Microarchitecture Is Severely Compromised but Motor Protein Function Is Preserved in Dystrophic mdx Skeletal Muscle

Progressive force loss in Duchenne muscular dystrophy is characterized by degeneration/regeneration cycles and fibrosis. Disease progression may involve structural remodeling of muscle tissue. An effect on molecular motorprotein function may also be possible. We used second harmonic generation imaging to reveal vastly altered subcellular sarcomere microarchitecture in intact single dystrophic mdx muscle cells (∼1 year old). Myofibril tilting, twisting, and local axis deviations explain at least up to 20% of force drop during unsynchronized contractile activation as judged from cosine angle sums of myofibril orientations within mdx fibers. In contrast, in vitro motility assays showed unaltered sliding velocities of single mdx fiber myosin extracts. Closer quantification of the microarchitecture revealed that dystrophic fibers had significantly more Y-shaped sarcomere irregularities (“verniers”) than wild-type fibers (∼130/1000 μm³ vs. ∼36/1000 μm³). In transgenic mini-dystrophin-expressing fibers, ultrastructure was restored (∼38/1000 μm³ counts). We suggest that in aged dystrophic toe muscle, progressive force loss is reflected by a vastly deranged micromorphology that prevents a coordinated and aligned contraction. Second harmonic generation imaging may soon be available in routine clinical diagnostics, and in this work we provide valuable imaging tools to track and quantify ultrastructural worsening in Duchenne muscular dystrophy, and to judge the beneficial effects of possible drug or gene therapies.     

Diffusion Coefficients of Endogenous Cytosolic Proteins from Rabbit Skinned Muscle Fibers

Efflux time courses of endogenous cytosolic proteins were obtained from rabbit psoas muscle fibers skinned in oil and transferred to physiological salt solution. Proteins were separated by gel electrophoresis and compared to load-matched standards for quantitative analysis. A radial diffusion model incorporating the dissociation and dissipation of supramolecular complexes accounts for an initial lag and subsequent efflux of glycolytic and glycogenolytic enzymes. The model includes terms representing protein crowding, myofilament lattice hindrance, and binding to the cytomatrix. Optimization algorithms returned estimates of the apparent diffusion coefficients, D(r,t), that were very low at the onset of diffusion (∼10⁻¹⁰ cm² s⁻¹) but increased with time as cytosolic protein density, which was initially high, decreased. D(r,t) at later times ranged from 2.11 × 10⁻⁷ cm² s⁻¹ (parvalbumin) to 0.20 × 10⁻⁷ cm² s⁻¹ (phosphofructose kinase), values that are 3.6- to 12.3-fold lower than those predicted in bulk water. The low initial values are consistent with the presence of complexes in situ; the higher later values are consistent with molecular sieving and transient binding of dissociated proteins. Channeling of metabolic intermediates via enzyme complexes may enhance production of adenosine triphosphate at rates beyond that possible with randomly and/or sparsely distributed enzymes, thereby matching supply with demand.     

Functions of the spermathecal muscle of the boll weevil,Anthonomus grandis

In female boll weevils, Anthonomus grandis, spermathecal filling was not affected by severing the spermathecal muscles. Females whose spermathecal muscles were severed 2 to 4 weeks after mating laid infertile eggs and resumed virginal ovipositional behaviour indicating the importance of this muscle in supplying sperm for egg fertilization. The presence of normal active sperm within the spermatheca in no way influenced ovipositional behaviour. In females whose spermathecal muscles had been severed, 22 per cent sperm displacement occurred after a second mating compared with 66 per cent for normal females. The physical displacement of sperm was thus largely dependent on a functional spermathecal muscle.     

