The Creatine Phosphoryltransfer Reaction in Iodoacetate-Poisoned Muscle

The iodoacetate-nitrogen-poisoned muscle offers the possibility of studying the stoichiometry of the single muscle twitch since metabolic resynthesis by glycolysis and oxidative phosphorylation are blocked, and there remains as an energy source only the creatine phosphoryltransfer system, creatine phosphate reacting with adenosinediphosphate to give the triphosphate and creatine. It is shown, preparatory to a determination of the amount of phosphocreatine split in a single twitch, that iodoacetate does not inhibit creatine phosphoryltransferase at concentrations which block glycolysis. An analysis is developed which assumes that the transferase maintains the creatine phosphoryl transfer reaction in equilibrium following contraction, and further that the creatine phosporyltransfer reaction and the myokinase reaction are isolated in muscle. On the basis of this analysis and the data obtained, an estimate of the equilibrium constant of the creatine phosphoryl reaction in muscle is obtained which agrees with values determined in vitro. Using the estimated equilibrium constant, and the concentrations of creatine, creatine phosphate, and adenosinetriphosphate found, a value for the concentration of free adenosinediphosphate is obtained which is considerably less than that found by direct chemical analysis.     

Multiscale modeling of twitch contractions in cardiac trabeculae

Understanding the dynamics of a cardiac muscle twitch contraction is complex because it requires a detailed understanding of the kinetic processes of the Ca²⁺ transient, thin-filament activation, and the myosin–actin cross-bridge chemomechanical cycle. Each of these steps has been well defined individually, but understanding how all three of the processes operate in combination is a far more complex problem. Computational modeling has the potential to provide detailed insight into each of these processes, how the dynamics of each process affect the complexity of contractile behavior, and how perturbations such as mutations in sarcomere proteins affect the complex interactions of all of these processes. The mechanisms involved in relaxation of tension during a cardiac twitch have been particularly difficult to discern due to nonhomogeneous sarcomere lengthening during relaxation. Here we use the multiscale MUSICO platform to model rat trabecular twitches. Validation of computational models is dependent on being able to simulate different experimental datasets, but there has been a paucity of data that can provide all of the required parameters in a single experiment, such as simultaneous measurements of force, intracellular Ca²⁺ transients, and sarcomere length dynamics. In this study, we used data from different studies collected under similar experimental conditions to provide information for all the required parameters. Our simulations established that twitches either in an isometric sarcomere or in fixed-length, multiple-sarcomere trabeculae replicate the experimental observations if models incorporate a length–tension relationship for the nonlinear series elasticity of muscle preparations and a scheme for thick-filament regulation. The thick-filament regulation assumes an off state in which myosin heads are parked onto the thick-filament backbone and are unable to interact with actin, a state analogous to the super-relaxed state. Including these two mechanisms provided simulations that accurately predict twitch contractions over a range of different conditions.     

A comparison of quantitative ultrastructural and contractile characteristics of muscle fibre types of the perch,Perca fluviatilis L.

Summary Quantitative ultrastructural and physiological parameters were investigated in three types of muscle fibres ofPerca fluviatilis: white fibres from the m. levator operculi anterior, pink (intermediate) fibres of the m. hyohyoideus and deep red fibres of the m. levator operculi anterior. Times to peak tension and half relaxation times of isometric twitches increased in the mentioned order. The extent of contact between the T system and the sarcoplasmic reticulum and the relative volume and surface area of the terminal cisternae showed an inverse relation with the time to peak tension of the twitch. The maximal isometric tetanic force per unit cross section area was similar for all three investigated types. The inverse relation between the time to peak tension of the twitch and the relative length of contact between T system and SR is in agreement with data obtained for fast- and slow twitch muscle fibres of the carp,Cyprinus carpio L.Abbreviations LOPA musculus levator operculi anterior - HH musculus hyohyoideus - SR Sarcoplasmic reticulum     

Generation of force by single-headed myosin.

