Filament Sliding Research Articles

Ultrastructural and kinetic evidence support that thick filaments slide through the Z-disc.

How myofilaments operate at short mammalian skeletal muscle lengths is unknown. A common assumption is that thick (myosin-containing) filaments get compressed at the Z-disc. We provide ultrastructural evidence of sarcomeres contracting down to 0.44 µm-approximately a quarter of thick filament resting length-in long-lasting contractions while apparently keeping a regular, parallel thick filament arrangement. Sarcomeres produced force at such extremely short lengths. Furthermore, sarcomeres adopted a bimodal length distribution with both modes below lengths where sarcomeres are expected to generate force in classic force-length measurements. Mammalian fibres did not restore resting length but remained short after deactivation, as previously reported for amphibian fibres, and showed increased forces during passive re-elongation. These findings are incompatible with viscoelastic thick filament compression but agree with predictions of a model incorporating thick filament sliding through the Z-disc. This more coherent picture of mechanical mammalian skeletal fibre functioning opens new perspectives on muscle physiology.

Directly Measuring Forces within Reconstituted Active Microtubule Bundles.

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as specialized arrays during mitosis to regulate chromosome segregation. Proteins that interact with microtubules include motors such as kinesins and dynein, which can generate active forces and directional motion, as well as non-motor proteins that crosslink filaments into higher-order networks or regulate filament dynamics. To date, biophysical studies of microtubule-associated proteins have overwhelmingly focused on the role of single motor proteins needed for vesicle transport, and significant progress has been made in elucidating the force-generating properties and mechanochemical regulation of kinesins and dyneins. However, for processes in which microtubules act both as cargo and track, such as during filament sliding within the mitotic spindle, much less is understood about the biophysical regulation of ensembles of the crosslinking proteins involved. Here, we detail our methodology for directly probing force generation and response within crosslinked microtubule minimal networks reconstituted from purified microtubules and mitotic proteins. Microtubule pairs are crosslinked by proteins of interest, one microtubule is immobilized to a microscope coverslip, and the second microtubule is manipulated by an optical trap. Simultaneous total internal reflection fluorescence microscopy allows for multichannel visualization of all the components of this microtubule network as the filaments slide apart to generate force. We also demonstrate how these techniques can be used to probe pushing forces exerted by kinesin-5 ensembles and how viscous braking forces arise between sliding microtubule pairs crosslinked by the mitotic MAP PRC1. These assays provide insights into the mechanisms of spindle assembly and function and can be more broadly adapted to study dense microtubule network mechanics in diverse contexts, such as the axon and dendrites of neurons and polar epithelial cells.

Molecular Motions in AIEgen Crystals: Turning on Photoluminescence by Force-Induced Filament Sliding.

Life process is amazing, and it proceeds against the eternal law of entropy increase through molecular motion and takes energy from the environment to build high-order complexity from chaos to achieve evolution with more sophisticated architectures. Inspired from the elegance of life process and also to effectively exploit the undeveloped solid-state molecular motion, two unique chiral Au(I) complexes were elaborately developed in this study, in which their powders could realize a dramatic transformation from nonemissive isolated crystallites to emissive well-defined microcrystals under the stimulation of mechanical force. Such an unusual crystallization was presumed to be caused by molecular motions driven by the formation of strong aurophilic interactions as well as multiple C-H···F and π-π interactions. Such a prominent macroscopic off/on luminescent switching could also be achieved through extremely subtle molecular motions in the crystal state and presented a filament sliding that occurred in a layer-by-layer molecular stacking fashion with no involvement of any crystal phase transition. Additionally, it had been demonstrated that the manipulation of the solid-state molecular motions could result in the generation of circularly polarized luminescence.