The Effect of Myofilament Compliance on Kinetics of Force Generation by Myosin Motors in Muscle

We use the inhibitor of isometric force of skeletal muscle N-benzyl-p-toluene sulfonamide (BTS) to decrease, in a dose dependent way, the number of myosin motors attached to actin during the steady isometric contraction of single fibers from frog skeletal muscle (4°C, 2.1 μm sarcomere length). In this way we can reduce the strain in the myofilament compliance during the isometric tetanus (T₀) from 3.54 nm in the control solution (T_0,NR) to ∼0.5 nm in 1 μM BTS, where T₀ is reduced to ∼0.15 T_0,NR. The quick force recovery after a step release (1-3 nm per half-sarcomere) becomes faster with the increase of BTS concentration and the decrease of T₀. The simulation of quick force recovery with a multistate model of force generation, that adapts Huxley and Simmons model to account for both the high stiffness of the myosin motor (∼3 pN/nm) and the myofilament compliance, shows that the increase in the rate of quick force recovery by BTS is explained by the reduced strain in the myofilaments, consequent to the decrease in half-sarcomere force. The model estimates that i), for the same half-sarcomere release the state transition kinetics in the myosin motor are five times faster in the absence of filament compliance than in the control; and ii), the rate of force recovery from zero to T₀ is ∼6000/s in the absence of filament compliance.     

Sodium, Potassium, and Chloride Fluxes in Intercostal Muscle from Normal Goats and Goats with Hereditary Myotonia

In isolated bundles of external intercostal muscle from normal goats and goats with hereditary myotonia the following were determined: concentrations and unidirectional fluxes of Na⁺, K⁺, and Cl^-, extracellular volume, water content, fiber geometry, and core-conductor constants. No significant difference between the two groups of preparations was found with respect to distribution of fiber size, intracellular concentrations of Na⁺ or Cl^-, fiber water, resting membrane potential, or overshoot of action potential. The intracellular Cl^- concentration in both groups of preparations was 4 to 7 times that expected if Cl^- were distributed passively between intracellular and extracellular water. The membrane permeability to K (P_K) calculated from efflux data was (a) at 38°C, 0.365 x 10^-6 cm sec^-1 for normal and 0.492 x 10^-6 for myotonic muscle, and (b) at 25°C, 0.219 x 10^-6 for normal and 0.199 x 10^-6 for myotonic muscle. From Cl^- washout curves of normal muscle usually only three exponential functions could be extracted, but in every experiment with myotonic muscle there was an additional, intermediate component. From these data PP_cl could be calculated; it was 0.413 x 10^-6 cm sec^-1 for myotonic fibers and was 0.815 x 10^-6 cm sec^-1 for normal fibers. The resting membrane resistance of myotonic fibers was 4 to 6 times greater than that of normal fibers.     

Viscous Dynamics of Lyme Disease and Syphilis Spirochetes Reveal Flagellar Torque and Drag

The spirochetes that cause Lyme disease (Borrelia burgdorferi) and syphilis (Treponema pallidum) swim through viscous fluids, such as blood and interstitial fluid, by undulating their bodies as traveling, planar waves. These undulations are driven by rotation of the flagella within the periplasmic space, the narrow (∼20–40 nm in width) compartment between the inner and outer membranes. We show here that the swimming speeds of B. burgdorferi and T. pallidum decrease with increases in viscosity of the external aqueous milieu, even though the flagella are entirely intracellular. We then use mathematical modeling to show that the measured changes in speed are consistent with the exertion of constant torque by the spirochetal flagellar motors. Comparison of simulations, experiments, and a simple model for power dissipation allows us to estimate the torque and resistive drag that act on the flagella of these major spirochetal pathogens.     

Monovalent Cationic Channel Activity in the Inner Membrane of Nuclei from Skeletal Muscle Fibers

Nuclear ion channels remain among the least studied and biophysically characterized channels. Although considerable progress has been made in characterizing calcium release channels in the nuclear membrane, very little is known regarding the properties of nuclear monovalent cationic channels. Here, we describe a method to isolate nuclei from adult skeletal muscle fibers that are suitable for electrophysiological experiments. Using this approach, we show for the first time, to our knowledge, that a nuclear monovalent cationic channel (NMCC) is prominently expressed in the inner membrane of nuclei isolated from flexor digitorum brevis skeletal muscle fibers of adult mice. In isotonic 140 mM KCl, the skeletal muscle NMCC exhibits a unitary conductance of ∼160 pS and high, voltage-independent open probability. Based on single-channel reversal potential measurements, NMCCs are slightly more permeable to potassium ions over sodium (P_K/P_Na = 2.68 ± 0.21) and cesium (P_K/P_Cs = 1.39 ± 0.03) ions. In addition, NMCCs do not permeate divalent cations, are inhibited by calcium ions, and demonstrate weak rectification in asymmetric Ca²⁺-containing solutions. Together, these studies characterize a voltage-independent NMCC in skeletal muscle, the properties of which are ideally suited to serve as a countercurrent mechanism during calcium release from the nuclear envelope.     

