Tom Irving

The myosin motor in muscle generates a smaller and slower working stroke at higher load

Muscle contraction is driven by the motor protein myosin II, which binds transiently to an actin filament, generates a unitary filament displacement or ‘working stroke’, then detaches and repeats the cycle. The stroke size has been measured previously using isolated myosin II molecules at low load, with rather variable results, but not at the higher loads that the motor works against during muscle contraction. Here we used a novel X-ray-interference technique to measure the working stroke of myosin II at constant load in an intact muscle cell, preserving the native structure and function of the motor. We show that the stroke is smaller and slower at higher load. The stroke size at low load is likely to be set by a structural limit; at higher loads, the motor detaches from actin before reaching this limit. The load dependence of the myosin II stroke is the primary molecular determinant of the mechanical performance and efficiency of skeletal muscle.

The BioCAT undulator beamline 18ID: a facility for biological non-crystalline diffraction and X-ray absorption spectroscopy at the Advanced Photon Source.

The 18ID undulator beamline of the Biophysics Collaborative Access Team at the Advanced Photon Source, Argonne, IL, USA, is a high-performance instrument designed for, and dedicated to, the study of partially ordered and disordered biological materials using the techniques of small-angle X-ray scattering, fiber diffraction, and X-ray absorption spectroscopy. The beamline and associated instrumentation are described in detail and examples of the representative experimental results are presented.

X-Ray Diffraction Indicates That Active Cross-Bridges Bind to Actin Target Zones in Insect Flight Muscle

We report the first time-resolved study of the two-dimensional x-ray diffraction pattern during active contraction in insect flight muscle (IFM). Activation of demembranated Lethocerus IFM was triggered by 1.5-2.5% step stretches (risetime 10 ms; held for 1.5 s) giving delayed active tension that peaked at 100-200 ms. Bundles of 8-12 fibers were stretch-activated on SRS synchrotron x-ray beamline 16.1, and time-resolved changes in diffraction were monitored with a SRS 2-D multiwire detector. As active tension rose, the 14.5- and 7.2-nm meridionals fell, the first row line dropped at the 38.7 nm layer line while gaining a new peak at 19.3 nm, and three outer peaks on the 38.7-nm layer line rose. The first row line changes suggest restricted binding of active myosin heads to the helically preferred region in each actin target zone, where, in rigor, two-headed lead bridges bind, midway between troponin bulges that repeat every 38.7 nm. Halving this troponin repeat by binding of single active heads explains the intensity rise at 19.3 nm being coupled to a loss at 38.7 nm. The meridional changes signal movement of at least 30% of all myosin heads away from their axially ordered positions on the myosin helix. The 38.7- and 19.3-nm layer line changes signal stereoselective attachment of 7-23% of the myosin heads to the actin helix, although with too little ordering at 6-nm resolution to affect the 5.9-nm actin layer line. We conclude that stretch-activated tension of IFM is produced by cross-bridges that bind to rigors lead-bridge target zones, comprising < or = 1/3 of the 75-80% that attach in rigor.

In Vivo X-Ray Diffraction of Indirect Flight Muscle from Drosophila melanogaster

Small-angle x-ray diffraction from isolated muscle preparations is commonly used to obtain time-resolved structural information during contraction. We extended this technique to the thoracic flight muscles of living fruit flies, at rest and during tethered flight. Precise measurements at 1-ms time resolution indicate that the myofilament lattice spacing does not change significantly during oscillatory contraction. This result is consistent with the notion that a net radial force maintains the thick filaments at an equilibrium interfilament spacing of approximately 56 nm throughout the contractile cycle. Transgenic flies with amino-acid substitutions in the conserved phosphorylation site of the myosin regulatory light chain (RLC) exhibit structural abnormalities that can explain their flight impairment. The I(20)/I(10) equatorial intensity ratio of the mutant fly is 35% less than that of wild type, supporting the hypothesis that myosin heads that lack phosphorylated RLC remain close to the thick filament backbone. This new experimental system facilitates investigation of the relation between molecular structure and muscle function in living organisms.

Reverse actin sliding triggers strong myosin binding that moves tropomyosin

Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the “steric blocking” mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca2+ with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence that TM in insect flight muscle (IFM) moves in a manner consistent with the steric blocking mechanism. We find that both isometric contraction, at high [Ca2+], and stretch activation, at lower [Ca2+], develop similarly high x-ray intensities on the IFM fourth actin layer line because of TM movement, coinciding with x-ray signals of strong-binding cross-bridge attachment to helically favored “actin target zones.” Vanadate (Vi), a phosphate analog that inhibits active cross-bridge cycling, abolishes all active force in IFM, allowing high [Ca2+] to elicit initial TM movement without cross-bridge attachment or other changes from relaxed structure. However, when stretched in high [Ca2+], Vi-“paralyzed” fibers produce force substantially above passive response at pCa ∼ 9, concurrent with full conversion from resting to active x-ray pattern, including x-ray signals of cross-bridge strong-binding and TM movement. This argues that myosin heads can be recruited as strong-binding “brakes” by backward-sliding, calcium-activated thin filaments, and are as effective in moving TM as actively force-producing cross-bridges. Such recruitment of myosin as brakes may be the major mechanism resisting extension during lengthening contractions.

