Trail Head Research Articles

Exploiting cryo-EM structures of actomyosin-5a to reveal the physical properties of its lever.

Myosin 5a (Myo5a) is a dimeric processive motor protein that transports cellular cargos along filamentous actin (F-actin). Its long lever is responsible for its large power-stroke, step size, and load-bearing ability. Little is known about the levers' structure and physical properties, and how they contribute to walking mechanics. Using cryoelectron microscopy (cryo-EM) and molecular dynamics (MD) simulations, we resolved the structure of monomeric Myo5a, comprising the motor domain and full-length lever, bound to F-actin. The range of its lever conformations revealed its physical properties, how stiffness varies along its length and predicts a large, 35nm, working stroke. Thus, the newly released trail head in a dimeric Myo5a would only need to perform a small diffusive search for its new binding site on F-actin, and stress would only be generated across the dimer once phosphate is released from the lead head, revealing new insight into the walking behavior of Myo5a.

Standardised inventories of spiders (Arachnida, Araneae) on touristic trails of the native forests of the Azores (Portugal).

BackgroundThe sharp increase in tourist visitation of the Azores Archipelago from 2015 onwards raised concerns about the impacts of recreational tourism on native habitats. In response, a project was financed by the Azorean Government to investigate the drivers of biodiversity erosion associated with recreational tourism. Here, we present the data on spider biodiversity found on trails located within the native Azorean forests as they are home to several endemic species of great conservation value. We applied an optimised and standardised sampling protocol (COBRA) in twenty-three plots located in five trails on Terceira and São Miguel Islands and assessed diversity and abundance of spider species at different distances from the trail head and the trail itself.New informationOf the 45 species (12435 specimens) collected, 13 were endemic to the Azores (9690 specimens), 10 native non-endemic (2047 specimens) and 22 introduced (698 specimens). This database will be the baseline of a long-term monitoring project for the assessment of touristic impacts on native forest trails. This methodology can also be used on other habitats and biogeograhical regions.

Biased Binding Mechanism Alone Can Explain the Preferential Forward Stepping of Kinesin-1

Kinesin-1 moves along microtubule by alternately moving two motor domains (“heads”); the unbound trailing head is prohibited from rebinding to the rear-binding site and is allowed to binds to the forward-binding site 16-nm ahead. The “neck linker-docking (or biased-diffusion) model” has been proposed to explain the preferential forward stepping, however this model cannot explain how the rebinding of the unbound head is prohibited while kinesin waits for ATP-binding. Alternate “biased-binding model” proposes that an unfavorable increase in the tension on the neck linker prohibits the unbound head from rebinding to the rear-binding site. To distinguish between these two models, we previously engineered an asymmetrically joined two-headed monomer, in which the neck linker of the first head (N-head) is connected to N-terminus of second head (C-head), such that this motor alternately uses biased-binding (when N-head steps forward) and biased-diffusion (when C-head steps) mechanisms to move in a hand-over-hand manner. In this study we observed the motion of each heads during the processive movement of the two-headed monomer using the total internal reflection-based dark-field microscopy with 50 µs temporal resolution. We found that the stepping of C-head was less efficient because the unbound C-head frequently rebound to the rear-binding site even at saturating ATP condition, while N-head stepped forward efficiently because the unbound N-head rarely showed rebinding under various ATP conditions. These results indicate that the diffusional-biased of the unbound head is not necessary, and that the biased-binding mechanism that prohibits rebinding of the unbound head is sufficient, for the preferential forward stepping of kinesin-1, at least under low external load.

Parsing the roles of neck-linker docking and tethered head diffusion in the stepping dynamics of kinesin

Kinesin walks processively on microtubules (MTs) in an asymmetric hand-over-hand manner consuming one ATP molecule per 16-nm step. The individual contributions due to docking of the approximately 13-residue neck linker to the leading head (deemed to be the power stroke) and diffusion of the trailing head (TH) that contributes in propelling the motor by 16 nm have not been quantified. We use molecular simulations by creating a coarse-grained model of the MT-kinesin complex, which reproduces the measured stall force as well as the force required to dislodge the motor head from the MT, to show that nearly three-quarters of the step occurs by bidirectional stochastic motion of the TH. However, docking of the neck linker to the leading head constrains the extent of diffusion and minimizes the probability that kinesin takes side steps, implying that both the events are necessary in the motility of kinesin and for the maintenance of processivity. Surprisingly, we find that during a single step, the TH stochastically hops multiple times between the geometrically accessible neighboring sites on the MT before forming a stable interaction with the target binding site with correct orientation between the motor head and the [Formula: see text] tubulin dimer.

