Electron tomography of fast frozen, stretched rigor fibers reveals elastic distortions in the myosin crossbridges

Li, Jun; Reedy, Mary C.; Goldman, Yale E.; Franzini‐Armstrong, Clara; Sasaki, Hiroyuki; Tregear, R. T.; Lucaveche, Carmen; Winkler, Hanspeter; Baumann, Bruce A.J.; Squire, John M.; Irving, Thomas C.; Reedy, Michael K.; Taylor, Kenneth A.

doi:10.1016/j.jsb.2004.03.008

Cited by 48 publications

(58 citation statements)

References 48 publications

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“…This suggests that the rod-like tail of troponin, which we now know to consist mainly of TnT1, is positioned closer to the M-band than the Z-band. A similar interpretation of troponin polarity can also be inferred from the shape of the thin filament in an electron tomogram of fast frozen stretched rigor insect flight muscle fibers (47). We consider below whether this reverse polarity, as observed in our reconstruction, can be reconciled with the earlier observations.…”

Section: Discussionsupporting

confidence: 81%

Structure and Orientation of Troponin in the Thin Filament

Paul

Morris

Kensler

et al. 2009

Journal of Biological Chemistry

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The troponin complex on the thin filament plays a crucial role in the regulation of muscle contraction. However, the precise location of troponin relative to actin and tropomyosin remains uncertain. We have developed a method of reconstructing thin filaments using single particle analysis that does not impose the helical symmetry of actin and is independent of a starting model. We present a single particle three-dimensional reconstruction of the thin filament. Atomic models of the F-actin filament were fitted into the electron density maps and troponin and tropomyosin located. The structure provides evidence that the globular head region of troponin labels the two strands of actin with a 27.5-Å axial stagger. The density attributed to troponin appears tapered with the widest point toward the barbed end. This leads us to interpret the polarity of the troponin complex in the thin filament as reversed with respect to the widely accepted model.Regulation of actin filament function is a fundamental biological process with implications ranging from cell migration to muscle contraction. Skeletal and cardiac muscle thin filaments consist of actin and the regulatory proteins troponin and tropomyosin. Contraction is initiated by release of Ca 2ϩ into the sarcomere and the consequent binding of Ca 2ϩ to regulatory sites on troponin. Troponin is believed to undergo a conformational change leading to an azimuthal movement of tropomyosin, which allows myosin heads to interact with actin, hydrolyze ATP, and generate force. The molecular basis by which troponin acts to regulate muscle contraction is only partly understood. It is essential that the structure of troponin in the thin filament at high and low Ca 2ϩ is determined to properly understand the mechanism of regulation.The basic structure of the thin filament was described by Ebashi in 1972 (1). In this structure each tropomyosin molecule covers seven actin monomers, and there is a 27.5-Å stagger between troponin molecules. The 7-Å tropomyosin structure (2), the atomic model of F-actin (3), and the troponin "core domain" (4) have recently been used to generate atomic models of the thin filament in low and high Ca 2ϩ states (5). While the position of troponin in these models was constrained by known distance measurements between filament components, the exact arrangement of the complex on the filament has not been determined a priori. Although recently published crystal structures of partial troponin complexes (4, 6) have provided valuable insights into the arrangement of the globular head or core domain, the complex in its entirety has not been crystallized.Troponin is believed to consist of a globular core domain with an extended tail (7). The globular core contains the Ca 2ϩ -binding subunit (TnC), 2 the inhibitory subunit (TnI), and the C-terminal part (residues 156 -262) of the tropomyosin-binding subunit (TnT). The extended tail consists of the N-terminal part of TnT (residues 1-155). A structural rearrangement associated with Ca 2ϩ dissociation from the troponin cor...

