery stroke that involves a change in conformation at the relay/converter domain interface. Significantly, the double mutant restored the hydrolysis step to values similar to the wild-type myosin. Modeling the relay/converter interface suggests a possible interaction between converter residue 759 and relay residue 509 in the actin-detached conformation, which is lost in R759E but is restored in N509K/R759E. This detailed kinetic analysis of Drosophila myosin carrying the R759E mutation shows that the interface between the relay loop and converter domain is important for fine-tuning myosin kinetics, in particular ATP binding and hydrolysis.
Geeves, M., Hitchcock-DeGregori, S. and Gunning, P. (2015). A systematic nomenclature for mammalian tropomyosin isoforms. Journal of Muscle Research and Cell Motility [Online] 36:147-153. Available at: http://doi.org/10.1007/s10974-014-9389-6.Tropomyosin, a ubiquitous protein in animals and fungi, is associated with the actin cytoskeleton and is involved with stabilising actin filaments and regulating the interaction of the filament with other actin binding proteins. The protein is best known for its role in regulating the interaction between actin and myosin in muscle contraction but in recent years its role as a major player in the organisation and dynamics of the cytoskeleton has been increasingly recognised. In mammals Tpm is expressed from four distinct genes and alternate splicing of each gene can produce a total of up to 40 different mRNA variants most of which are expressed as proteins. We are expecting a renaissance in the study of tropomyosins as the roles of these different isoforms are beginning to be deciphered. However, it is our belief that such a renaissance is being limited by confusion over the naming systems for the tropomyosin isoforms. These result in even experienced workers struggling to reconcile work done in different laboratories and at different times. We propose here a systematic nomenclature for tropomyosin based on the best current practice. We recommend the adoption of these names and a cross-reference to the table of alternate names and accession numbers for protein sequences is included here. The National Center for Biotechnology Information (NCBI) website has been amended to include the nomenclature for the human, mouse and rat genes.
Desai, R., Geeves, M. and Kad, N. (2015). Using Fluorescent Myosin to Directly Visualize Cooperative Activation of Thin Filaments. Journal of Biological Chemistry [Online]:jbc.M114.609743-jbc.M114.609743. Available at: http://dx.doi.org/10.1074/jbc.M114.609743.Contraction of striated muscle is tightly regulated by the release and sequestration of calcium within myocytes. At the molecular level, calcium modulates myosin’s access to the thin filament. Once bound, myosin is hypothesized to potentiate the binding of further myosins. Here we directly image single molecules of myosin binding to and activating thin filaments. Using this approach the cooperative binding of myosin along thin filaments has been quantified. We have found that two myosin heads are required to laterally activate a regulatory unit of thin filament. The regulatory unit is found to be capable of accommodating 11 further myosins. Three thin filament activation states possessing differential myosin binding capacities are also visible. To describe this system we have formulated a simple chemical kinetic model of cooperative activation that holds across a wide range of solution conditions. The stochastic nature of activation is strongly highlighted by data obtained in sub-optimal activation conditions where the generation of activation waves and their catastrophic collapse can be observed. This suggests that the thin filament has the potential to be turned fully on or off on a binary fashion.
Lehman, W. et al. (2015). Phosphorylation of Ser283 enhances the stiffness of the tropomyosin head-to-tail overlap domain. Archives of Biochemistry and Biophysics [Online] 571:10-15. Available at: http://doi.org/10.1016/j.abb.2015.02.026.The ends of coiled-coil tropomyosin molecules are joined together by nine to ten residue-long head-to-tail “overlapping domains”. These short four-chained interconnections ensure formation of continuous tropomyosin cables that wrap around actin filaments. Molecular Dynamics simulations indicate that the curvature and bending flexibility at the overlap is 10–20% greater than over the rest of the molecule, which might affect head-to-tail filament assembly on F-actin. Since the penultimate residue of striated muscle tropomyosin, Ser283, is a natural target of phosphorylating enzymes, we have assessed here if phosphorylation adjusts the mechanical properties of the tropomyosin overlap domain. MD simulations show that phosphorylation straightens the overlap to match the curvature of the remainder of tropomyosin while stiffening it to equal or exceed the rigidity of canonical coiled-coil regions. Corresponding EM data on phosphomimetic tropomyosin S283D corroborate these findings. The phosphorylation-induced change in mechanical properties of tropomyosin likely results from electrostatic interactions between C-terminal phosphoSer283 and N-terminal Lys12 in the four-chain overlap bundle, while promoting stronger interactions among surrounding residues and thus facilitating tropomyosin cable assembly. The stiffening effect of D283-tropomyosin noted correlates with previously observed enhanced actin–tropomyosin activation of myosin S1-ATPase, suggesting a role for the tropomyosin phosphorylation in potentiating muscle contraction.