Mechanical amplification mechanism of kinesin’s [formula omitted]-domain

Conventional kinesin’s force generation process always takes place on the leading head and the generated force is transmitted to the trailing head through two neck linkers. To guarantee a strong force to be transmitted to the trailing head so that it can be detached from microtubule surface, the neck linker of the leading head must have a large enough forward displacement, which is proposed to be achieved by the amplifying function of the β-domain. However, the experimental result shows that the forward displacement of the β-domain itself appears too small. To elucidate the function of the β-domain, we make a detailed analysis of the mechanical relationship between the two motor heads and, based on the results of molecular dynamics simulation and mechanical analysis, we calculate the forward displacement of the neck linker of the leading head during the ATP binding induced motor head rotation. We show that β-domain achieves its amplifying function together with β0, so that neck linker can have a forward displacement during motor head rotation. This displacement of neck linker is large enough to cause detachment of the trailing head. Based on these results, a possible initiation mechanism of neck linker docking is proposed.

Dissecting the Kinematics of the Kinesin Step

Kinesin walks processively on microtubules in an asymmetric hand-over-hand manner with each step spanning 16 nm. We used molecular simulations to determine the fraction of a single step due to conformational changes in the neck linker, and that due to diffusion of the tethered head. Stepping is determined largely by two energy scales, one favoring neck-linker docking and the other, ε(h)(MT-TH), between the trailing head (TH) and the microtubule. Neck-linker docking and an optimal value of ε(h)(MT-TH) are needed to minimize the probability that the TH takes side steps. There are three major stages in the kinematics of a step. In the first, the neck linker docks, resulting in ∼(5-6) nm movements of the trailing head. The TH moves an additional (6-8) nm in stage II by anisotropic translational diffusion. In the third stage, spanning ∼(3-4) nm, the step is complete with the TH binding to the αβ-tubulin binding site.

Simultaneous Observation of the Three-Dimensional Orientation and Position of a Single Fluorescent Probe

We have developed a new microscopy system for simultaneously measuring the three-dimensional orientation and position of a fluorescent probe based on the principal of polarization analysis. This method requires only that one collect fluorescence counts from a single fluorescent probe at four different polarizations such that only relatively simple optics and mathematical equations are required to yield the orientation. Orientations can be determined with less than 10° accuracy at 33ms time resolution except for a situation where a probe is nearly parallel to optical axis. We used the newly developed microscopy and quantum rods as our fluorescent probes to simultaneously measure myosin V movement along an actin filament and rotation around its own axis. Myosin V was seen to rotate 90° around its own axis for each step. From this result, we suggest that in the two-headed bound state the necks are perpendicular to one another, while in the one-headed bound state the detached trailing head is biased forward in part by rotating its lever arm about its own axis. This new microscopy system should have particular applicability to biophysical studies that investigate single molecule orientation dynamics.

富士登山者の満足度の登山口別比較

Mt. Fuji has 4 major paths to the top and most climbers use 4 trailheads located at the mid-slope of the mountain (5th step) presently. It goes without saying that climbers’ experience on 4 routes differs. In order to have climbers deeply satisfied with climbing experience, it is important to clarify the quality of experience and the attractiveness of route and to convey the information of them to potential climbers who have willingness to climb. This study aimed at evaluating the quality of climbing on the routes by using indicators of satisfaction level and employed a questionnaire survey. In concrete terms, the purpose of this study is 1) to grasp the characteristics of climbing on each route, 2) to evaluate the quality of climbing on the routes through climbers’ satisfaction classified by use of trail head and 3) to make clear the relationship between the attribute of climbers and the satisfaction level. As a result, satisfaction level with some expectations related to landscape from the route, rural beauty and solitude and others differed. In other words, differences of the quality of climbing existed on 4 routes at Mt. Fuji.