Myosin has two heads, each of which can interact with actin and ATP. We have investigated the possibility that co-operative interactions occur between the heads by measuring the force generated by single-headed myosin in reconstituted actomyosin threads. Myofibrils were digested with papain, actomyosin was extracted from the myofibrils, and one-headed myosin was purified by cycles of sedimentation with actin. The one-headed myosin was approximately 90 to 95% pure as determined by densitometer scans of polyacrylamide gels run in 20 mm-PP₁ (impurities consisted of 1 to 5% of myosin and 1 to 5% myosin rod). The ATPase activity per mole of single-headed myosin was one half that of myosin under conditions where the activity was activated by Ca²⁺, K⁺ or actin. One-headed myosin could also participate in superprecipitation, although with a rate that was at least one order of magnitude slower than that for myosin. Myosin or one-headed myosin was mixed with actin, threads were formed via extrusion into low ionic strength, and the isometric forces and isotonic velocities generated by the threads were measured. The ratio of the isometric tension produced per head by the one-headed myosin to the isometric tension produced per head by myosin was 1·0 ± 0·1. The maximum velocity of thread contraction for the one-headed myosin was also not different from the control myosin. Thus, the absence of one head does not appear to impair the generation of force or motion by the remaining head.     

Energetics of Isometric and Isotonic Twitches in Toad Sartorius

Contractile energetics have been studied in twitches of toad sartorius muscle at 6-7°C. Isometric and isotonic energy production has been measured and plotted against a wide range of developed tensions and tension-time integrals. These parameters were varied by altering the isotonic load or by changing the preset isometric length. The isometric tension-independent heat was 1.12 ±0.18 (SD) mcal/g. The isometric heat coefficient Pl₀/H was 12.0 ±1.4 in muscles having twitch to tetanus ratios ranging from 0.4 to 0.6. Isometric enthalpy increased monotonically with tension or tension-time integral but the correlation between isometric heat and these parameters was poor. Isotonic enthalpy consumption was always higher than isometric enthalpy for any given tension or tension-time integral; however, isotonic heat production was consistently less than isometric heat production. The isotonic heat for the highest load (3 g) was not significantly different from the isometric tension-independent heat. Thus isotonic heat production first decreased and then increased with increasing tension or tension-time integral. In the discussion it is shown that the results conflict with all current interpretations of muscle energetics.     

Muscle Volume Changes

Measurements have been made of the volume changes accompanying single isometric and isotonic twitches of frog sartorius muscle. The volume change consists of a rapid increase, a subsequent decrease, and a return to the initial volume; the order of magnitude of increase and decrease is 10^-5 cc/g of muscle. This volume change is length-dependent: the initial increase becomes more pronounced as the initial length of the muscle is decreased, while the volume decrease is greatest at reference length and is diminished for longer and shorter initial lengths. Muscle volume changes are also dependent upon temperature and amount of shortening: the return phase is prolonged as the temperature is lowered; and, in an isotonic twitch, a volume increase accompanying muscle shortening is superimposed upon the volume change described for an isometric twitch. These "shortening volume changes" may correspond to the volume decrease observed in frog muscle under a passive stretch. If the active state is prolonged by the use of a frog Ringer solution in which iodide ions have been substituted for chloride ions, the time course of the volume decrease is likewise prolonged; this suggests a relationship between the volume decrease and the active state of the muscle.     

Potentiation in mouse lumbrical muscle without myosin light chain phosphorylation: Is resting calcium responsible?

The increase in isometric twitch force observed in fast-twitch rodent muscles during or after activity, known universally as potentiation, is normally associated with myosin regulatory light chain (RLC) phosphorylation. Interestingly, fast muscles from mice devoid of detectable skeletal myosin light chain kinase (skMLCK) retain a reduced ability to potentiate twitch force, indicating the presence of a secondary origin for this characteristic feature of the fast muscle phenotype. The purpose of this study was to assess changes in intracellular cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) after a potentiating stimulus in mouse lumbrical muscle (37°C). Lumbricals were loaded with the Ca²⁺-sensitive fluorescent indicators fura-2 or furaptra to detect changes in resting and peak, respectively, intracellular Ca²⁺ levels caused by 2.5 s of 20-Hz stimulation. Although this protocol produced an immediate increase in twitch force of 17 ± 3% (all data are n = 10) (P < 0.01), this potentiation dissipated quickly and was absent 30 s afterward. Fura-2 fluorescence signals at rest were increased by 11.1 ± 1.3% (P < 0.01) during potentiation, indicating a significant increase in resting [Ca²⁺]_i. Interestingly, furaptra signals showed no change to either the amplitude or the duration of the intracellular Ca²⁺ transients (ICTs) that triggered potentiated twitches during this time (P < 0.50). Immunofluorescence work showed that 77% of lumbrical fibers expressed myosin heavy chain isoform IIx and/or IIb, but with low expression of skMLCK and high expression of myosin phosphatase targeting subunit 2. As a result, lumbrical muscles displayed no detectable RLC phosphorylation either at rest or after stimulation. We conclude that stimulation-induced elevations in resting [Ca²⁺]_i, in the absence of change in the ICT, are responsible for a small-magnitude, short-lived potentiation of isometric twitch force. If operative in other fast-twitch muscles, this mechanism may complement the potentiating influence of myosin RLC phosphorylation.     