Walklate, J. and Geeves, M. (2015). Temperature manifold for a stopped-flow machine to allow measurements from ?10 to +40°C. Analytical Biochemistry [Online] 476:11-16. Available at: http://doi.org/10.1016/j.ab.2015.01.020.Conducting enzymatic stopped-flow experiments at temperatures far removed from ambient can be very problematic because extremes in temperature (<10 °C or >30 °C) can damage the machine or the enzyme. We have devised a simple manifold that can be attached to most commercial stopped-flow systems that is independently heated or cooled separate from the main stopped-flow system. Careful calibration of the flow circuit allows the sample to be heated or cooled to the measurement temperature (?8 to +40 °C) 1 to 2 s before mixing in the reaction chamber. This approach allows measurements at temperatures where the stopped flow or the protein is normally unstable. To validate the manifold, we investigated the well-defined ATP-induced dissociation of rabbit muscle myosin subfragment 1 (S1) from its complex with pyrene-labeled actin. This process has both temperature-dependent and -independent components. Use of ethylene glycol allowed us to measure the reaction below 0 °C and up to 42 °C, and as expected the second-order rate constant (K1k+2) and the maximum rate of dissociation (k+2) both increased with temperature, whereas 1/K1 is unaffected by the change in temperature.
Geeves, M. and Lehrer, S. (2014). Cross-Talk, Cross-Bridges, and Calcium Activation of Cardiac Contraction. Biophysical Journal [Online] 107:543-545. Available at: http://dx.doi.org/10.1016/j.bpj.2014.06.019.
Lehrer, S. and Geeves, M. (2014). The myosin-activated thin filament regulatory state, M ? -open: a link to hypertrophic cardiomyopathy (HCM). Journal of Muscle Research and Cell Motility [Online] 35:153-160. Available at: http://doi.org/10.1007/s10974-014-9383-z.
Bloemink, M. et al. (2014). The Hypertrophic Cardiomyopathy Myosin Mutation R453C Alters ATP Binding and Hydrolysis of Human Cardiac beta-Myosin. Journal of Biological Chemistry [Online] 289:5158-5167. Available at: http://dx.doi.org/10.1074/jbc.M113.511204.The human hypertrophic cardiomyopathy mutation R453C results in one of the more severe forms of the myopathy. Arg-453 is found in a conserved surface loop of the upper 50-kDa domain of the myosin motor domain and lies between the nucleotide binding pocket and the actin binding site. It connects to the cardiomyopathy loop via a long ?-helix, helix O, and to Switch-2 via the fifth strand of the central ?-sheet. The mutation is, therefore, in a position to perturb a wide range of myosin molecular activities. We report here the first detailed biochemical kinetic analysis of the motor domain of the human ?-cardiac myosin carrying the R453C mutation. A recent report of the same mutation (Sommese, R. F., Sung, J., Nag, S., Sutton, S., Deacon, J. C., Choe, E., Leinwand, L. A., Ruppel, K., and Spudich, J. A. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 12607–12612) found reduced ATPase and in vitro motility but increased force production using an optical trap. Surprisingly, our results show that the mutation alters few biochemical kinetic parameters significantly. The exceptions are the rate constants for ATP binding to the motor domain (reduced by 35%) and the ATP hydrolysis step/recovery stroke (slowed 3-fold), which could be the rate-limiting step for the ATPase cycle. Effects of the mutation on the recovery stroke are consistent with a perturbation of Switch-2 closure, which is required for the recovery stroke and the subsequent ATP hydrolysis.
Lawrence, A. et al. (2014). FAD binding, cobinamide binding and active site communication in the corrin reductase (CobR). Bioscience Reports [Online] 34:345-355. Available at: http://dx.doi.org/10.1042/BSR20140060.Adenosylcobalamin, the coenzyme form of vitamin B12, is one Nature's most complex coenzyme whose de novo biogenesis proceeds along either an anaerobic or aerobic metabolic pathway. The aerobic synthesis involves reduction of the centrally chelated cobalt metal ion of the corrin ring from Co(II) to Co(I) before adenosylation can take place. A corrin reductase (CobR) enzyme has been identified as the likely agent to catalyse this reduction of the metal ion. Herein, we reveal how Brucella melitensis CobR binds its coenzyme FAD (flavin dinucleotide) and we also show that the enzyme can bind a corrin substrate consistent with its role in reduction of the cobalt of the corrin ring. Stopped-flow kinetics and EPR reveal a mechanistic asymmetry in CobR dimer that provides a potential link between the two electron reduction by NADH to the single electron reduction of Co(II) to Co(I).
