K. J. V. Poole

Tomographic 3D Reconstruction of Quick-Frozen, Ca2+-Activated Contracting Insect Flight Muscle

Motor actions of myosin were directly visualized by electron tomography of insect flight muscle quick-frozen during contraction. In 3D images, active cross-bridges are usually single myosin heads, bound preferentially to actin target zones sited midway between troponins. Active attached bridges (approximately 30% of all heads) depart markedly in axial and azimuthal angles from Rayments rigor acto-S1 model, one-third requiring motor domain (MD) tilting on actin, and two-thirds keeping rigor contact with actin while the light chain domain (LCD) tilts axially from approximately 105 degrees to approximately 70 degrees. The results suggest the MD tilts and slews on actin from weak to strong binding, followed by swinging of the LCD through an approximately 35 degrees axial angle, giving an approximately 13 nm interaction distance and an approximately 4-6 nm working stroke.

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.

Time-Resolved Structural Studies on Insect Flight Muscle after Photolysis of Caged-ATP.

The time course of structural changes occurring on ATP-induced relaxation of glycerinated insect flight muscle from the rigor state has been investigated using synchrotron radiation as a source of high intensity x rays and photolysis of caged-ATP to produce a rapid rise in ATP concentration. Temporal resolutions of 1 ms for the strongest equatorial reflections and 5 ms for the 14.5 nm meridional reflection are attainable from single events (i.e., without averaging over several cycles). The equatorial intensity changes completely, the meridional intensity partially, towards their respective relaxed values on a much faster time scale than relaxation of tension. The results suggest that actively cycling bridges present shortly after ATP-release are either too few in number to be detected in the equatorial diffraction pattern or that their structure is different from that of rigor bridges.

Dynamic X-ray diffraction measurements following photolytic relaxation and activation of skinned rabbit psoas fibres

1) The ATP binding and crossbridge dissociation in muscle fibres is as fast as in solution, has a Q10 ca. 2-3, and is not measurably strain sensitive. 2) The final ADP release from the AM.ADP state achieved by adding ADP to rigor fibres must be greater than or equal to 69 sec-1 at 10 degrees C, and the combination of this rate and the ADP rebinding rate at 1 mM ADP limits the ATP induced crossbridge dissociation rate at greater than 2 mM ATP, but these kinetics were not strain sensitive. The strain sensitive steps must occur earlier on the attached pathway. 3) On activation, the equatorial changes thought to reflect crossbridge attachment are faster than tension production. The 10 intensity may change slightly ahead of the 11. This rate was not very temperature sensitive unlike the tension producing step in the mechanism. 4) The re-equilibration of equatorial intensity levels was much faster on activation from the rigor state than from the relaxed state. We conclude that crossbridges do not necessarily move far from the thin filaments when they detach in a fully activated thin filament system. 5) The 14.3 nm meridional intensity increases greater than 200% on fibre activation at 24 degrees C. The structural reorganisation of the heads responsible for this increase is associated with the tension generating step in the ATPase mechanism rather than the initial binding of bridges.

Time-resolved X-ray diffraction studies on stretch-activated insect flight muscle.

SummaryThe specific feature of stretch activation of the indirect flight muscle of the tropical waterbugLethocerus was used to correlate mechanical and structural aspects of muscle contraction. The time courses of the changes in intensities of the strongest equatorial reflections, the (10) and (20) and of the first meridional reflection at 14.5 nm−1 were monitored using synchrotron radiation as a high intensity X-ray source. The ratio of the intensities of the equatorial reflections, (I20/I10), which reflects the mass distribution within the filament lattice array, increases by about 10% relative to the Ca2+-activated level when a rapid stretch is imposed, compared with a 200% change seen when fibres change from the relaxed to the rigor state, while the spacing of the lattice planes decreases by about 1%. The intensity of the first meridional reflection at 14.5 nm−1 decreases by about 35% during stretch activation with a slightly faster time course than the delayed tension increase. The results suggest that the average structure of cycling crossbridges is different from that present in the rigor state.

Synchrotron Radiation Studies on Insect Flight Muscle

Insect flight muscle has proved particularly suitable for the structural investigation of the contractile proteins and their interactions. Here we review the low angle x-ray scattering experiments performed on this muscle using synchrotron radiation as a source. We briefly outline the present concensus on the cross-bridge mechanism of muscle contraction, and introduce insect muscle structure and muscle fibre diffraction. Details of x-ray beamline and camera configurations are considered with particular reference to the requirements necessary for small specimen work. A brief summary of the early experimental work on equilibrium or pseudoequilibrium states, such as that trapped by the ATP analogue AMP-PNP, is followed by a more detailed account of recent time-resolved experiments. These include measurements of the intensities of particular low-angle reflections during stretch activation and following the rapid, photolytic release of ATP from caged-ATP.