John Sleep

Elasticity of the Red Cell Membrane and Its Relation to Hemolytic Disorders: An Optical Tweezers Study

We have used optical tweezers to study the elasticity of red cell membranes; force was applied to a bead attached to a permeabilized spherical ghost and the force-extension relation was obtained from the response of a second bead bound at a diametrically opposite position. Interruption of the skeletal network by dissociation of spectrin tetramers or extraction of the actin junctions engendered a fourfold reduction in stiffness at low applied force, but only a twofold change at larger extensions. Proteolytic scission of the ankyrin, which links the membrane skeleton to the integral membrane protein, band 3, induced a similar effect. The modified, unlike the native membranes, showed plastic relaxation under a prolonged stretch. Flaccid giant liposomes showed no measurable elasticity. Our observations indicate that the elastic character is at least as much a consequence of the attachment of spectrin as of a continuous membrane-bound network, and they offer a rationale for formation of elliptocytes in genetic conditions associated with membrane-skeletal perturbations. The theory of Parker and Winlove for elastic deformation of axisymmetric shells (accompanying paper) allows us to determine the function BH(2) for the spherical saponin-permeabilized ghost membranes (where B is the bending modulus and H the shear modulus); taking the literature value of 2 x 10(-19) Nm for B, H then emerges as 2 x 10(-6) Nm(-1). This is an order of magnitude higher than the value reported for intact cells from micropipette aspiration. Reasons for the difference are discussed.

Mapping the actin filament with myosin

Structural studies have shown that the heads of the myosin motor molecule bind preferentially to “target zones” of favorably oriented sites on the helices of the actin filament. We present direct evidence for target zones from the interactions of a single myosin head with an actin filament held between two optically trapped beads. With compliant traps, thermal motions of the filament allow the fixed myosin-S1 to interact with at least two zones, observed as a bi-modal distribution of filament displacements due to myosin binding, whose spacing is near the 36-nm helix repeat distance. The number of binding events and the “apparent working stroke” (mean displacement with myosin bound), vary periodically as the filament is moved past the fixed myosin by displacing the traps; observed periods are close to 36 nm and the apparent stroke varies from 0–10 nm. We also observe a strong modulation at the 5.5-nm actin monomer repeat confirming that myosin interacts with a single strand and that the actin is not free to rotate. Each interaction can be assigned to an actin monomer and each active zone on the helix is made up of three actin monomers.

Fluctuations of the Red Blood Cell Membrane: Relation to Mechanical Properties and Lack of ATP Dependence

We have analyzed the fluctuations of the red blood cell membrane in both the temporal ((omega(s(-1))) and spatial (q(m(-1))) frequency domains. The cells were examined over a range of osmolarities leading to cell volumes from 50% to 170% of that in the isotonic state. The fluctuations of the isotonic cell showed an approximately q(-3)-dependence, indicative of a motion dominated by bending, with an inferred bending modulus of approximately 9 x 10(-19) J. When the cells were osmotically swollen to just below the point of lysis (166% of physiological volume), a q(-1)-dependence of the fluctuations supervened, implying that the motion was now dominated by membrane tension; estimated as approximately 1.3 x 10(-4) nm(-1). When, on the other hand, the cells were osmotically dehydrated, the fluctuation amplitude progressively decreased. This was caused by a rise in internal viscosity, as shown by measurements on resealed ghosts containing a reduced hemoglobin concentration, which displayed no such effect. We examined, in addition, cells depleted of ATP, before the onset of echinocytosis, and could observe no change in fluctuation amplitude. We conclude that the membrane fluctuations of the red cell are governed by bending modulus, membrane tension, and cytosolic viscosity, with little or no dependence on the presence or absence of ATP.

In vitro motility speed of slow myosin extracted from single soleus fibres from young and old rats

1 Isolated soleus muscle fibres from aged rats contract more slowly than those from young rats. To determine whether this effect is due to a difference between the myosin molecules, we measured the rate at which actin filaments are driven over a myosin coated surface in the presence of ATP by using a novel in vitro motility assay where myosin is extracted from single muscle fibre segments. 2 Motility was dependent on the myosin density on the coverslip. In regions of high myosin density, actin motility was orientated parallel and anti‐parallel to the direction of flow during myosin adhesion to the coverslip. In contrast, in regions of lower myosin density, actin motility was more random. The speed was about 20% higher in the high density regions (P < 0.001). Further, the speed of filaments in the high density region, moving away or towards the fibre was less variable (P < 0.05) than that of more randomly moving filaments in the low density region. 3 The speed with myosin from slow soleus fibres of young adult rats (3–6 months old; v= 1.43 ± 0.23 μm s−1; mean ±s.d.) was faster (P < 0.001) than with myosin from aged rats (20–24 months old; v= 1.27 ± 0.23 μm s−1). 4 No difference in myosin isoforms between young adult and aged fibres could be detected using electrophoretic and immunocytochemical techniques. Fibres of both ages expressed the β/slow myosin heavy chain (MyHC) isoform and slow isoforms of essential and regulatory myosin light chains (MyLCs). 5 It is concluded that an age‐related alteration in myosin contributes to the slowing of the maximum shortening velocity (V0) observed in soleus muscle fibres expressing the β/slow MyHC isoform.