Bloemink, M. et al. (2013). The Superfast Human Extraocular Myosin Is Kinetically Distinct from the Fast Skeletal IIa, IIb, and IId Isoforms. Journal of Biological Chemistry [Online] 288:27469-27479. Available at: http://dx.doi.org/10.1074/jbc.M113.488130.
Janco, M. et al. (2013). Polymorphism in tropomyosin structure and function. Journal of Muscle Research and Cell Motility [Online] 34:177-187. Available at: http://dx.doi.org/10.1007/s10974-013-9353-x.Tropomyosins (Tm) in humans are expressed from four distinct genes and by alternate splicing >40 different Tm polypeptide chains can be made. The functional Tm unit is a dimer of two parallel polypeptide chains and these can be assembled from identical (homodimer) or different (heterodimer) polypeptide chains provided both chains are of the same length. Since most cells express multiple isoforms of Tm, the number of different homo and heterodimers that can be assembled becomes very large. We review the mechanism of dimer assembly and how preferential assembly of some heterodimers is driven by thermodynamic stability. We examine how in vitro studies can reveal functional differences between Tm homo and heterodimers (stability, actin affinity, flexibility) and the implication for how there could be selection of Tm isomers in the assembly on to an actin filament. The role of Tm heterodimers becomes more complex when mutations in Tm are considered, such as those associated with cardiomyopathies, since mutations can appear in only one of the chains.
Geeves, M. and Ranatunga, K. (2012). Tuning the Calcium Sensitivity of Cardiac Muscle. Biophysical Journal [Online] 103:849-850. Available at: http://dx.doi.org/10.1016/j.bpj.2012.07.039.
Deery, E. et al. (2012). An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis. Nature Chemical Biology [Online] 8:933-940. Available at: http://dx.doi.org/10.1038/nchembio.1086.The biosynthesis of many vitamins and coenzymes has often proven difficult to elucidate owing to a combination of low abundance and kinetic lability of the pathway intermediates. Through a serial reconstruction of the cobalamin (vitamin B 12) pathway in Escherichia coli and by His tagging the terminal enzyme in the reaction sequence, we have observed that many unstable intermediates can be isolated as tightly bound enzyme-product complexes. Together, these approaches have been used to extract intermediates between precorrin-4 and hydrogenobyrinic acid in their free acid form and permitted the delineation of the overall reaction catalyzed by CobL, including the formal elucidation of precorrin-7 as a metabolite. Furthermore, a substrate-carrier protein, CobE, that can also be used to stabilize some of the transient metabolic intermediates and enhance their onward transformation, has been identified. The tight association of pathway intermediates with enzymes provides evidence for a form of metabolite channeling.
Deacon, J. et al. (2012). Identification of functional differences between recombinant human ? and ? cardiac myosin motors. Cellular and Molecular Life Sciences [Online] 69:2261-2277. Available at: http://dx.doi.org/10.1007/s00018-012-0927-3.The myosin isoform composition of the heart is
dynamic in health and disease and has been shown to affect
contractile velocity and force generation. While different
mammalian species express different proportions of a and
b myosin heavy chain, healthy human heart ventricles
express these isoforms in a ratio of about 1:9 (a:b) while
failing human ventricles express no detectable a-myosin.
We report here fast-kinetic analysis of recombinant human
a and b myosin heavy chain motor domains. This represents the ?rst such analysis of any human muscle myosin
motor and the ?rst of a-myosin from any species. Our
?ndings reveal substantial isoform differences in individual
kinetic parameters, overall contractile character, and predicted cycle times. For these parameters, a-subfragment 1
(S1) is far more similar to adult fast skeletal muscle myosin
isoforms than to the slow b isoform despite 91% sequence
identity between the motor domains of a- and b-myosin.
Among the features that differentiate a- from b-S1: the
ATP hydrolysis step of a-S1 is *ten-fold faster than b-S1,
a-S1 exhibits *?ve-fold weaker actin af?nity than b-S1,
and actin a-S1 exhibits rapid ADP release, which is > tenfold faster than ADP release for b-S1. Overall, the cycle
times are ten-fold faster for a-S1 but the portion of time
each myosin spends tightly bound to actin (the duty ratio)
is similar. Sequence analysis points to regions that might
underlie the basis for this ?nding.