Force and Power of a Synthetic Myosin II-Based Machine

The function as a collective motor of skeletal muscle myosin II is studied in vitro with a synthetic bio-machine, consisting of an ensemble of myosin motors interacting with a single actin filament attached with the correct polarity to a bead trapped in the focus of a Dual Laser Optical Tweezers (DLOT, Bianco et al. Biophys. J.101:866-874, 2011). The motor ensemble provides the condition for cyclic interactions with the actin filament, allowing the development of steady force and filament sliding. The mechanical outputs of the machine are measured by means of the DLOT, which acts as a force transducer (range 0.5-200 pN and resolution ∼0.3 pN), and a piezoelectric nano-positioner carrying the support for the myosin motors, which acts as a length transducer (range 1-75.000 nm and resolution ∼1 nm). Here is reported the performance of a first version of the machine, consisting of an ensemble of myosin motors purified from frog skeletal muscle randomly adsorbed on the surface of a chemically etched single-mode optical fibre with diameter 4 µm. Isometric and isotonic contractions are reproduced by the motor ensemble in solution with physiological [ATP] (2 mM) and temperature 21 °C. Following a drop in force from the maximum isometric value (F0) to a lower value (F), the actin filament slides at a constant velocity (V) which is larger the smaller the force, as expected from the in vivo force-velocity relation. Up to five F-V points for each interaction can be determined, allowing the definition of the maximum power at F ∼0.3 F0 (V ∼2 µm/s) and demonstrating the unequalled ability of the synthetic machine to define the power of native and engineered myosin II motors from striated muscle. Supported by IIT-SEED, Genova and ECRF, 2015 (Italy).

The Kinetics Underlying the Velocity of Smooth Muscle Myosin Filament Sliding on Actin Filaments in vitro

The Kinetics Underlying the Velocity of Smooth Muscle Myosin Filament Sliding on Actin Filaments in vitro

The Kinetics Underlying the Velocity of Smooth Muscle Myosin Filament Sliding on Actin Filaments in Vitro

Actin-myosin interactions are well studied using soluble myosin fragments, but little is known about effects of myosin filament structure on mechanochemistry. We stabilized unphosphorylated smooth muscle myosin (SMM) and phosphorylated smooth muscle myosin (pSMM) filaments against ATP-induced depolymerization using a cross-linker and attached fluorescent rhodamine (XL-Rh-SMM). Electron micrographs showed that these side polar filaments are very similar to unmodified filaments. They are ~0.63 μm long and contain ~176 molecules. Rate constants for ATP-induced dissociation and ADP release from acto-myosin for filaments and S1 heads were similar. Actin-activated ATPases of SMM and XL-Rh-SMM were similarly regulated. XL-Rh-pSMM filaments moved processively on F-actin that was bound to a PEG brush surface. ATP dependence of filament velocities was similar to that for solution ATPases at high [actin], suggesting that both processes are limited by the same kinetic step (weak to strong transition) and therefore are attachment- limited. This differs from actin sliding over myosin monomers, which is primarily detachment-limited. Fitting filament data to an attachment-limited model showed that approximately half of the heads are available to move the filament, consistent with a side polar structure. We suggest the low stiffness subfragment 2 (S2) domain remains unhindered during filament motion in our assay. Actin-bound negatively displaced heads will impart minimal drag force because of S2 buckling. Given the ADP release rate, the velocity, and the length of S2, these heads will detach from actin before slack is taken up into a backwardly displaced high stiffness position. This mechanism explains the lack of detachment- limited kinetics at physiological [ATP]. These findings address how nonlinear elasticity in assemblies of motors leads to efficient collective force generation.

Ca2+-Regulatory Function of the Inhibitory Peptide Region of Cardiac Troponin I is Aided by the C-Terminus of Cardiac Troponin T: Effects of FHC Mutations Ctni R145G and Ctnt R278C, Alone and in Combination, on Filament Sliding

Investigations of cardiomyopathy mutations in Ca2+ regulatory proteins troponin and tropomyosin provide crucial information about cardiac disease mechanisms, and provide insights into functional domains in the affected polypeptides. Hypertrophic cardiomyopathy-associated mutations TnI R145G, located within the inhibitory peptide (Ip) of human cardiac troponin I (hcTnI), and TnT R278C, located immediately C-terminal to the IT arm in human cardiac troponin T (hcTnT), share remarkable features: structurally, biochemically, and pathologically. Using bioinformatics, we find compelling evidence that affected regions of hcTnI and hcTnT, may be related_not just structurally_but also evolutionarily. Because alignment of TnI and TnT coincides with the known structure of the IT-arm, and both Arg mutations are located close to the C-terminal end of the IT-arm, we investigated functional relationships between hcTnI R145G and hcTnT R278C. We hypothesized that if the mutations affected function independently, then their effects would be additive in a double mutant complex. We characterized Tn complexes containing either mutation alone, or both mutations simultaneously, using in vitro motility assays run with varying [Ca2+], temperature, or HMM density. Our most significant findings show that TnT R278C rescued some deleterious effects of TnI R145G at high Ca2+, but exacerbated the loss of function (i.e., switching off the actomyosin interaction) at low Ca2+. Taken together, our results raise the likelihood that cTnI's Ip sequence might share a common evolutionary origin with, and thus be structurally and functionally related to, the C-terminus of cTnT. In accord with this prediction, our experimental results suggest that the C-terminus of cTnT aids Ca2+-regulatory function of cTnI Ip within the troponin complex.