Orthovanadate and orthophosphate inhibit muscle force via two different pathways of the myosin ATPase cycle

Measurements of the half-sarcomere stiffness during activation of skinned fibers from rabbit psoas (sarcomere length 2.5 μm, temperature 12°C) indicate that addition of 0.1 mM orthovanadate (Vi) to the solution produces a drop to ∼1/2 in number of force-generating myosin motors, proportional to the drop in steady isometric force (T₀), an effect similar to that produced by the addition of 10 mM phosphate (Pi). However, in contrast to Pi, Vi does not change the rate of isometric force development. The depression of T₀ in a series of activations in presence of Vi is consistent with an apparent second-order rate constant of ∼1 × 10³ M⁻¹ s⁻¹. The rate constant of T₀ recovery in a series of activations after removal of Vi is 3.5 × 10⁻² s⁻¹. These results, together with the finding in the literature that the ATPase rate is reduced by Vi in proportion to isometric force, are reproduced with a kinetic model of the acto-myosin cross-bridge cycle where binding of Vi to the force-generating actomyosin-ADP state induces detachment from actin to form a stable myosin-ADP-Vi complex that is not able to complete the hydrolysis cycle and reenters the cycle only via reattachment to actin upon activation in Vi-free solution.     

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca²⁺ signaling is a fundamental physiological process in living cells. Ca²⁺ sparks are the elementary units of Ca²⁺ signaling in the striated muscle fibers that appear as highly localized Ca²⁺ release events mediated by ryanodine receptor (RyR) Ca²⁺ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca²⁺ sparks could provide information on the intracellular Ca²⁺ handling properties of healthy and diseased striated muscles. Although Ca²⁺ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.Detailed here is an experimental protocol for measuring Ca²⁺ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca²⁺ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca²⁺ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca²⁺ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP₃R) and RyR contributes to Ca²⁺ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca²⁺ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).     

Nanomechanics of Native Thick Filaments from Indirect Flight Muscles

During flight, the wings of Drosophila melanogaster beat nearly 200 times per second. The indirect flight muscle fibers that power this movement have evolved to resist the repetitive mechanical stress that results from the 5-ms wing beat cycle at a strain amplitude of 3.5%. In order to understand how this is achieved at the sarcomere level, we have analyzed the mechanical properties of native thick filaments isolated from indirect flight muscle. Single filaments adsorbed onto a solid support were manipulated in physiological buffer using an atomic force microscope. Images taken after the manipulation revealed that segments were stretched, on average, to 150%, with a maximum at 385% extension. The lateral-force-versus-displacement curve associated with each manipulation contained information about the bending and tensile properties of each filament. The bending process was dominated by shearing between myosin dimers and yielded a shear modulus between 3 and 13 MPa. Maximum tension along the stretched filaments was observed at ∼ 200% extension and varied between 8 and 17 nN. Based on current models of thick filament structure, these variations can be attributed to cross-links between myosin dimers distributed along the filament.     

Pre-Steady-State Kinetics of Ba-Ca Exchange Reveals a Second Electrogenic Step Involved in Ca Translocation by the Na-Ca Exchanger

