Insect flight muscle (IFM) is the preferred model system for visualizing actin-myosin interactions due to its highly ordered lattice of actin and myosin filaments. Electron tomography
(ET) of fast-frozen, actively contracting Lethocerus IFM has recently resulted in a model for the weak to strong transition in the myosin crossbridges that produce force (Wu et al., 2010).
These myosin molecules consist of a motor domain (MD), a lever arm and a coiled-coil rod domain that forms the filament backbone. The MD and lever arm region together constitute the
subfragment 1 (S1) domain. The MD contains the ATP catalytic site and the actin-binding site. The myosin lever arm contains the essential and regulatory light chain bound to a long alpha
helix. The first 50 nm of the rod domain consists of the subfragment 2 (S2) region, which acts both as a linker and adapter to transmit force produced by the MD and is sufficient to form the
myosin dimer. Atomic models of myosin heads remodeled to fit the cross-bridge density show a distinctly straightened appearance when compared to the crystal structures of myosin subfragment 1
(S1). This implies that there is an aspect of the structural changes that occurs in force production that has not been recognized in myosin crystal structures of various intermediates. A
weak-to-strong binding transition involving an azimuthal reorientation of the myosin MD on actin could explain this observation provided that myosin's α-helical coiled-coil S2 domain emerged
from the thick filament backbone at a particular location. Previous studies did not visualize the S2 domain in either the raw tomogram or in subvolume averages. Here we have used ET of IFM
fibers in rigor, in which the filament lattice has been swollen in low ionic strength buffer, to view where S2 emerges from the thick filament backbone as a test of the weak to strong
transition. The results show that the S2 origins of those rigor myosin heads bound to the target zone of active muscle originate from the same region of the thick filament as implied by the
position of the S1/S2 junction observed in active muscle. This shows the myosin heads in clear agreement with the previously proposed weak to strong transition model. In order to visualize
IFM by ET, crossbridge samples must be sectioned because they are otherwise too thick. Sample preparation methods include fixation and embedding followed by sectioning and staining. These
preparation steps can potentially induce artifacts. The most notable sectioning artifacts are compression and shearing of the specimen. We examined ~80 nm thick transverse sections
(cross-sections) cut with both a vibrating knife (sonic-knife) and static knife and explored different knife settings. We examined the mitigating effects of these sectioning parameters on
both compression during cutting and shear distortions on the filaments as seen in tomograms of rigor muscle swollen in low ionic strength medium. Separate from specimen preparation
challenges, data collection also presents with a set of artifacts. Mass loss in plastic sections in conventional ET can reduce section thickness by as much as 30%. We evaluated the benefit of
collecting tilt series at -190°C with < 60 e⁻/Å2 total exposure, a value that is 50% of the dose typically used in cryo-ET for frozen hydrated specimen, in order to minimize
radiation-induced mass loss that results in section thinning. Reducing the artifacts in our sample facilitated reconstruction of the IFM lattice making it possible to probe aspects of muscle
contraction in greater detail than previously possible. ET is most useful for imaging biological structures in situ. A sample with reduced artifacts opens the door for better reconstructions.
We used subvolume averages of both thin and thick filaments to reassemble the filament lattice with high signal-to-noise ratio averages. We used the improved samples with the intent of
resolving the subfilament structures in the thick filament backbones as well as the subunit structure in the actin thin filaments. The information obtained from these averages provided two
separate frames of reference for deciphering the relationship between crossbridge origins on the thick filament with crossbridge binding sites on actin. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester, 2014. / October 27, 2014. / Actin, Insect Flight Muscle, Lethocerus, Myosin, Thick filament, Thin filament / Includes bibliographical references. / Kenneth A. Taylor, Professor Directing Dissertation; Timothy Logan, University Representative; P. Bryant Chase, Committee Member; Thomas C. S. Keller,
Committee Member; Scott Stagg, Committee Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_252796 |
| Contributors | Arakelian, Claudia (authoraut), Taylor, Kenneth A. (professor directing dissertation), Logan, Timothy M., 1961- (university representative), Chase, P. Bryant (committee member), Keller, Thomas C. S. (committee member), Stagg, Scott (committee member), Florida State University (degree granting institution), College of Arts and Sciences (degree granting college), Department of Biological Science (degree granting department) |
| Publisher | Florida State University, Florida State University |
| Source Sets | Florida State University |
| Language | English, English |
| Detected Language | English |
| Type | Text, text |
| Format | 1 online resource (130 pages), computer, application/pdf |
| Rights | This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). The copyright in theses and dissertations completed at Florida State University is held by the students who author them. |
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