Towards a Unified Theory of Muscle Contraction. I: Foundations

Molecular models of contractility in striated muscle require an integrated description of the action of myosin motors, firstly in the filament lattice of the half-sarcomere. Existing models do not adequately reflect the biochemistry of the myosin motor and its sarcomeric environment. The biochemical actin–myosin–ATP cycle is reviewed, and we propose a model cycle with two 4- to 5-nm working strokes, where phosphate is released slowly after the first stroke. A smaller third stroke is associated with ATP-induced detachment from actin. A comprehensive model is defined by applying such a cycle to all myosin-S1 heads in the half-sarcomere, subject to generic constraints as follows: (a) all strain-dependent kinetics required for actin–myosin interactions are derived from reaction-energy landscapes and applied to dimeric myosin, (b) actin–myosin interactions in the half-sarcomere are controlled by matching rules derived from the structure of the filaments, so that each dimer may be associated with a target zone of three actin sites, and (c) the myosin and actin filaments are treated as elastically extensible. Numerical predictions for such a model are presented in the following paper.

The ATP hydrolysis and phosphate release steps control the time course of force development in rabbit skeletal muscle

The time course of isometric force development following photolytic release of ATP in the presence of Ca2+ was characterized in single skinned fibres from rabbit psoas muscle. Pre‐photolysis force was minimized using apyrase to remove contaminating ATP and ADP. After the initial force rise induced by ATP release, a rapid shortening ramp terminated by a step stretch to the original length was imposed, and the time course of the subsequent force redevelopment was again characterized. Force development after ATP release was accurately described by a lag phase followed by one or two exponential components. At 20°C, the lag was 5.6 ± 0.4 ms (s.e.m., n= 11), and the force rise was well fitted by a single exponential with rate constant 71 ± 4 s−1. Force redevelopment after shortening–restretch began from about half the plateau force level, and its single‐exponential rate constant was 68 ± 3 s−1, very similar to that following ATP release. When fibres were activated by the addition of Ca2+ in ATP‐containing solution, force developed more slowly, and the rate constant for force redevelopment following shortening–restretch reached a maximum value of 38 ± 4 s−1 (n= 6) after about 6 s of activation. This lower value may be associated with progressive sarcomere disorder at elevated temperature. Force development following ATP release was much slower at 5°C than at 20°C. The rate constant of a single‐exponential fit to the force rise was 4.3 ± 0.4 s−1 (n= 22), and this was again similar to that after shortening–restretch in the same activation at this temperature, 3.8 ± 0.2 s−1. We conclude that force development after ATP release and shortening–restretch are controlled by the same steps in the actin–myosin ATPase cycle. The present results and much previous work on mechanical–chemical coupling in muscle can be explained by a kinetic scheme in which force is generated by a rapid conformational change bracketed by two biochemical steps with similar rate constants – ATP hydrolysis and the release of inorganic phosphate – both of which combine to control the rate of force development.

The working stroke upon myosin–nucleotide complexes binding to actin

For many years, it has been known that myosin binds to actin tightly, but it had not been possible to devise a muscle fiber experiment to determine whether this binding energy is directly coupled to the working stroke of the actomyosin crossbridge cycle. Addressing the question at the single-molecule level with optical tweezers allows the problem to be resolved. We have compared the working stroke on the binding of four myosin complexes (myosin, myosin-ADP, myosin-pyrophosphate, and myosin-adenyl-5′yl imidodiphosphate) with that observed while hydrolyzing ATP. None of the four was observed to give a working stroke significantly different from zero. A working stroke (5.4 nm) was observed only with ATP, which indicates that the other states bind to actin in a rigor-like conformation and that myosin products (M.ADP.Pi), the state that binds to actin during ATPase activity, binds in a different, prestroke conformation. We conclude that myosin, while dissociated from actin, must be able to take up at least two mechanical conformations and show that our results are consistent with these conformations corresponding to the two states characterized at high resolution, which are commonly referred to in terms of having open and closed nucleotide binding pockets.