Sarcomeric dysfunction contributes to muscle weakness in facioscapulohumeral muscular dystrophy

Objective: To investigate whether sarcomeric dysfunction contributes to muscle weakness in facioscapulohumeral muscular dystrophy (FSHD). Methods: Sarcomeric function was evaluated by contractile studies on demembranated single muscle fibers obtained from quadriceps muscle biopsies of 4 patients with FSHD and 4 healthy controls. The sarcomere length dependency of force was determined together with measurements of thin filament length using immunofluorescence confocal scanning laser microscopy. X-ray diffraction techniques were used to study myofilament lattice spacing. Results: FSHD muscle fibers produced only 70% of active force compared to healthy controls, a reduction which was exclusive to type II muscle fibers. Changes in force were not due to changes in thin filament length or sarcomere length. Passive force was increased 5- to 12-fold in both fiber types, with increased calcium sensitivity of force generation and decreased myofilament lattice spacing, indicating compensation by the sarcomeric protein titin. Conclusions: We have demonstrated a reduction in sarcomeric force in type II FSHD muscle fibers, and suggest compensatory mechanisms through titin stiffening. Based on these findings, we propose that sarcomeric dysfunction plays a critical role in the development of muscle weakness in FSHD.

Structure‐Function Relation of the Myosin Motor in Striated Muscle

Abstract: Force and shortening in striated muscle are driven by a structural working stroke in the globular portion of the myosin molecules—the myosin head—that cross‐links the myosin‐containing filaments and the actin‐containing filaments. We use time‐resolved X‐ray diffraction in single fibers from frog skeletal muscle to link the conformational changes in the myosin head determined at atomic resolution in crystallographic studies with the kinetic and mechanical features of the molecular motor in the preserved sarcomeric structure. Our approach exploits the improved brightness and collimation of the X‐ray beams of the third generation synchrotrons by using X‐ray interference between the two arrays of myosin heads in each bipolar myosin filament to measure with Å sensitivity the axial motions of myosin heads in situ during the synchronous execution of the working stroke elicited by rapid decreases in length or load imposed during an active isometric contraction. Changes in the intensity and interference‐fine structure of the axial X‐ray reflections following the mechanical perturbation allowed to establish the average conformation of the myosin heads during the active isometric contraction and the extent of tilt during the elastic response and during the subsequent working stroke. The myosin working stroke is 12 nm at low loads, which is consistent with crystallographic studies, while it is smaller and slower at higher loads. The load dependence of the size and speed of the myosin working stroke is the molecular determinant of the macroscopic performance and efficiency of muscle.

Fast-scanning high-flux microprobe for biological X-ray fluorescence microscopy and microXAS

There is a growing interest in the biomedical community in obtaining information concerning the distribution and local chemical environment of metals in tissues and cells. Recently, biological X-ray fluorescence microscopy (XFM) has emerged as the tool of choice to address these questions. A fast-scanning high-flux X-ray microprobe, built around a recently commissioned pair of 200 mm-long Rh-coated silicon Kirkpatrick-Baez mirrors, has been constructed at BioCAT beamline 18ID at the Advanced Photon Source. The new optical system delivers a flux of 1.3 x 10(12) photons s(-1) into a minimum focal spot size of approximately 3-5 microm FWHM. A set of Si drift detectors and bent Laue crystal analyzers may be used in combination with standard ionization chambers for X-ray fluorescence measurements. BioCATs scanning software allows fast continuous scans to be performed while acquiring and storing full multichannel analyzer spectra per pixel on-the-fly with minimal overhead time (<20 ms per pixel). Together, the high-flux X-ray microbeam and the rapid-scanning capabilities of the BioCAT beamline allow the collection of XFM and micro X-ray absorption spectroscopy (microXAS) measurements from as many as 48 tissue sections per day. This paper reports the commissioning results of the new instrument with representative XFM and microXAS results from tissue samples.

X-Ray Interference Evidence Concerning the Range of Crossbridge Movement, and Backbone Contributions to the Meridional Pattern

Interference fringes on the 14.5 nm meridional reflection (M3) are generated by diffraction from the arrays of myosin crossbridges in the two halves of each thick filament. The thick filaments are all constructed -in identical fashion (or nearly so), and all have H-zones of the same width, so that the interference distance between the two diffracting arrays in each filament is the same, and so is the pattern each gives. The separation between the fringes is inversely proportional to this interference distance (about 900 nm) so that a very high-resolution camera is necessary to resolve them. At the M3 reflection, one is seeing fringes of approximately the 62nd order, so that small changes in interference distance are magnified by this factor, and, for instance, a 1% change in distance will shift the position of the fringes by more than one half a fringe width.

Spacing Changes in the Actin and Myosin Filaments during Activation, and Their Implications

Small but important axial spacing changes have been found to occur in the X-ray reflections from both the actin and myosin filaments when a muscle changes from the resting state to a state of isometric contraction1,2. The overall spacing change in the actin is an increase of approximately 0.25–0.30%, measured on the first actin meridional reflection at 27.3 A. This implies a length increase of approximately 2.5–3.0 nm in the actin filaments in each half-sarcomere. This is an appreciable fraction of the total compliance measured in quick release experiments3, and therefore has a considerable effect on their interpretation.