On the Cooperativity of Microtubule Motors during Cargo Transport and Directional Filament Sliding

The stepping behavior of single microtubule-based motor proteins has been studied in great detail. However, in cells, these motors often do not work alone but rather function in groups when they transport cellular cargo or slide microtubules against each other. Until now, the cooperative interactions between motors in such groups are poorly understood. We will report on in vitro experiments specifically designed to study the activity of coupled motors. With respect to cargo transport by rigidly-coupled kinesin-1 motors, we investigated (i) the step-size distribution of small groups of motors, (ii) the motility behavior in the presence of obstacles, and (iii) the tug-of-war between antagonistically acting force generators. With respect to the activity of diffusively-coupled kinesin-14 motors during cell division, we studied (i) the directional sliding of microtubules, (ii) the adaptive braking by passive crosslinkers, and (iii) the generation of torsional motion. All observed behaviors result from the collaborative action of multiple motors - none of the described properties is found in the activity of single motors.

A Nonmotor Microtubule Binding Site in Kinesin-5 Is Required for Filament Crosslinking and Sliding

Kinesin-5, a widely conserved motor protein required for assembly of the bipolar mitotic spindle in eukaryotes, forms homotetramers with two pairs of motor domains positioned at opposite ends of a dumbbell-shaped molecule [1-3]. It has long been assumed that this configuration of motor domains is the basis of kinesin-5's ability to drive relative sliding of microtubules [2, 4, 5]. Recently, it was suggested that in addition to the N-terminal motor domain, kinesin-5 also has a nonmotor microtubule binding site in its C terminus [6]. However, it is not known how the nonmotor domain contributes to motor activity, or how a kinesin-5 tetramer utilizes a combination of four motor and four nonmotor microtubule binding sites for its microtubule organizing functions. Here we show, in single molecule assays, that kinesin-5 homotetramers require the nonmotor C terminus for crosslinking and relative sliding of two microtubules. Remarkably, this domain enhances kinesin-5's microtubule binding without substantially reducing motor activity. Ourresults suggest that tetramerization of kinesin-5's low-processivity motor domains is not sufficient for microtubule sliding because the motor domains alone are unlikely tomaintain persistent microtubule crosslinks. Rather, kinesin-5 utilizes nonmotor microtubule binding sites to tune its microtubule attachment dynamics, enabling it to efficiently align and sort microtubules during metaphase spindle assembly and function.

Effects of Actin-Myosin Kinetics on the Calcium Sensitivity of Regulated Thin Filaments

Activation of thin filaments in striated muscle occurs when tropomyosin exposes myosin binding sites on actin either through calcium-troponin (Ca-Tn) binding or by actin-myosin (A-M) strong binding. However, the extent to which these binding events contributes to thin filament activation remains unclear. Here we propose a simple analytical model in which strong A-M binding and Ca-Tn binding independently activates the rate of A-M weak-to-strong binding. The model predicts how the level of activation varies with pCa as well as A-M attachment, N·k(att), and detachment, k(det), kinetics. To test the model, we use an in vitro motility assay to measure the myosin-based sliding velocities of thin filaments at different pCa, N·k(att), and k(det) values. We observe that the combined effects of varying pCa, N·k(att), and k(det) are accurately fit by the analytical model. The model and supporting data imply that changes in attachment and detachment kinetics predictably affect the calcium sensitivity of striated muscle mechanics, providing a novel A-M kinetic-based interpretation for perturbations (e.g. disease-related mutations) that alter calcium sensitivity.