Influence of lever structure on myosin 5a walking

Using electron microscopy and image processing, we have observed myosin 5a modified with lever arms of different lengths (four, six, and eight calmodulin-binding IQ domains) and orientations walking along actin filaments. Step lengths were dependent on lever length: 8IQ > 6IQ > 4IQ, which is consistent with myosin 5a having evolved to walk straight along actin. Lead heads were mostly in the prepowerstroke state, tethered there by the trail head. However, improved image processing showed that in 5-10% of molecules the lead motor was in the postpowerstroke state. This is a unique attached state of myosin, where the motor domain has completed its powerstroke at the expense of severe lever distortion, but with little cargo movement. Postpowerstroke lead heads were seen in both wild-type and modified lever molecules, mostly where there was least strain. These data allow the strain dependence of the equilibrium between pre- and postpowerstroke conformations to be measured. Slow rates of ADP dissociation observed from lead heads of these molecules can be explained by the unfavorable equilibrium between the pre- and postpowerstroke conformations preceding ADP loss.

Myosin 5A Walking Mechanism: The Structural Basis of Slow ADP Dissociatin from the Lead Head

Using electron microscopy and image averaging, we have observed myosin 5a walking along actin filaments in the presence of low concentrations of ATP. Most molecules are attached with 13 actin subunits between heads but ∼10% are bound with 11 or 15 subunit spacings. Most lead heads are in the pre-powerstroke conformation, but some post-powerstroke lead heads are observed, especially at smaller separations where there is less strain in the myosin. Post-powerstroke lead heads have the converter at the front of the motor domain with its lever bent strongly backwards. Lead heads attached at the 13 subunit spacing are 98 % in pre-powerstroke state, tethered there by the trail head. However, heads spaced by 11 subunits are more evenly distributed (60:40) pre- to post-powerstroke. No post-powerstroke lead heads are seen in heads spaced by 15 actin subunits. These results are consistent with an energy difference of 10 kJ/mole between the pre- and post-power stroke conformations at 11 and 13 actin subunit separation. The post-powerstoke lead head is a new attached state of myosin: the motor domain has completed its powerstroke at the expense of severe lever distortion, but with little cargo movement. The rate of ADP dissociation from lead heads measured by stopped-flow fluorescence is >30 fold slower than from trail heads. The slower rate can be explained by a mechanism in which ADP only dissociates from the post-powerstroke state. ADP dissociation from the lead head is therefore inhibited by an unfavorable equilibrium between the pre-and post-powerstroke conformations. Supported by NIH EB00209.

Direct observation of the mechanochemical coupling in myosin Va during processive movement

Myosin Va transports intracellular cargoes along actin filaments in cells. This processive, two-headed motor takes multiple 36-nm steps in which the two heads swing forward alternately towards the barbed end of actin driven by ATP hydrolysis. The ability of myosin Va to move processively is a function of its long lever arm, the high duty ratio of its kinetic cycle and the gating of the kinetics between the two heads such that ADP release from the lead head is greatly retarded. Mechanical studies at the multiple- and the single-molecule level suggest that there is tight coupling (that is, one ATP is hydrolysed per power stroke), but this has not been directly demonstrated. We therefore investigated the coordination between the ATPase mechanism of the two heads of myosin Va and directly visualized the binding and dissociation of single fluorescently labelled nucleotide molecules, while simultaneously observing the stepping motion of the fluorescently labelled myosin Va as it moved along an actin filament. Here we show that preferential ADP dissociation from the trail head of mouse myosin Va is followed by ATP binding and a synchronous 36-nm step. Even at low ATP concentrations, the myosin Va molecule retained at least one nucleotide (ADP in the lead head position) when moving. Thus, we directly demonstrate tight coupling between myosin Va movement and the binding and dissociation of nucleotide by simultaneously imaging with near nanometre precision.

Myosin V Passing over Arp2/3 Junctions: Branching Ratio Calculated from the Elastic Lever Arm Model

Myosin V is a two-headed processive motor protein that walks in a hand-over-hand fashion along actin filaments. When it encounters a filament branch, formed by the Arp2/3 complex, it can either stay on the straight mother filament, or switch to the daughter filament. We study both probabilities using the elastic lever arm model for myosin V. We calculate the shapes and bending energies of all relevant configurations in which the trail head is bound to the actin filament before Arp2/3 and the lead head is bound either to the mother or to the daughter filament. Based on the assumption that the probability for a head to bind to a certain actin subunit is proportional to the Boltzmann factor obtained from the elastic energy, we calculate the mother/daughter filament branching ratio. Our model predicts a value of 27% for the daughter and 73% for the mother filament. This result is in good agreement with recent experimental data.