Janco, M. et al. (2012). ?-Tropomyosin with a D175N or E180G Mutation in Only One Chain Differs from Tropomyosin with Mutations in Both Chains. Biochemistry [Online] 51:9880-9890. Available at: http://dx.doi.org/10.1021/bi301323n.?-Tropomyosin (Tm) carrying hypertrophic cardiomyopathy mutation D175N or E180G was expressed in Escherichia coli. We have assembled dimers of two polypeptide chains in vitro that carry one (??*) or two (?*?*) copies of the mutation. We found that the presence of the mutation has little effect on dimer assembly, thereby predicting that individuals heterozygous for the Tm mutations are likely to express both ??* and ?*?* Tm. Depending on the expression level, the heterodimer may be the predominant form in individuals carrying the mutation. Thus, it is important to define differences in the properties of Tm molecules carrying one or two copies of the mutation. We examined the Tm homo- and heterodimer properties: actin affinity, thermal stability, calcium regulation of myosin subfragment 1 binding, and calcium regulation of myofibril force. We report that the properties of the heterodimer may be similar to those of the wild-type homodimer (actin affinity, thermal stability, D175N ??*), similar to those of the mutant homodimer (calcium sensitivity, D175N ??*), intermediate between the two (actin affinity, E180G ??*), or different from both (thermal stability, E180G ??*). Thus, the properties of the homodimer are not a completely reliable guide to the properties of the heterodimer.
Kalyva, A., Schmidtmann, A. and Geeves, M. (2012). In Vitro Formation and Characterization of the Skeletal Muscle ?·? Tropomyosin Heterodimers. Biochemistry [Online] 51:6388-6399. Available at: http://dx.doi.org/10.1021/bi300340r.Tropomyosin (Tm) is a dimer made of two alpha helical chains associated into a parallel coiled-coil. In mammalian skeletal and cardiac muscle, the Tm is expressed from two separate genes to give the ?- and ?-Tm isoforms. These associate in vivo to form homo- (?2) and heterodimers (?·?) with little ?2 normally observed. The proportion of ?2 vs ?·? varies across species and across muscle types from almost 100% ?2- to 50% ?·?-Tm. The ratio can also vary during development and in disease. The functional significance of the presence of these two isoforms has not been defined because it is difficult to isolate or purify the ?·? dimer for functional studies. Here we report an effective method for purifying bacterially expressed Tm as ?·? dimers using a cleavable N-terminal tag on one of the two chains. The same method can be used to isolate Tm dimers in which one chain carries a mutation. We go on to show that the ?·? dimers differ in key properties (actin affinity, thermal stability) from either the ?2- or ?2-Tm. However, the ability to regulate myosin binding when combined with cardiac troponin appears unaffected.
Mijailovich, S. et al. (2012). Cooperative regulation of myosin-S1 binding to actin filaments by a continuous flexible Tm–Tn chain. European Biophysics Journal [Online] 41:1015-1032. Available at: http://dx.doi.org/10.1007/s00249-012-0859-8.The regulation of striated muscle contraction involves cooperative interactions between actin filaments, myosin-S1 (S1), tropomyosin (Tm), troponin (Tn), and calcium. These interactions are modeled by treating overlapping tropomyosins as a continuous flexible chain (CFC), weakly confined by electrostatic interactions with actin. The CFC is displaced locally in opposite directions on the actin surface by the binding of either S1 or Troponin I (TnI) to actin. The apparent rate constants for myosin and TnI binding to and detachment from actin are then intrinsically coupled via the CFC model to the presence of neighboring bound S1s and TnIs. Monte Carlo simulations at prescribed values of the CFC stiffness, the CFC’s degree of azimuthal confinement, and the angular displacements caused by the bound proteins were able to predict the stopped-flow transients of S1 binding to regulated F-actin. The transients collected over a large range of calcium concentrations could be well described by adjusting a single calcium-dependent parameter, the rate constant of TnI detachment from actin, k ?I. The resulting equilibrium constant KB?1/KI varied sigmoidally with the free calcium, increasing from 0.12 at low calcium (pCa >7) to 12 at high calcium (pCa <5.5) with a Hill coefficient of ~2.15. The similarity of the curves for excess-actin and excess-myosin data confirms their allosteric relationship. The spatially explicit calculations confirmed variable sizes for the cooperative units and clustering of bound myosins at low calcium concentrations. Moreover, inclusion of negative cooperativity between myosin units predicted the observed slowing of myosin binding at excess-myosin concentrations.