Nonacceptance of innervation by innervated neonatal rat muscle

Denervated adult muscle accepts innervation and has high levels of extrajunctional acetylcholine (ACh) receptor, compared to innervated adult muscle. If the high receptor density or any externally oriented part of the receptor molecule permitted or triggered functional synaptogenesis, then innervated neonatal muscle, with its known high extrajunctional sensitivity, should also accept extra synapses from implanted motor nerves. This prediction was tested by implanting the common peroneal nerve into innervated lateral gastrocnemius muscle in 25 neonatal rats and studying the innervation achieved 1–8 weeks later. With one exception, zero or negligible twitch tensions were obtained when the implanted nerve was stimulated. Intracellular recording in two cases showed no evidence of subthresholdevoked potentials in surface muscle fibers. In contrast, when the original motor nerve was cut at the time of common peroneal nerve implantation, reinnervation occurred as soon as 4 days later, and substantial indirect twitches (most observed qualitatively) were invariably found 6–7 days after operation. Four to eight weeks after nerve implantation into denervated muscle, substantial twitch tensions were obtained upon stimulation of the implanted nerve. α-Bungarotoxin binding to extrajunctional ACh receptors per unit surface area was similar in innervated neonatal and denervated adult muscle. Therefore, nonacceptance of additional functional innervation in neonatal muscle implies that a high average density of extrajunctional ACh receptor is not sufficient to permit or trigger functional neuromuscular junction formation.     

Spectrophotometric studies on the pH of frog skeletal muscle. PH change during and after contractile activity

The spectral characteristics of the pH-sensitive dyes neutral red (NR) and bromcresol purple (BCP) were utilized for studies of the changing intracellular pH (pHi) of sartorius muscles from Rana pipiens, both during the course of an isometric twitch and during recovery metabolism subsequent to a train of twitches. The information from the two dissimilar dyes correlated to confirm the methodology. Neither the fast realkalinization observed during a twitch nor the slow alkalizing phase of recovery metabolism was affected in an obvious manner when phosphocreatine (PC) hydrolysis was blocked by 1-fluoro-2,4-dinitrobenzene (FDNB). Iodoacetic acid (IAA) did inhibit the slow acidic phase of recovery metabolism. The conclusion is made that alkalizing reactions other than PC breakdown must be considered as operative at these levels of activity. Hypertonic solutions altered twitch tension and time course without altering the pHi shifts observed until approximately 75% of the twitch amplitude was abolished. Multiple effects of hypertonic solutions as the muscle approach tonic equilibrium are proposed.     

Juxtaposition of the changes in intracellular calcium and force during staircase potentiation at 30 and 37°C

Ca²⁺ entry during the action potential stimulates muscle contraction. During repetitive low frequency stimulation, skeletal muscle undergoes staircase potentiation (SP), a progressive increase in the peak twitch force induced by each successive stimulus. Multiple mechanisms, including myosin regulatory light chain phosphorylation, likely contribute to SP, a temperature-dependent process. Here, we used the Ca²⁺-sensitive fluorescence indicators acetoxymethyl (AM)-furaptra and AM-fura-2 to examine the intracellular Ca²⁺ transient (ICT) and the baseline Ca²⁺ level at the onset of each ICT during SP at 30 and 37°C in mouse lumbrical muscle. The stimulation protocol, 8 Hz for 8 s, resulted in a 27 ± 3% increase in twitch force at 37°C and a 7 ± 2% decrease in twitch force at 30°C (P < 0.05). Regardless of temperature, the peak rate of force production (+df/dt) was higher in all twitches relative to the first twitch (P < 0.05). Consistent with the differential effects of stimulation on twitch force at the two temperatures, raw ICT amplitude decreased during repetitive stimulation at 30°C (P < 0.05) but not at 37°C. Cytosolic Ca²⁺ accumulated during SP such that baseline Ca²⁺ at the onset of ICTs occurring late in the train was higher (P < 0.05) than that of those occurring early in the train. ICT duration increased progressively at both temperatures. This effect was not entirely proportional to the changes in twitch duration, as twitch duration characteristically decreased before increasing late in the protocol. This is the first study identifying a changing ICT as an important, and temperature-sensitive, modulator of muscle force during repetitive stimulation. Moreover, we extend previous observations by demonstrating that contraction-induced increases in baseline Ca²⁺ coincide with greater +df/dt but not necessarily with higher twitch force.     