Kinetics of ADP Dissociation from the Trail and Lead Heads of Actomyosin V following the Power Stroke

Myosin V is a cellular motor protein, which transports cargos along actin filaments. It moves processively by 36-nm steps that require at least one of the two heads to be tightly bound to actin throughout the catalytic cycle. To elucidate the kinetic mechanism of processivity, we measured the rate of product release from the double-headed myosin V-HMM using a new ATP analogue, 3'-(7-diethylaminocoumarin-3-carbonylamino)-3'-deoxy-ATP (deac-aminoATP), which undergoes a 20-fold increase in fluorescence emission intensity when bound to the active site of myosin V (Forgacs, E., Cartwright, S., Kovács, M., Sakamoto, T., Sellers, J. R., Corrie, J. E. T., Webb, M. R., and White, H. D. (2006) Biochemistry 45, 13035-13045). The kinetics of ADP and deac-aminoADP dissociation from actomyosin V-HMM, following the power stroke, were determined using double-mixing stopped-flow fluorescence. These used either deac-aminoATP as the substrate with ADP or ATP chase or alternatively ATP as the substrate with either a deac-aminoADP or deac-aminoATP chase. Both sets of experiments show that the observed rate of ADP or deac-aminoADP dissociation from the trail head of actomyosin V-HMM is the same as from actomyosin V-S1. The dissociation of ADP from the lead head is decreased by up to 250-fold.

Within Sight

FEATURED AUTHOR—MEMOIR Within Sight Darnell Arnoult "What possessed him to ask you to do that?" my daughter Beth said when I called her to tell her the plan. "Is he out of his mind?" Beth immediately hung up the phone and called her brother at work in Delaware. "What possessed you to do that? Are you out of your mind?" "I know! I know!" Chad replied. "It just popped out and I couldn't take it back." It was late June, 1994. My son Chad, older than Beth by twenty-one months, had called me earlier at work. He was one month into his summer internship with MBNA in Delaware. He hadn't had time to stop in North Carolina for a visit on his way north from Rollins College in Winter Park, Florida, where he was a rising junior. He would have only a few days at the end of the internship before he needed to report back to Rollins. Both my children were athletes, abilities they inherited from their father. Chad had captained in football and baseball in high school and had just finished his second year on the Rollins baseball team. I was an overweight, out of shape, early middle-aged mother who had spent more than ten years sitting in the stands at nearly every athletic event my children participated in, cheering them on and developing bleacher hips. For fifteen years I'd been a single mother; my children were at the center of my life and they knew it. So Chad knew I would be crushed if he didn't stop to visit me on his way back to school. Chad also had a new found passion—backpacking. He really wanted to get in a good backpacking trip before school started, and he knew better than to go by himself. Chad had a dilemma. I'm still not sure what possessed Chad to ask me to come along on a four day hike in George Washington National Forest in Virginia. But mothers don't turn down such offers from adult sons. At least, this one didn't. The theme from Rocky started playing in my head. Chad instructed me to get my fanny on the stair-climbing machine every day until the hike, and to invest in a good pair ofhiking boots and startbreaking them in immediately. Veteran hikers in my office at Duke, once they asked me to repeat my good news so they could be sure they had heard me right, warned me about the importance of breaking in 34 those boots. I went right out and spent $150 on hiking boots and wore them every day, everywhere, with everything: dresses, skirts, culottes, pajamas, everything. I still wear those boots. I wasn't as diligent about using the Stair Master. Needless to say, I don't own it anymore. Like in an old movie, the calendar turned rapidly and the end of summer arrived in no time. I understood we were going into the woods at Harrisonburg. Chad stopped at a Waffle House pay phone near our trail head to call his grandmother and tell her for certain where we would enter the woods, in case there was a problem and someone had to come looking for us. She would then meet us at the same Waffle House when we came out four days later to take me back to my car, which I had left at her house in Bassett. Chad would go on to his father's house in Alexandria, for a brief visit, then head south to Rollins. While Chad talked on the pay phone, I looked around and realized we were in Staunton, Virginia. I hadn't been paying attention to the signs when we exited the interstate. Stunned, I recognized the boxy red brick rectangle buildings cast across the hillside across the road. It was the state mental hospital where my mother was hospitalized in 1963, when I was eight and she was forty—the same age I was that day, sitting in the Jeep outside the Waffle House in my hiking boots waiting for my son to lead me into the wilderness. The smell inside those...