Inhibition of unloaded shortening velocity in permeabilized muscle fibres by caged ATP compounds

SummaryThe effects of both the P3-1-(2-nitrophenyl)ethyl ester of adenosine 5′-triphosphate (NPE-caged ATP) and its separate diastereoisomers, and the P3-3′,5′-dimethoxybenzoin ester of ATP (DMB-caged ATP) were studied on the unloaded shortening velocity of glycerinated rabbit psoas muscle fibres. The unloaded shortening velocities of the active fibres were measured as a function of ATP concentration (0.1–5 mm) using the ‘slack-test’ with and without 2 mm caged ATP. Shortening velocity followed a Michaelis-Menten relationship with ATP concentration, the Km for ATP being 170 μm. The caged ATP compounds inhibited shortening velocity, in a manner consistent with competitive inhibition, with a Ki of 1–2 mm. The R- and S-diastereoisomers of NPE-caged ATP showed the same degree of competitive inhibition of the shortening velocity, as did DMB-caged ATP. These observations suggest that caged ATP compounds bind to the ATPase site of the actomyosin where they compete with the substrate, Mg2+ATP.

Kinetics of muscle contraction and actomyosin NTP hydrolysis from rabbit using a series of metal–nucleotide substrates

Mechanical properties of skinned single fibres from rabbit psoas muscle have been correlated with biochemical steps in the cross‐bridge cycle using a series of metal–nucleotide (Me·NTP) substrates (Mn2+ or Ni2+ substituted for Mg2+; CTP or ITP for ATP) and inorganic phosphate. Measurements were made of the rate of force redevelopment following (1) slack tests in which force recovery followed a period of unloaded shortening, or (2) ramp shortening at low load terminated by a rapid restretch. The form and rate of force recovery were described as the sum of two exponential functions. Actomyosin‐Subfragment 1 (acto‐S1) Me·NTPase activity and Me·NDP release were monitored under the same conditions as the fibre experiments. Mn·ATP and Mg·CTP both supported contraction well and maintained good striation order. Relative to Mg·ATP, they increased the rates and Me·NTPase activity of cross‐linked acto‐S1 and the fast component of a double‐exponential fit to force recovery by ∼50% and 10–35%, respectively, while shortening velocity was moderately reduced (by 20–30%). Phosphate also increased the rate of the fast component of force recovery. In contrast to Mn2+ and CTP, Ni·ATP and Mg·ITP did not support contraction well and caused striations to become disordered. The rates of force recovery and Me·NTPase activity were less than for Mg·ATP (by 40–80% and 50–85%, respectively), while shortening velocity was greatly reduced (by ∼80%). Dissociation of ADP from acto‐S1 was little affected by Ni2+, suggesting that Ni·ADP dissociation does not account for the large reduction in shortening velocity. The different effects of Ni2+ and Mn2+ were also observed during brief activations elicited by photolytic release of ATP. These results confirm that at least one rate‐limiting step is shared by acto‐S1 ATPase activity and force development. Our results are consistent with a dual rate‐limitation model in which the rate of force recovery is limited by both NTP cleavage and phosphate release, with their relative contributions and apparent rate constants influenced by an intervening rapid force‐generating transition.

Kinetics of force recovery following length changes in active skinned single fibres from rabbit psoas muscle

Redevelopment of isometric force following shortening of skeletal muscle is thought to result from a redistribution of cross‐bridge states. We varied the initial force and cross‐bridge distribution by applying various length‐change protocols to active skinned single fibres from rabbit psoas muscle, and observed the effect on the slowest phase of recovery (‘late recovery’) that follows transient changes. In response to step releases that reduced force to near zero (∼8 nm (half sarcomere)−1) or prolonged shortening at high velocity, late recovery was well described by two exponentials of approximately equal amplitude and rate constants of ∼2 s−1 and ∼9 s−1 at 5°C. When a large restretch was applied at the end of rapid shortening, recovery was accelerated by (1) the introduction of a slow falling component that truncated the rise in force, and (2) a relative increase in the contribution of the fast exponential component. The rate of the slow fall was similar to that observed after a small isometric step stretch, with a rate of 0.4–0.8 s−1, and its effects could be reversed by reducing force to near zero immediately after the stretch. Force at the start of late recovery was varied in a series of shortening steps or ramps in order to probe the effect of cross‐bridge strain on force redevelopment. The rate constants of the two components fell by 40–50% as initial force was raised to 75–80% of steady isometric force. As initial force increased, the relative contribution of the fast component decreased, and this was associated with a length constant of about 2 nm. The results are consistent with a two‐state strain‐dependent cross‐bridge model. In the model there is a continuous distribution of recovery rate constants, but two‐exponential fits show that the fast component results from cross‐bridges initially at moderate positive strain and the slow component from cross‐bridges at high positive strain.