The reaction of myosin with N-ethylmaleimide in the presence of ADP

Myosin reacted with N-ethylmaleimide in the presence of ADP lost its ability to be activated by actin. Subfragment 1 behaved similarly. About 2 moles of N-ethylmaleimide per mole of Subfragment 1 were required to eliminate actin activation of the Mg²⁺-ATPase activity. At the point at which actin activation was lost the K⁺-EDTA-ATPase activity was also lost, but the Ca²⁺-activated ATPase activity was increased. Kinetic measurements indicated that the labelling with N-ethylmaleimide in the presence of ADP reduced V (the ATPase activity at infinite actin concentration) but did not effect K_app (which is related to the dissociation constant of the actin-Subfragment 1 complex). The Mg²⁺-activated activity of the reacted myosin alone remained unaltered and the ability to bind actin was retained. We propose that the N-ethylmaleimide labelling blocked the actin activation by preventing the accelerated release of hydrolysis products from the myosin.     

The nature and reactivity of the ‘essential’ thiol in rabbit muscle creatine kinase III (EC 2.7.3.2)

Rabbit muscle creatine kinase III (EC 2.7.3.2) can be reacted with 2-chloromercuri-4-nitrophenol and this results in the incorporation of two moles of mercurial per mole of enzyme subunit in a biphasic reaction. The second-order rate constant for the slow reaction is 475 ± 42 M^?1 s^?1. S-Carboxamidomethyl-creatine kinase reacts with a single mole of mercurial per mole of subunit. The rate constant, 466 ± 57 M^?1 s^?1, is almost identical to that for the slow reaction of the native enzyme. The reaction between 3-carboxy-4-nitrophenylthio-creatine kinase and 2-chloromercuri-4-nitrophenol has a second-order rate constant of 449 ± 56 M^?1 s^?1. The results may be explained if the mercurial reacts very rapidly with that cysteine residue which reacts independently with iodoacetamide or 5,5′-dithiobis(2-nitrobenzoic acid). However, 2-chloromercuri-4-nitrophenol also reacts more slowly with a second cysteine residue. Definition of the essentiality of thiol groups in enzymes by reaction with labile ligands, here represented by organomercurials, clearly must be approached with caution.     

Radiocalcium Release by Stimulated and Potassium-Treated Sartorius Muscles of the Frog

Stimulation of frog (Rana pipiens) sartorius muscle accelerates release of Ca⁴⁵, but only during the period of stimulation. No appreciable difference is obtained in the calcium released per impulse whether stimulation is at a rate of 20/sec. or 0.5/sec. However, prior stimulation may appreciably increase the loss per impulse. In unfatigued muscles, the minimum amount of calcium liberated during an isotonic twitch is estimated to be about that previously calculated to enter, viz. 0.2 µµmole/cm². The time course of radiocalcium release during potassium depolarization depends on the nature of the contracture. When contracture is isometric, the rate of escape is doubled and declines only slowly; if isotonic, the rate is quadrupled but declines in a few minutes to a level maintained at about double that before potassium. The minimal calcium release during the first 10 minutes of potassium treatment is estimated to be about the same in both cases and about one-half to one-third the uptake. This, and especially the close equality of calcium entry and exit during electrical stimulation, are pointed out as not necessarily inconsistent with a transitory net entry of calcium, comparable to the influx, into restricted regions of the individual fibers.     