Kinetic properties of the Na-Ca exchanger (guinea pig NCX1) expressed in Xenopus oocytes were investigated with excised membrane patches in the inside-out configuration and photolytic Ca²⁺ concentration jumps with either 5 mM extracellular Sr²⁺ or Ba²⁺. After a Ca²⁺ concentration jump on the cytoplasmic side, the exchanger performed Sr-Ca or Ba-Ca exchange. In the Sr-Ca mode, currents are transient and decay in a monoexponential manner similar to that of currents in the Ca-Ca exchange mode described before. Currents recorded in the Ba-Ca mode are also transient, but the decay is biphasic. In the Sr-Ca mode the amount of charge translocated increases at negative potentials in agreement with experiments performed in the Ca-Ca mode. In the Ba-Ca mode the total amount of charge translocated after a Ca²⁺ concentration jump is ∼4 to 5 times that in Ca-Ca or Sr-Ca mode. In the Ba-Ca mode the voltage dependence of charge translocation depends on the Ca²⁺ concentration on the cytosolic side before the Ca²⁺ concentration jump. At low initial Ca²⁺ levels (∼0.5 μM), charge translocation is voltage independent. At a higher initial concentration (1 μM Ca²⁺), the amount of charge translocated increases at positive potentials. Biphasic relaxation of the current was also observed in the Ca-Ca mode if the external Ca²⁺ concentration was reduced to ≤0.5 mM. The results reported here and in previous publications can be described by using a 6-state model with two voltage-dependent conformational transitions.     

Control of muscle degeneration following autotomy of a hindleg in the grasshopper,Barytettix humphreysii

When the grasshopper, Barrytettix humphreysii, sheds a hindlimb during autotomy, certain thoracic muscles degenerate although they are neither directly damaged nor denervated. Muscle degeneration is induced when a leg nerve (N5) that does not innervate the thoracic muscles is severed. Together these results suggest that transneuronal mechanisms influence muscle survival. To further characterize this autotomy-induced process, we studied the degeneration of a thoracic tergotrochanteral muscle (M#133b,c) following autotomy or experimental manipulation in adult animals. Its degeneration is correlated with reduced activity of its neural input and occurs by programmed cell death (PCD). PCD onset is variable between individual muscle fibers, indicating that the trigger of degeneration is fiber specific. Muscle degeneration appears to be triggered by the loss of proprioceptive input from the autotomized limb, since severing of axons from proprioceptive organs, but not exteroceptive chemo- or mechanoreceptors, leads to muscle degeneration. Muscle disuse, neuronal degeneration, or changes in juvenile hormone titer do not appear to play a role in autotomy-induced degeneration. We propose that the loss of proprioceptive input from proximal campaniform sensilla on the tibia deafferents the thoracic muscle motor neurons and leads to a decrease in their activity. Muscle degeneration is ultimately triggered by the loss of normal neural activity.     

Oxidative and Nitrosative Modifications of Tropoelastin Prevent Elastic Fiber Assembly in Vitro

Elastic fibers are extracellular structures that provide stretch and recoil properties of tissues, such as lungs, arteries, and skin. Elastin is the predominant component of elastic fibers. Tropoelastin (TE), the precursor of elastin, is synthesized mainly during late fetal and early postnatal stages. The turnover of elastin in normal adult tissues is minimal. However, in several pathological conditions often associated with inflammation and oxidative stress, elastogenesis is re-initiated, but newly synthesized elastic fibers appear abnormal. We sought to determine the effects of reactive oxygen and nitrogen species (ROS/RNS) on the assembly of TE into elastic fibers. Immunoblot analyses showed that TE is oxidatively and nitrosatively modified by peroxynitrite (ONOO⁻) and hypochlorous acid (HOCl) and by activated monocytes and macrophages via release of ONOO⁻ and HOCl. In an in vitro elastic fiber assembly model, oxidatively modified TE was unable to form elastic fibers. Oxidation of TE enhanced coacervation, an early step in elastic fiber assembly, but reduced cross-linking and interactions with other proteins required for elastic fiber assembly, including fibulin-4, fibulin-5, and fibrillin-2. These findings establish that ROS/RNS can modify TE and that these modifications affect the assembly of elastic fibers. Thus, we speculate that oxidative stress may contribute to the abnormal structure and function of elastic fibers in pathological conditions.     