Load-dependent kinetics of myosin-V can explain its high processivity

Recent studies provide strong evidence that single myosin class V molecules transport vesicles and organelles processively along F-actin, taking several 36-nm steps, 'hand over hand', for each diffusional encounter. The mechanisms regulating myosin-V's processivity remain unknown. Here, we have used an optical-tweezers-based transducer to measure the effect of load on the mechanical interactions between rabbit skeletal F-actin and a single head of mouse brain myosin-V, which produces its working stroke in two phases. We found that the lifetimes of the first phase of the working stroke changed exponentially and about 10-fold over a range of pushing and pulling forces of +/- 1.5 pN. Stiffness measurements suggest that intramolecular forces could approach 3.6 pN when both heads are bound to F-actin, in which case extrapolation would predict the detachment kinetics of the front head to slow down 50-fold and the kinetics of the rear head to accelerate respectively. This synchronizing effect on the chemo-mechanical cycles of the heads increases the probability of the trail head detaching first and causes a strong increase in the number of forward steps per diffusional encounter over a system with no strain dependence.

Elastic Lever-Arm Model for Myosin V

We present a mechanochemical model for myosin V, a two-headed processive motor protein. We derive the properties of a dimer from those of an individual head, which we model both with a four-state cycle (detached; attached with ADP.Pi; attached with ADP; and attached without nucleotide) and alternatively with a five-state cycle (where the powerstroke is not tightly coupled to the phosphate release). In each state the lever arm leaves the head at a different, but fixed, angle. The lever arm itself is described as an elastic rod. The chemical cycles of both heads are coordinated exclusively by the mechanical connection between the two lever arms. The model explains head coordination by showing that the lead head only binds to actin after the powerstroke in the trail head and that it only undergoes its powerstroke after the trail head unbinds from actin. Both models (four- and five-state) reproduce the observed hand-over-hand motion and fit the measured force-velocity relations. The main difference between the two models concerns the load dependence of the run length, which is much weaker in the five-state model. We show how systematic processivity measurement under varying conditions could be used to distinguish between both models and to determine the kinetic parameters.

Mechanochemical coupling of two substeps in a single myosin V motor.

Myosin V is a double-headed processive molecular motor that moves along an actin filament by taking 36-nm steps. Using optical trapping nanometry with high spatiotemporal resolution, we discovered that there are two possible pathways for the 36-nm steps, one with 12- and 24-nm substeps, in this order, and the other without substeps. Based on the analyses of effects of ATP, ADP and 2,3-butanedione 2-monoxime (a reagent shown here to slow ADP release from actomyosin V) on the dwell time and the occurrence frequency of the main and the intermediate states, we propose that the 12-nm substep occurs after ATP binding to the bound trailing head and the 24-nm substep results from a mechanical step following the isomerization of an actomyosin-ADP state on the bound leading head. When the isomerization precedes the 12-nm substep, the 36-nm step occurs without substeps.

What kinesin does at roadblocks: the coordination mechanism for molecular walking.

Competing models for the coordination of processive stepping in kinesin can be tested by introducing a roadblock to prevent lead head attachment. We used T93N, an irreversibly binding mutant monomer, as a roadblock, and measured the rates of nucleotide-induced detachment of kinesin monomers or dimers with and without the T93N roadblock using microflash photolysis combined with stopped flow. Control nucleotide-induced monomer (rK340) unbinding was 73.6 s(-1) for ATP and 40.5 s(-1) for ADP. Control ADP-induced dimer (rK430) unbinding was 18.6 s(-1). Added 20 mM Pi slowed both monomer and dimer unbinding. With the roadblock in place, lead head attachment of dimers is prevented and ATP-induced trail head unbinding was then 42 s(-1). This is less than two-fold slower than the stepping rate of unimpeded rK430 dimers (50-70 s(-1)), indicating that during walking, lead head attachment induces at most only a slight (less than two-fold) acceleration of trail head detachment. As we discuss, this implies a coordination model having very fast (>2000 s(-1)) ATP-induced attachment of the lead head, followed by slower, strain-sensitive ADP release from the lead head.