Mijailovich, S. et al. (2012). The Hill Model for Binding Myosin S1 to Regulated Actin Is not Equivalent to the McKillop–Geeves Model. Journal of Molecular Biology [Online] 417:112-128. Available at: http://dx.doi.org/10.1016/j.jmb.2012.01.011.The Hill two-state cooperativity model and the McKillop–Geeves (McK–G) three-state model predict very similar binding traces of myosin subfragment 1 (S1) binding to regulated actin filaments in the presence and absence of calcium, and both fit the experimental data reasonably well [Chen et al., Biophys. J., 80, 2338–2349]. Here, we compared the Hill model and the McK–G model for binding myosin S1 to regulated actin against three sets of experimental data: the titration of regulated actin with S1 and the kinetics of S1 binding of regulated actin with either excess S1 to actin or excess actin to S1. Each data set was collected for a wide range of specified calcium concentrations. Both models were able to generate reasonable fits to the time course data and to titration data. The McK–G model can fit all three data sets with the same calcium-concentration-sensitive parameters. Only KB and KT show significant calcium dependence, and the parameters have a classic pCa curve. A unique set of the Hill model parameters was extremely difficult to estimate from the best fits of multiple sets of data. In summary, the McK–G cooperativity model more uniquely resolves parameters estimated from kinetic and titration data than the Hill model, predicts a sigmoidal dependence of key parameters with calcium concentration, and is simpler and more suitable for practical use.
Canepari, M. et al. (2012). Actomyosin kinetics of pure fast and slow rat myosin isoforms studied by in vitro motility assay approach. Experimental Physiology [Online] 97:873-881. Available at: http://dx.doi.org/10.1113/expphysiol.2012.064576.An in vitro motility assay approach was used to investigate the mechanisms of the functional differences between myosin isoforms, by studying the effect of MgATP and MgADP on actin sliding velocity (Vf) of pure slow and fast rat skeletal myosin at different temperatures. The value of Vf depended on [MgATP] according to Michaelis–Menten kinetics, with an apparent constant (Km) of 54.2, 64.4 and 200 ?M for the fast isoform and 18.6, 36.5 and 45.5 ?M for the slow isoform at 20, 25 and 35°C, respectively. The presence of 2 mM MgADP decreased Vf and yielded an inhibition constant (Ki) of 377, 463 and 533 ?M for the fast isoform at 20, 25 and 35°C, respectively, and 120 and 355 ?M for the slow isoform at 25 and 35°C, respectively. The analysis of Km and Ki suggested that slow and fast isoforms differ in the kinetics limiting Vf. Moreover, the higher sensitivity of the fast myosin isoform to a drop in [MgATP] is consistent with the higher fatigability of fast fibres than slow fibres. From the Michaelis–Menten relation in the absence of MgADP, we calculated the rate of actomyosin dissociation by MgATP (k+ATP) and the rate of MgADP release (k-ADP). We found values of k+ATP of 4.8 × 106, 6.5 × 106 and 6.6 × 106 M?1 s?1 for the fast isoform and 3.3 × 106, 2.9 × 106 and 6.7 × 106 M?1 s?1 for the slow isoform and values of k-ADP of 263, 420 and 1320 s?1 for the fast isoform and 62, 107 and 306 s?1 for the slow isoform at 20, 25 and 35°C, respectively. The results suggest that k-ADP could be the major determinant of functional differences between the fast and slow myosin isoforms at physiological temperatures.
Deacon, J. et al. (2012). Erratum to: Identification of functional differences between recombinant human ? and ? cardiac myosin motors. Cellular and Molecular Life Sciences [Online] 69:4239-4255. Available at: http://dx.doi.org/10.1007/s00018-012-1111-5.The myosin isoform composition of the heart is dynamic in health and disease and has been shown to affect contractile velocity and force generation. While different mammalian species express different proportions of ? and ? myosin heavy chain, healthy human heart ventricles express these isoforms in a ratio of about 1:9 (?:?) while failing human ventricles express no detectable ?-myosin. We report here fast-kinetic analysis of recombinant human ? and ? myosin heavy chain motor domains. This represents the first such analysis of any human muscle myosin motor and the first of ?-myosin from any species. Our findings reveal substantial isoform differences in individual kinetic parameters, overall contractile character, and predicted cycle times. For these parameters, ?-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow ? isoform despite 91% sequence identity between the motor domains of ?- and ?-myosin. Among the features that differentiate ?- from ?-S1: the ATP hydrolysis step of ?-S1 is ~ten-fold faster than ?-S1, ?-S1 exhibits ~five-fold weaker actin affinity than ?-S1, and actin·?-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for ?-S1. Overall, the cycle times are ten-fold faster for ?-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar. Sequence analysis points to regions that might underlie the basis for this finding.