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Section: Discussionsupporting

confidence: 81%

Structure and Orientation of Troponin in the Thin Filament

Paul

Morris

Kensler

et al. 2009

Journal of Biological Chemistry

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“…Phase 1 is characterized by an increase in force. As the attached cross-bridges and myofilaments are extended, they resist against the elastic distortion [22]. During phase 2, a drop in force occurs because some crossbridges are detached [13].…”

Section: Discussionmentioning

confidence: 99%

“…Single muscle fibers appear to be more compliant in older men. The decrease in ΔT 1 can only be caused by a higher total compliance of the attached crossbridges and myofilaments [22]. This higher compliance can be due to either a lower number of strongly bound actin-myosin cross-bridges with the same compliance per cross-bridge and myofilaments or the same number of strongly bound actin-myosin cross-bridges with a lower compliance per cross-bridge and myofilaments.…”

Section: Discussionmentioning

confidence: 99%

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Single skeletal muscle fiber behavior after a quick stretch in young and older men: a possible explanation of the relative preservation of eccentric force in old age

Ochala

Dorer

Frontera

et al. 2006

Pflugers Arch - Eur J Physiol

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The origins of the smaller age-related decrease in eccentric force compared to isometric and concentric conditions in vivo remain unclear. Could this originate from contractile elements of muscle cells? The main intent of the current investigation was to assess the force behavior of muscle cells with aging, during lengthening. Chemically skinned single muscle fibers (n=235) from m. vastus lateralis of six young (mean age 31.6 years) and six older men (mean age 66.1 years) were maximally activated with pCa 4.5 at 15 degrees C. Maximal isometric force and cross-sectional area were measured allowing the calculation of the tension (T (0)). A quick stretch (2 nm per half-sarcomere length) was applied and caused an immediate increase in tension followed by a decrease and a secondary delayed and transient rise in tension (phase 3); finally, the tension recovered a steady state value (phase 4). The tension enhancements during phase 3 (DeltaT (3)) and phase 4 (DeltaT (4)) were evaluated. The myosin heavy-chain isoform composition of each single fiber was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. DeltaT (3) and DeltaT (4) were preserved in older men for both type I and IIa fibers despite a reduction in T (0). Therefore, the age-related preservation of the tension increments after a quick stretch in single muscle fibers could explain in part the smaller decrease in force during eccentric contractions compared to isometric and concentric conditions in vivo with aging usually observed.

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“…All classes have a pronounced headpiece and weaker leg piece. Thirteen classes show the membrane clearly (23,29,43,44,47,49,(51)(52)(53)(54)(55)(56) and 59) while another seven (9,11,12,35,36,42, and 57) showed some weak membrane density ( Figure 5A). (The complete set of 60 class averages can be seen in Supplemental Figure S2.)…”

Section: Molecular Images Of Membrane Incorporated Integrinsmentioning

confidence: 99%

Integrin αIIbβ3 in a Membrane Environment Remains the Same Height after Mn2+ Activation when Observed by Cryoelectron Tomography

Winkler

et al. 2008

Journal of Molecular Biology

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SummaryIntegrins perform the critical function of signalling cell attachment to the extracellular matrix or to other cells. This signalling is done through a structural change propagated bidirectionally across the plasma membrane. Integrin activation has been extensively studied with ectodomain constructs, but the structural change within intact, membrane-bound molecules remains a subject of live debate. Using cryoelectron tomography, we examined the simplest predication of the different integrin activation models, i.e. the change in height of the molecules. Analysis using techniques that compensate for the missing wedge during alignment and averaging and that search for patterns in the structure of the aligned molecular subvolumes extracted from the tomogram reveals that the vast majority of molecules show no dramatic height change upon Mn 2+ induced activation of membrane bound integrins when compared with an inactive integrin control group. Thus, the result is inconsistent with the switchblade activation model.

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Electron tomography of fast frozen, stretched rigor fibers reveals elastic distortions in the myosin crossbridges

Cited by 48 publications

References 48 publications

Structure and Orientation of Troponin in the Thin Filament

Structure and Orientation of Troponin in the Thin Filament

Single skeletal muscle fiber behavior after a quick stretch in young and older men: a possible explanation of the relative preservation of eccentric force in old age

Integrin αIIbβ3 in a Membrane Environment Remains the Same Height after Mn2+ Activation when Observed by Cryoelectron Tomography

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