The myosin head can bind two actin monomers

Force impulse is thought to be generated in muscle when myosin head (S-1), while weakly bound to actin filament, undergoes orientational change to form a strong (rigor) bond with actin. There is ample evidence that this bond involves interaction of 1 myosin head with 1 actin monomer. However, X-ray diffraction data of muscle decorated with S-1, as well as recently proposed model of the thin filaments, suggested that each S-1 molecule interacted with two actin monomers. We reinvestigated this controversy and found that the stoichiometry of acto-S-1 bond depended on the relative amounts of actin and myosin present during titrations: when increasing amounts of actin were added to a fixed amount of S-1 (i.e. when myosin heads were initially in excess over actin), the saturating stoichiometry was 1 mol of S-1 per 1 mol of actin. However, when increasing amounts of S-1 were added slowly to a fixed amount of F-actin (i.e. when actin was initially in excess over S-1), the stoichiometry at saturation was 1 mol of S-1 per 2 mols of actin. The ability of S-1 to bind either one or two actin monomers suggests a way that force could be generated during muscle contraction.     

The localization of the MM isozyme of creatine phosphokinase on the surface membrane of myocardial cells and its functional coupling to ouabain-inhibited (Na+,K+)-ATPase

A rat heart plasma membrane preparation isolated in a sucrose medium and some of its enzymatic properties have been investigated.It has been shown that a rat heart plasma membrane fraction contains high creatine phosphokinase activity which can not be diminished by repeated washing with sucrose solution. Creatine phosphokinase extracted from a plasma membrane fraction with potassium chloride and 0.01% deoxycholate solution is electrophoretically identical to MM isoenzyme of creatine phosphokinase. Under the conditions where (Na⁺,K⁺)-ATPase is activated by addition of Na⁺,K⁺ and MgATP, creatine phosphokinase of plasma membrane fraction is able to maintain a low ADP concentration in the medium if creatine phosphate is present. The rate of creatine release is dependent upon MgATP concentration in accordance with the kinetic parameters of the (Na⁺,K⁺)-ATPase and is significantly inhibited by ouabain (0.5 mM). The rate of creatine release is also dependent on creatine phosphate concentration in conformance with the kinetic parameters of MM isozyme of creatine phosphokinase,It is concluded that in intact heart cells the plasma membrane creatine phosphokinase may ensure effective utilization of creatine phosphate for immediate rephosphorylation of ADP produced in the (Na⁺,K⁺)-ATPase reaction.     

Arp2/3- and Cofilin-coordinated Actin Dynamics Is Required for Insulin-mediated GLUT4 Translocation to the Surface of Muscle Cells

GLUT4 vesicles are actively recruited to the muscle cell surface upon insulin stimulation. Key to this process is Rac-dependent reorganization of filamentous actin beneath the plasma membrane, but the underlying molecular mechanisms have yet to be elucidated. Using L6 rat skeletal myoblasts stably expressing myc-tagged GLUT4, we found that Arp2/3, acting downstream of Rac GTPase, is responsible for the cortical actin polymerization evoked by insulin. siRNA-mediated silencing of either Arp3 or p34 subunits of the Arp2/3 complex abrogated actin remodeling and impaired GLUT4 translocation. Insulin also led to dephosphorylation of the actin-severing protein cofilin on Ser-3, mediated by the phosphatase slingshot. Cofilin dephosphorylation was prevented by strategies depolymerizing remodeled actin (latrunculin B or p34 silencing), suggesting that accumulation of polymerized actin drives severing to enact a dynamic actin cycling. Cofilin knockdown via siRNA caused overwhelming actin polymerization that subsequently inhibited GLUT4 translocation. This inhibition was relieved by reexpressing Xenopus wild-type cofilin-GFP but not the S3E-cofilin-GFP mutant that emulates permanent phosphorylation. Transferrin recycling was not affected by depleting Arp2/3 or cofilin. These results suggest that cofilin dephosphorylation is required for GLUT4 translocation. We propose that Arp2/3 and cofilin coordinate a dynamic cycle of actin branching and severing at the cell cortex, essential for insulin-mediated GLUT4 translocation in muscle cells.     