Li, X. et al. (2012). The flexibility of two tropomyosin mutants, D175N and E180G, that cause hypertrophic cardiomyopathy. Biochemical and Biophysical Research Communications [Online] 424:493-496. Available at: http://dx.doi.org/10.1016/j.bbrc.2012.06.141.
Geeves, M. (2012). 4.13 Thin Filament Regulation. Comprehensive Biophysics [Online] 4:251-267. Available at: http://dx.doi.org/10.1016/B978-0-12-374920-8.00416-1.The review summarizes the current state of knowledge of the calcium regulation of striated muscle contraction via the thin filament proteins, tropomyosin and troponin. The description focuses on in vitro studies of the thin filament and covers structural, biochemical and dynamic aspects of the thin filament's response to calcium binding. A reductionist approach has allowed many of the transitions to be defined at the level of a single structural unit. Here an emphasis is placed on the co-operative nature of the structural and biochemical transitions of the thin filament and the allosteric relationship between calcium and myosin binding to the thin filament.
Preller, M. et al. (2011). Structural Basis for the Allosteric Interference of Myosin Function by Reactive Thiol Region Mutations G680A and G680V. Journal of Biological Chemistry [Online] 286:35051-35060. Available at: http://dx.doi.org/10.1074/jbc.M111.265298.The cold-sensitive single-residue mutation of glycine 680 in the reactive thiol region of Dictyostelium discoideum myosin-2 or the corresponding conserved glycine in other myosin isoforms has been reported to interfere with motor function. Here we present the x-ray structures of myosin motor domain mutants G680A in the absence and presence of nucleotide as well as the apo structure of mutant G680V. Our results show that the Gly-680 mutations lead to uncoupling of the reactive thiol region from the surrounding structural elements. Structural and functional data indicate that the mutations induce the preferential population of a state that resembles the ADP-bound state. Moreover, the Gly-680 mutants display greatly reduced dynamic properties, which appear to be related to the recovery of myosin motor function at elevated temperatures.
Adamek, N., Geeves, M. and Coluccio, L. (2011). Myo1c mutations associated with hearing loss cause defects in the interaction with nucleotide and actin. Cellular and Molecular Life Sciences [Online] 68:139-150. Available at: http://dx.doi.org/10.1007/s00018-010-0448-x.Three heterozygous missense mutations in the motor domain of myosin 1c (Myo1c), which mediates adaptation in the inner ear, are associated with bilateral sensorineural hearing loss in humans. With transient kinetic analyses, steady-state ATPase and motility assays, and homology modeling, we studied the interaction of these mutants with nucleotide and actin using a truncated construct, Myo1c(1IQ-SAH), which includes an artificial lever arm. Results indicate that mutation R156W, near switch 1, affects the nucleotide-binding pocket and the calcium binding by disrupting switch 1 movement. Mutation V252A, in the K helix of the upper 50 kDa domain, showed reduced actin affinity consistent with disruption of communication between the actin- and nucleotide-binding sites. T380M, in a Myo1c-specific insert in the HO linker, displayed aberrant changes in most kinetic parameters and uncoupling of the ATPase from motility. These data allow for an interpretation of how these mutations might affect adaptation.