Ca2+ Regulation of Rabbit Skeletal Muscle Thin Filament Sliding: Role of Cross-Bridge Number

We investigated how strong cross-bridge number affects sliding speed of regulated Ca2+-activated, thin filaments. First, using in vitro motility assays, sliding speed decreased nonlinearly with reduced density of heavy meromyosin (HMM) for regulated (and unregulated) F-actin at maximal Ca2+. Second, we varied the number of Ca2+-activatable troponin complexes at maximal Ca2+ using mixtures of recombinant rabbit skeletal troponin (WT sTn) and sTn containing sTnC(D27A,D63A), a mutant deficient in Ca2+ binding at both N-terminal, low affinity Ca2+-binding sites (xxsTnC-sTn). Sliding speed decreased nonlinearly as the proportion of WT sTn decreased. Speed of regulated thin filaments varied with pCa when filaments contained WT sTn but filaments containing only xxsTnC-sTn did not move. pCa50 decreased by 0.12–0.18 when either heavy meromyosin density was reduced to ∼60% or the fraction of Ca2+-activatable regulatory units was reduced to ∼33%. Third, we exchanged mixtures of sTnC and xxsTnC into single, permeabilized fibers from rabbit psoas. As the proportion of xxsTnC increased, unloaded shortening velocity decreased nonlinearly at maximal Ca2+. These data are consistent with unloaded filament sliding speed being limited by the number of cycling cross-bridges so that maximal speed is attained with a critical, low level of actomyosin interactions.

Orientation Changes of the Myosin Light Chain Domain During Filament Sliding in Active and Rigor Muscle

Structural changes in myosin power many types of cell motility including muscle contraction. Tilting of the myosin light chain domain (LCD) seems to be the final step in transducing the energy of ATP hydrolysis, amplifying small structural changes near the ATP binding site into nanometer-scale motions of the filaments. Here we used polarized fluorescence measurements from bifunctional rhodamine probes attached at known orientations in the LCD to describe the distribution of orientations of the LCD in active contraction and rigor. We applied rapid length steps to perturb the orientations of the population of myosin heads that are attached to actin, and thereby characterized the motions of these force-bearing myosin heads. During active contraction, this population is a small fraction of the total. When the filaments slide in the shortening direction in active contraction, the long axis of LCD tilts towards its nucleotide-free orientation with no significant twisting around this axis. In contrast, filament sliding in rigor produces coordinated tilting and twisting motions.

In vitro Motility Analysis of Thin Filaments from Failing and Non-failing Human Heart: Troponin from Failing Human Hearts Induces Slower Filament Sliding and Higher Ca2+ Sensitivity

Contractility of the myocardium is altered in end-stage heart failure. We investigated whether this was related to functional changes in troponin. We isolated troponin from 1 g samples of end-stage failing, non-failing and foetal human heart and studied its regulation of actin-tropomyosin movement over immobilised HMM by in vitro motility assay. At pCa5.4 the sliding velocity of thin filaments reconstituted with non-failing heart troponin was 52±4% more than actin-tropomyosin, with failing heart troponin velocity increased by 35±2% and with foetal heart troponin velocity increased by 11±4%. Thin filaments containing troponin from failing hearts were more Ca2+-sensitive than non-failing heart troponin. EC50 for the fraction of filaments motile and filament velocity decreased 1.76±0.20 and 1.89±0.62-fold respectively relative to non-failing heart troponin. With foetal heart troponin the EC50 decreased 2.16±0.23 and 3.50±1.73-fold for fraction and velocity respectively. Western blots revealed no difference in troponin T or troponin I isoform expression in troponin from failing and non-failing adult hearts but foetal isoforms of troponin I and T were observed in troponin from foetal heart. The level of PKA phosphorylation of troponin from failing and non-failing heart was not significantly different, however, complete non-specific dephosphorylation of troponin abolished most of the difference between failing and non-failing heart troponin. These findings show functional alterations in troponin in failing hearts which could account for the reduced contractile function but there is no change in troponin isoform expression or PKA phosphorylation. Differential phosphorylation by other kinases may account for altered troponin function.