Influence of ryanodine receptor agonist and antagonist on development of potassium contracture in phasic (twitch) and tonic fibers of frog skeletal muscle

We compared the influence of external calcium and the inhibitor (dantrolene) and activator (4-chloro-m-cresol) of ryanodine-sensitive Ca channels of the sarcoplasmic reticulum on the characteristics of potassium contracture in phasic and tonic frog skeletal muscle fibers. The duration of contracture in tonic fibers, as contrasted to the phasic ones, is not limited by the presence of Ca²⁺. The tonic contractile response is virtually indifferent to dantrolene and is much less sensitive to chlorocresol than the phasic one (1 mM vs. 0.25 mM). In phasic fibers, the K⁺ contracture on the chlorocresol background is quite similar in amplitude and dynamics to that in control, whereas tonic fibers exhibit response summation without relaxation upon removal of excessive K⁺. One can suggest that in phasic fibers the Ca²⁺ influx can directly create a level sufficient to sustain contraction, while in tonic fibers its effect is mediated by Ca-dependent activation of the beta isoform of the ryanodine-sensitive channel.     

Laser ablation reveals the impact of Cdc15p on the stiffness of the contractile ring

The mechanics that govern the constriction of the contractile ring remain poorly understood yet are critical to understanding the forces that drive cytokinesis. We used laser ablation in fission yeast cells to unravel these mechanics focusing on the role of Cdc15p as a putative anchoring protein. Our work shows that the severed constricting contractile ring recoils to a finite point leaving a gap that can heal if less than ∼1 µm. Severed contractile rings in Cdc15p-depleted cells exhibit an exaggerated recoil, which suggests that the recoil is limited by the anchoring of the ring to the plasma membrane. Based on a physical model of the severed contractile ring, we propose that Cdc15p impacts the stiffness of the contractile ring more than the viscous drag.     

Dissecting regional variations in stress fiber mechanics in living cells with laser nanosurgery

The ability of a cell to distribute contractile stresses across the extracellular matrix in a spatially heterogeneous fashion underlies many cellular behaviors, including motility and tissue assembly. Here we investigate the biophysical basis of this phenomenon by using femtosecond laser nanosurgery to measure the viscoelastic recoil and cell-shape contributions of contractile stress fibers (SFs) located in specific compartments of living cells. Upon photodisruption and recoil, myosin light chain kinase-dependent SFs located along the cell periphery display much lower effective elasticities and higher plateau retraction distances than Rho-associated kinase-dependent SFs located in the cell center, with severing of peripheral fibers uniquely triggering a dramatic contraction of the entire cell within minutes of fiber irradiation. Image correlation spectroscopy reveals that when one population of SFs is pharmacologically dissipated, actin density flows toward the other population. Furthermore, dissipation of peripheral fibers reduces the elasticity and increases the plateau retraction distance of central fibers, and severing central fibers under these conditions triggers cellular contraction. Together, these findings show that SFs regulated by different myosin activators exhibit different mechanical properties and cell shape contributions. They also suggest that some fibers can absorb components and assume mechanical roles of other fibers to stabilize cell shape.     

Orientation of the essential light chain region of myosin in relaxed, active, and rigor muscle

The orientation of the ELC region of myosin in skeletal muscle was determined by polarized fluorescence from ELC mutants in which pairs of introduced cysteines were cross-linked by BSR. The purified ELC-BSRs were exchanged for native ELC in demembranated fibers from rabbit psoas muscle using a trifluoperazine-based protocol that preserved fiber function. In the absence of MgATP (in rigor) the ELC orientation distribution was narrow; in terms of crystallographic structures of the myosin head, the LCD long axis linking heavy-chain residues 707 and 843 makes an angle (β) of 120-125° with the filament axis. This is ∼30° larger than the broader distribution determined previously from RLC probes, suggesting that, relative to crystallographic structures, the LCD is bent between its ELC and RLC regions in rigor muscle. The ELC orientation distribution in relaxed muscle had two broad peaks with β ∼70° and ∼110°, which may correspond to the two head regions of each myosin molecule, in contrast with the single broad distribution of the RLC region in relaxed muscle. During isometric contraction the ELC orientation distribution peaked at β ∼105°, similar to that determined previously for the RLC region.     