Korte, F. et al. (2011). Upregulation of cardiomyocyte ribonucleotide reductase increases intracellular 2 deoxy-ATP, contractility, and relaxation. Journal of Molecular and Cellular Cardiology [Online] 51:894-901. Available at: http://dx.doi.org/10.1016/j.yjmcc.2011.08.026.We have previously demonstrated that substitution of ATP with 2 deoxy-ATP (dATP) increased the magnitude and rate of force production at all levels of Ca2+-mediated activation in demembranated cardiac muscle. In the current study we hypothesized that cellular [dATP] could be increased by viral-mediated overexpression of the ribonucleotide reductase (Rrm1 and Rrm2) complex, which would increase contractility of adult rat cardiomyocytes. Cell length and ratiometric (Fura2) Ca2+ fluorescence were monitored by video microscopy. At 0.5 Hz stimulation, the extent of shortening was increased ~ 40% and maximal rate of shortening was increased ~ 80% in cardiomyocytes overexpressing Rrm1 + Rrm2 as compared to non-transduced cardiomyocytes. The maximal rate of relaxation was also increased ~ 150% with Rrm1 + Rrm2 overexpression, resulting in decreased time to 50% relaxation over non-transduced cardiomyocytes. These differences were even more dramatic when compared to cardiomyocytes expressing GFP-only. Interestingly, Rrm1 + Rrm2 overexpression had no effect on minimal or maximal intracellular [Ca2+], indicating increased contractility is primarily due to increased myofilament activity without altering Ca2+ release from the sarcoplasmic reticulum. Additionally, functional potentiation was maintained with Rrm1 + Rrm2 overexpression as stimulation frequency was increased (1 Hz and 2 Hz). HPLC analysis indicated cellular [dATP] was increased by approximately 10-fold following transduction, becoming ~ 1.5% of the adenine nucleotide pool. Furthermore, 2% dATP was sufficient to significantly increase crossbridge binding and contractile force during sub-maximal Ca2+ activation in demembranated cardiac muscle. These experiments demonstrate the feasibility of directly targeting the actin–myosin chemomechanical crossbridge cycle to enhance cardiac contractility and relaxation without affecting minimal or maximal Ca2+. This article is part of a Special issue entitled "Possible Editorial".
Bloemink, M. and Geeves, M. (2011). Shaking the myosin family tree: Biochemical kinetics defines four types of myosin motor. Seminars in Cell & Developmental Biology [Online] 22:961-967. Available at: http://dx.doi.org/10.1016/j.semcdb.2011.09.015.Although all myosin motors follow the same basic cross-bridge cycle, they display a large variety in the rates of transition between different states in the cycle, allowing each myosin to be finely tuned for a specific task. Traditionally, myosins have been classified by sequence analysis into a large number of sub-families (?35). Here we use a different method to classify the myosin family members which is based on biochemical and mechanical properties. The key properties that define the type of mechanical activity of the motor are duty ratio (defined as the fraction of the time myosin remains attached to actin during each cycle), thermodynamic coupling of actin and nucleotide binding to myosin and the degree of strain-sensitivity of the ADP release step. Based on these properties we propose to classify myosins into four different groups: (I) fast movers, (II) slow/efficient force holders, (III) strain sensors and (IV) gates.
Rayes, R. et al. (2011). Dynamics of tropomyosin in muscle fibers as monitored by saturation transfer EPR of bi-functional probe. PLoS ONE [Online] 6:e21277. Available at: http://dx.doi.org/10.1371/journal.pone.0021277.The dynamics of four regions of tropomyosin was assessed using saturation transfer electron paramagnetic resonance in the muscle fiber. In order to fully immobilize the spin probe on the surface of tropomyosin, a bi-functional spin label was attached to i,i+4 positions via cysteine mutagenesis. The dynamics of bi-functionally labeled tropomyosin mutants decreased by three orders of magnitude when reconstituted into "ghost muscle fibers". The rates of motion varied along the length of tropomyosin with the C-terminus position 268/272 being one order of magnitude slower then N-terminal domain or the center of the molecule. Introduction of troponin decreases the dynamics of all four sites in the muscle fiber, but there was no significant effect upon addition of calcium or myosin subfragment-1.
Bloemink, M. et al. (2011). Two Drosophila myosin transducer mutants with distinct cardiomyopathies have divergent ADP and actin affinities. Journal of Biological Chemistry [Online] 286:28435-28443. Available at: http://dx.doi.org/10.1074/jbc.M111.258228.Two Drosophila myosin II point mutations (D45 and Mhc(5)) generate Drosophila cardiac phenotypes that are similar to dilated or restrictive human cardiomyopathies. Our homology models suggest that the mutations (A261T in D45, G200D in Mhc(5)) could stabilize (D45) or destabilize (Mhc(5)) loop 1 of myosin, a region known to influence ADP release. To gain insight into the molecular mechanism that causes the cardiomyopathic phenotypes to develop, we determined whether the kinetic properties of the mutant molecules have been altered. We used myosin subfragment 1 (S1) carrying either of the two mutations (S1(A261T) and S1(G200D)) from the indirect flight muscles of Drosophila. The kinetic data show that the two point mutations have an opposite effect on the enzymatic activity of S1. S1(A261T) is less active (reduced ATPase, higher ADP affinity for S1 and actomyosin subfragment 1 (actin · S1), and reduced ATP-induced dissociation of actin · S1), whereas S1(G200D) shows increased enzymatic activity (enhanced ATPase, reduced ADP affinity for both S1 and actin · S1). The opposite changes in the myosin properties are consistent with the induced cardiac phenotypes for S1(A261T) (dilated) and S1(G200D) (restrictive). Our results provide novel insights into the molecular mechanisms that cause different cardiomyopathy phenotypes for these mutants. In addition, we report that S1(A261T) weakens the affinity of S1 · ADP for actin, whereas S1(G200D) increases it. This may account for the suppression (A261T) or enhancement (G200D) of the skeletal muscle hypercontraction phenotype induced by the troponin I held-up(2) mutation in Drosophila.