Cross-Bridge Attachment during High-Speed Active Shortening of Skinned Fibers of the Rabbit Psoas Muscle: Implications for Cross-Bridge Action during Maximum Velocity of Filament Sliding

To characterize the kinetics of cross-bridge attachment to actin during unloaded contraction (maximum velocity of filament sliding), ramp-shaped stretches with different stretch-velocities (2–40,000 nm per half-sarcomere per s) were applied to actively contracting skinned fibers of the rabbit psoas muscle. Apparent fiber stiffness observed during such stretches was plotted versus the speed of the imposed stretch (stiffness-speed relation) to derive the rate constants for cross-bridge dissociation from actin. The stiffness-speed relation obtained for unloaded shortening conditions was shifted by about two orders of magnitude to faster stretch velocities compared to isometric conditions and was almost identical to the stiffness-speed relation observed in the presence of MgATP γS at high Ca 2+ concentrations, i.e., under conditions where cross-bridges are weakly attached to the fully Ca 2+ activated thin filaments. These data together with several control experiments suggest that, in contrast to previous assumptions, most of the fiber stiffness observed during high-speed shortening results from weak cross-bridge attachment to actin. The fraction of strongly attached cross-bridges during unloaded shortening appears to be as low as some 1–5% of the fraction present during isometric contraction. This is about an order of magnitude less than previous estimates in which contribution of weak cross-bridge attachment to observed fiber stiffness was not considered. Our findings imply that 1) the interaction distance of strongly attached cross-bridges during high-speed shortening is well within the range consistent with conventional cross-bridge models, i.e., that no repetitive power strokes need to be assumed, and 2) that a significant part of the negative forces that limit the maximum speed of filament sliding might originate from weak cross-bridge interactions with actin.

Induced Potential Model for Muscular Contraction Mechanism, Including Two Attached States of Myosin Head

The model for myosin head motion along an actin filament as proposed by Mitsui & Chiba [(1996).J. theor. Biol.182,147–159] is here modified so that it can explain the isometric tension and isotonic velocity transients having the same parameter values as the stationary filament sliding. The modified model differs in that a myosin head forms a complex with two actin molecules in an actin filament and has two attached states in the complex instead of three. Thus an incremental step in the myosin head motion is equal to the F-actin monomer repeat (5.46 nm). Muscle properties concerning the stationary filament sliding are calculated with new parameters in a manner similar to that of Mitsui–Chiba, with the results being qualitatively similar to theirs.In studying the transient phenomena, a quantitative expression is given for the potential energy of the myosin head in the complex, and two rate constants are applied to the kinetics of the head. The time course of tension recovery after a quick length change is determined by calculating the statistical distribution of the head in the two attached states, which conforms to experimental observations by Fordet al. [(1977).J. Physiol.269,441–515]. The tension variationsT1/T0andT2/T/0calculated with parameters determined from the analysis of stationary filament slidings are in fairly good agreement with the experimental data by Fordet al. The model suggest that a large fluctuation exists in the relative position between the actin and myosin filaments even when the load on a muscle is kept constant. Taking this fluctuation into account explains the characteristics of the isotonic velocity transient observed by Civan & Podolsky [(1966).J. Physiol.184,511–534].

Modulation of Actin Filament Sliding by Mutations of the SH2 Cysteine inDictyosteliumMyosin II

The cysteine residue called SH2 in the skeletal myosin heavy chain is conserved among various species. Cys 678 inDictyosteliummyosin II is equivalent to SH2 in skeletal myosin. Using theDictyosteliummyosin II heavy chain gene, SH2 was mutated to Gly, Ala, Ser, or Thr. These mutant myosins were expressed inDictyosteliummyosin-null cells. To investigate how these mutations affect the motor functions of myosin, we examined the phenotypes of the transformed cells. We also purified the mutant myosins, and characterized them by measuring the actin-activated MgATPase activity, sliding velocity of actin filaments and force level. All of these mutant myosins complemented the myosin-specific defects of the myosin-null cells. Consistent with these observed phenotypes, all of the purified mutant myosins retained similar actin-activated MgATPase activities and force levels to those of the wild-type myosin (WT). However, the sliding velocities of actin filaments were significantly different (WT≧Ser>Ala⪢Thr>Gly). In particular, the Gly and Thr mutants exhibited a striking decrease in velocity, while the Ser mutant exhibited velocity comparable to that of the wild-type myosin. Thus, mutations of SH2 resulted in uncoupling of ATP hydrolysis and the sliding.