Whose West Is It Anyway? or, What's Myth Got to Do With It? The Role of “America” in the Creation of the Myth of the West

Little more than a year ago, was sitting in a darkened cineplex on a Saturday afternoon with four small boys (two of them mine) munching popcorn and drinking cherry slushes, waiting to see Shrek for the third time. Suddenly, on the screen appeared a trailer for an animated film due out last summer--a Dreamworks production called Spirit: Stallion of the Cimarron, directed by Kelly Asbury and Lorna Cook, with a screenplay by John Fusco, who has been credited with the retro Westerns, Young Guns and Young Guns II. trailer opens as a horse paws a cliff, before panning alongside an eagle flying over the plains, into steep canyons and mesas, finally tracking alongside a herd of wild horses running freely (with John Williams'-style music on the soundtrack). That's all the context we're given, with a voice-over reminding us of the countless stories told of how the was won, though never a story from the horse's point of view. I remember when the horses ran free ... this is that story. As the film's official website states: The movie features photo-realistic horses set against panoramic vistas and very little dialogue. whole story will be told from the viewpoint of the horse, with his thoughts heard as narrative. (1) producers were reputed to have searched far and wide for the supposedly perfect horse voice, finally settling on Matt Damon for his airy exuberance and innocent tone, though the site helpfully reminds us that horses don't actually talk, they just whine and make horse noises. Here is the rest of the website's description: Spirit: Stallion of the Cimarron follows the adventures of a wild and rambunctious mustang stallion as he journeys through the untamed American frontier. Encountering man for the first time, Spirit defies being broken, even as he develops a remarkable friendship with a young Lakota brave. courageous young stallion also finds love with a beautiful paint mare named Rain on his way to becoming one of the greatest unsung heroes of the Old West. Through the eyes of Spirit, we follow the story of the westward movement, as the mustang runs free across the American countryside and sees his life changed by the slow but inexorable push of civilization in the wild. film first introduces Spirit in the freedom of the wild, only to have him caught by the cavalry and trained to be a war horse. However grim this may seem as cartoon, the film offers only the most recent version of a recognizably American of the West, incorporating the idea of free land and spectacular landscapes; of a wild, untamed spirit resisting the encroachments and suasions of civilization; and finally of lost freedom as civilization advances and the claims of responsible citizenry are made. And while nothing about the materials per se precludes their being Canadian or Mexican, or for that matter Australian, Brazilian, or South African, something about the myth requires American citizens to imagine it as intrinsically American. In short, a recognizable of the West exists for America but not for Canada or Mexico; while am not an expert, the proximate causes for this are not hard to find, beginning with the fact that Canada continued much longer as a colonial dependency, as part of the British Empire. Or that the garden image of the American was more suitable for mythic appropriation, unlike the far bleaker, more severe, more terrifying landscapes of Mexico and Canada. Or that (notwithstanding the Spanish rodeo and the Calgary stampede) neither Mexico nor Canada had much experience of the nineteenth-century cattle trade, with those western activities that helped transform the cowboy into a mythic figure: the roundup and long drive, the battles with farmers and Indians, the drunken escapades and deadly confrontations in cow towns and trail heads. Popular culture has celebrated the cowboy, the mythic descendant of American frontiersmen, as an independent laborer posed against the industrial working stiff, ever a stranger to the factory floor, unsupervised during the working day, free to roam on horseback with a six-gun at his hip (the only worker actually allowed to do so). …

The prepower stroke conformation of myosin V.

eW have used electron microscopy and single-particle image processing to study head conformation in myosin V molecules. We find that in the presence of ATP, many heads have a sharply angled conformation that is rare in its absence. The sharply angled conformation is similar to a myosin II atomic structure proposed to mimic the prepower stroke state. The leading head in molecules attached to actin by both heads has a similar conformation, but is also sharply angled in a second plane by tethering through the trail head. The lead head lever joins the motor domain ∼5 nm axially from where it joins the trail motor. These positions locate the converter subdomain and show the lead motor is in the prepower stroke conformation. Tethering by the trail head places the lead head motor domain at the correct axial position along the actin for binding, but at the wrong orientation. Attachment is achieved either by bending the lead head lever throughout its length or at the pliant point. The microscopy shows that most of the walking stride is produced by changes in lever angle brought about by converter movement, but is augmented by distortion produced by thermal energy.