Geeves, M. et al. (2011). Cooperative [Ca²+]-dependent regulation of the rate of myosin binding to actin: solution data and the tropomyosin chain model. Biophysical Journal [Online] 100:2679-2687. Available at: http://dx.doi.org/10.1016/j.bpj.2011.04.020.The regulation of muscle contraction by calcium involves interactions among actin filaments, myosin-S1, tropomyosin (Tm), and troponin (Tn). We have extended our previous model in which the TmTn regulatory units are treated as a continuous flexible chain, and applied it to transient kinetic data. We have measured the time course of myosin-S1 binding to actin-Tm-Tn filaments in solution at various calcium levels with [actin]/[myosin] ratios of 10 and 0.1, which exhibit modest slowing as [Ca(2+)] is reduced and a lag phase at low calcium. These observations can be explained if myosin binds to actin in two steps, where the first step is rate-limiting and blocked by TmTnI at low calcium, and the second step is fast, reversible, and controlled by the neighboring configuration of coupled tropomyosin-troponin units. The model can describe the calcium dependence of the observed myosin binding reactions and predicts cooperative calcium binding to TnC with competition between actin and Ca-TnC for the binding of TnI. Implications for theories of thin-filament regulation in muscle are discussed.
Adamek, N. and Geeves, M. (2014). Use of pyrene-labelled actin to probe actin-Myosin interactions: kinetic and equilibrium studies. in: Fluorescent Methods Applied to Molecular Motors: from single molecules to whole cells. Springer, pp. 87-104. Available at: http://link.springer.com/book/10.1007%2F978-3-0348-0856-9.
Toseland, C. and Geeves, M. (2014). Rapid reaction kinetic techniques. in: Toseland, C. P. and Fili, N. eds. Fluorescent Methods Applied to Molecular Motors: from single molecules to whole cells. Springer, pp. 49-64. Available at: http://dx.doi.org/10.1007/978-3-0348-0856-9.
Johnson, M., Geeves, M. and Mulvihill, D. (2013). Production of Amino-Terminally Acetylated Recombinant Proteins in E. coli. in: Hake, S. and Janzen, C. eds. Protein Acetylation. Humana Press, pp. 193-200. Available at: http://dx.doi.org/10.1007/978-1-62703-305-3_15.The majority of proteins in eukaryote cells are subjected to amino-terminal acetylation. This co-translational modification can affect the stability of a protein and also regulate its biological function. Amino-terminally acetylated recombinant proteins cannot be produced using prokaryote expression systems, such as E. coli, as these cells lack the appropriate N-?-terminal acetyltransferase complexes. Here we describe a simple protocol that allows the recombinant expression and purification of NatB-dependent amino-terminally acetylated proteins from E. coli.
Geeves, M. and Pearson, D. (2013). Kinetics: Relaxation Methods. in: Roberts, G. C. K. ed. Encyclopedia of Biophysics. Springer Berlin Heidelberg, pp. 1207-1212. Available at: http://dx.doi.org/10.1007/978-3-642-16712-6_62.
Baker, K. et al. (2018). TORC2 dependent phosphorylation 1 modulates calcium regulation of fission yeast myosin. EMBO Journal.All cells have the ability to respond to changes in their environment. Signalling networks modulate cytoskeleton and membrane organisation to impact cell cycle progression, polarised cell growth and multicellular development according to the environmental setting. Using diverse in vitro, in vivo and single molecule techniques we have explored the role of myosin-1 signalling in
regulating endocytosis during both mitotic and meiotic cell cycles. We have established that a conserved serine within the neck region of the sole fission yeast myosin-1 is phosphorylated in a TORC2 dependent manner to modulate myosin function. Myo1 neck phosphorylation brings about a change in the conformation of the neck region and modifies its interaction with calmodulins, Myo1 dynamics at endocytic foci, and promotes calcium dependent switching between different calmodulin light chains. These data provide insight into a novel mechanism by which myosin neck phosphorylation modulates acto44
myosin dynamics to control polarised cell growth in response to mitotic and meiotic cell-cycle progression and the cellular environment.