Nucleoside triphosphate metabolism in the muscle tissue of Ascaris lumbricoides (Nematoda)

Barrett J. 1973. Nucleoside triphosphate metabolism in muscle tissue of Ascaris lumbricoides (Nematoda). International Journal for Parasitology3: 393–400. Nucleosidediphosphate kinase and adenylate kinase were found to be extremely active in Ascaris muscle. Apart from adenylate kinase, no other nucleosidemonophosphate kinases could be detected. There was no measurable AMP deaminase activity or arginine or creatine phosphokinase activity in Ascaris muscle. Analysis of perchlorate extracts of freeze clamped Ascaris muscle revealed no arginine or creatine phosphate and negligible amounts of acid labile phosphate. Adenosine tri-, di- and monophosphates were the major nucleotides, constituting 93 per cent of the total, with only small amounts of inosine and guanosine di- and triphosphates being detected. The significance of these results in the energy metabolism of Ascaris muscle is discussed.     

The Impact of Magnesium on Isometric Twitch Parameters and Resting Membrane Potential of the Skeletal Muscle in Diabetic Rats

To present the relationship between oral magnesium supplementation, blood glucose, and changes in isometric twitch parameters, resting membrane potential (RMP), in the gastrocnemius muscle in diabetic rats. Sixty rats were used in this study. The rats were divided into four groups: control (drinking tap water, Group I, n = 15), control with treated with magnesium sulfate (10 g/L) (Group II, n = 15), diabetic (Group III, n = 15), and diabetic with treated with magnesium sulfate (10 g/L) (Group IV, n = 15). In Group II and IV, the level of plasma magnesium was increased comparing to those of the control group (p < 0.05). Isometric twitch tensions were decreased significantly in the Group III, but Group IV isometric twitch tensions were increased significantly. Group IV RMP values were close to the Group I. Hyperglycemia decreases gastrocnemius muscle isometric twitch tension and increases RMP in diabetic rats. Magnesium treatment can prevent these diabetic complications.     

Force-Generating Capacity and Contractile Protein Content of Arterial Smooth Muscle

After correction for extracellular space (40%) determined from electron micrographs, the maximum isometric force developed by strips prepared from the media of the hog carotid artery (2.2 x 10⁶ dyn/cm²) can be extrapolated to give a value of 3.7 x 10⁶ dyn/cm² for the smooth muscle component of the strip. Three independent estimates of the myosin content of the smooth muscle cells were made based on (a) exhaustive extraction and purification with estimates of preparative losses, (b) the myosin catalyzed ATPase activity of media homogenates, and (c) quantitative densitometry of the peaks containing myosin, actin, and tropomyosin after disk electrophoresis of sodium dodecyl sulfate-treated media homogenates. The results were consistent and gave a myosin content of 5–10 mg/g media, or 8–17 mg/g cell. Method (c) gave myosin:actin:tropomyosin weight ratios of 1:3.2:0.8. Although measured force developed by the smooth muscle cell exceeds that of mammalian striated muscle, the myosin content in smooth muscle is about five times lower. The actin content of smooth muscle is relatively high. The actin and myosin contents are consistent with thick and thin filament ratios observed in electron micrographs of vascular smooth muscle.     

The location of ubiquitin in Lethocerus arthrin

Arthrin is a ubiquitinated actin that is present in flight muscles of some insects. In addition, it has been found in the malaria parasite Plasmodium falciparum. The role of this monoubiquitylation is not clear, and it does not appear to be associated with proteolytic degradation. The stoichiometry of arthrin to actin in Lethocerus indirect flight muscle, 1:6, suggests that there would be one arthrin molecule for each Tm-Tn (tropomyosin-troponin) complex. The appearance of arthrin after tropomyosin and troponin in Drosophila development is consistent with the Tm-Tn complex determining which actin subunit is targeted for conjugation with ubiquitin. We have used a new approach of three-dimensional reconstruction of helical filaments, the iterative helical real space reconstruction method, to extract segments of homogeneous arthrin out of long filaments where the conformation of the ubiquitin is more heterogeneous. Surprisingly, the location of the ubiquitin is on the face of actin subdomain 1, opposite to where tropomyosin binds in the “off” state, suggesting that there could not be a direct interaction between the ubiquitin and the tropomyosin. It is possible that the troponin complex in the “on” state that is bound to one actin strand makes an unfavorable contact with a ubiquitin molecule attached to the opposite actin strand. This might be the basis for a destabilization of the on state at rest length. Lys118 is the most likely residue to which the ubiquitin is conjugated, based upon fitting atomic structures of actin and ubiquitin into the reconstruction.