New actin-binding proteins from Dictyostelium discoideum

Dictyostelium discoideum contains a soluble actin-binding protein that caps actin filaments at their fast growing ends. The purified protein consists of two subunits with 34 kd and 32 kd apparent mol. wts. Like similar proteins from Acanthamoeba and bovine brain the capping protein from D. discoideum acts in a Ca²⁺ -independent manner. It lacks severing activity as indicated by its inability to disrupt the stress fibers and the microfilament network in detergent-extracted cells. Two actin-binding proteins from a plasma membrane-enriched fraction were labeled with [¹²⁵I]actin using a gel overlay technique. One of these proteins, with an apparent mol. wt. of 17 kd in SDS-polyacrylamide gels, has been purified from high-salt extracts, the other protein with an apparent mol. wt. of 31 kd has been purified from Triton X-100 extracted membranes. Monoclonal antibodies were raised against D. discoideum severin, α-actinin, the larger subunit of the capping protein, and the 17-kd membrane-associated protein. Immunoblotting of proteins from whole cell lysates showed that all these actin-binding proteins were present in both growth phase and aggregation-competent cells.     

Divalent cations permeation in a Ca2+ non-conducting skeletal muscle dihydropyridine receptor mouse model

In response to excitation of skeletal muscle fibers, trains of action potentials induce changes in the configuration of the dihydropyridine receptor (DHPR) anchored in the tubular membrane which opens the Ca²⁺ release channel in the sarcoplasmic reticulum membrane. The DHPR also functions as a voltage-gated Ca²⁺ channel that conducts L-type Ca²⁺ currents routinely recorded in mammalian muscle fibers, which role was debated for more than four decades. Recently, to allow a closer look into the role of DHPR Ca²⁺ influx in mammalian muscle, a knock-in (ki) mouse model (ncDHPR) carrying mutation N617D (adjacent to domain II selectivity filter E) in the DHPRα_1S subunit abolishing Ca²⁺ permeation through the channel was generated [Dayal et al., 2017]. In the present study, the Mn²⁺ quenching technique was initially intended to be used on voltage-clamped muscle fibers from this mouse to determine whether Ca²⁺ influx through a pathway distinct from DHPR may occur to compensate for the absence of DHPR Ca²⁺ influx. Surprisingly, while N617D DHPR muscle fibers of the ki mouse do not conduct Ca²⁺, Mn²⁺ entry and subsequent quenching did occur because Mn²⁺ was able to permeate and produce L-type currents through N617D DHPR. N617D DHPR was also found to conduct Ba²⁺ and Ba²⁺ currents were strongly blocked by external Ca²⁺. Ba²⁺ permeation was smaller, current kinetics slower and Ca²⁺ block more potent than in wild-type DHPR. These results indicate that residue N617 when replaced by the negatively charged residue D is suitably located at entrance of the pore to trap external Ca²⁺ impeding in this way permeation. Because Ba²⁺ binds with lower affinity to D, Ba²⁺ currents occur, but with reduced amplitudes as compared to Ba²⁺ currents through wild-type channels. We conclude that mutations located outside the selectivity filter influence channel permeation and possibly channel gating in a fully differentiated skeletal muscle environment.     

The drag and reconfiguration experienced by five macrophytes from a lowland river

Drag and flexibility of five macrophytes (fresh mass 3 g) collected from the same river were measured at velocities from 0 to 0.5 m s⁻¹ in a flume. Drag increased with increasing velocity for all five species examined. Sparganium emersum Rehmann, which has simple strap-like leaves experienced significantly less drag than the other, bushier species whilst there was no significant difference between the drag on Callitriche stagnalis Scop., Ranunculus penicillatus pseudofluitans (Syme) S.D. Webster, and Myriophyllum spicatum L. above 0.4 m s⁻¹. Potamogeton x zizii W.D.J. Koch ex Roth, which has large flat leaves, experienced significantly higher drag than all the other species. All the plants were very flexible but flexibility (as angle of bend) did not explain the drag experienced by the plants, e.g. S. emersum was the least flexible. The plants also changed shape and compressed (reconfigured) under increasing water velocity which reduced the rate at which drag increased. Reconfiguration capacity was assessed as E-values. There were no significant differences in E-values between species indicating that all the samples examined had a similar capacity to reconfigure. It is concluded that measurement of the drag experienced by plants is useful and may prove helpful in explaining the distribution of macrophytes in rivers.