Electron microscopy is an important technique for observing macromolecular structures, such as DNA and viruses, which would be
too small to see under light microscopy. This type of microscopy utilizes electrons as its illumination source, produced by an electron
gun, to generate an image of the specimen which is captured by either a charged coupled device (CCD) or direct electron detector (DED)
camera. Specimens in electron microscopy can either be stained with heavy atom, embedded in plastic, or embedded in vitrified ice.
Cryo-electron microscopy (cryo-EM) embeds specimens in a vitreous ice layer that resembles the specimen’s natural environment and
increases the overall resolution of the specimen. In conjunction with cryo-electron tomography (cryo-ET), cryo-EM specimens can be tilted
on a specimen holder to collect multiple 2D views in order to generate a 3D reconstruction through a weighted back-projection algorithm.
The projections are first corrected to counter the effects of contrast transfer function (CTF), which can filter out high resolution
information. The resulting tomogram undergoes cycles of image processing steps such as multivariate data analysis, classification, and
subvolume averaging to bring out the features of the specimen. At the borders of the striated muscle sarcomere, there exists an electron
dense structure called the Z-Disc. The arrangement of thin filaments in the Z-Disc differs between vertebrates and invertebrates. Z-Discs
of vertebrates have a tetragonal lattice that contrasts with the hexagonal lattice seen in the A-Band, which might be caused by
arrangement of an elastic protein named Titin from the A-Band to the Z-Disc. The tetragonal lattice in the vertebrate Z-Disc has two
structural states, small-square and basket-weave, depending on the contraction state of the muscle. Invertebrate Z-Discs have a hexagonal
lattice that contains five connecting densities that form large and small solvent channels. Z-Discs contain many proteins that are
important for structural stability and signaling functions. Three Z-Disc proteins that are structurally important in invertebrate Z-Discs
are α-actinin, an actin crosslinker, Kettin and Projectin, the latter being components of the elastic connecting filament. Alternative
Z-Disc isolation methods were explored using Wild-type (WT) Drosophila and Sls-RNAi knockdown Drosophila for the possibility of using the
specimens for cryo-EM. The insect flight muscle (IFM) was dissected from the thorax of WT Drosophila and the individual myofibrils were
obtained through a homogenization and cleaning process. The Z-Discs were isolated from the myofibrils by exposing them to high salt
buffers, to remove thick filaments, and gelsolin, to remove thin filaments. The isolated Z-Discs were negatively stained and observed
under an electron microscope. The lattice arrangement of the thin filaments could not be seen due to the stain, but this method produced
many Z-Discs on the EM grids. Cryo-EM samples of the isolated WT Drosophila Z-Discs could not be obtained due to problems pertaining to
the plunge freezing method. Sls-RNAi Drosophila was obtained using the GAL4/UAS method to generate smaller Z-Discs and decrease the width
of the myofibrils due to a decrease in the presence of Kettin. The IFM was extracted from the thorax, but the myofibrils were not exposed
to high salt buffers and gelsolin. Under negative stain, the myofibrils observed produced Z-Discs about 500 nm in width, which is ideal
for cryo-ET conditions. However, a width of 200-300 nm would produce higher resolution images for a 3D reconstruction. A cryo-EM 3D
reconstruction was generated from isolated Lethocerus Z-Discs to confirm the structural features seen in plastic-embedded sections of
Apis. Projections for cryo-ET were collected using a DED camera and underwent CTF correction. The tilt series images were coarse-aligned
and went through cycles of refinement using an Appion-based database with Protomo. A 3D reconstruction was generated using a weighted
back-projection algorithm, filtered to bring out structural features (subvolumes), and then the subvolumes were averaged through single-
and multi-reference alignment. The results were visualized in CHIMERA which confirmed the hexagonal lattice arrangement of thin filaments
as reported previously in Apis Z-Discs. The location of connecting densities, C1 and C2, were confirmed as forming apices and bases of the
large solvent channel, while C3 and C5 were confirmed to be connecting thin filaments of opposite orientation at the tapered end of the
small solvent channel. C4 connecting density/three-wheel spoke was seen linking the ends of three thin filaments in the same orientation
that form the small solvent channel. C1 and C2 were proposed to contain α-actinin, especially in C2 where an atomic model of F-actin with
CH1 domains closely interacted with an atomic model of α-actinin in the C2 density map. The results of this experiment confirmed what was
currently known about the invertebrate Z-Disc structure, but the locations of Z-Disc proteins, Kettin and Projectin, are yet to be
determined. / A Thesis submitted to the Department of Biological Science in partial fulfillment of the Master of
Science. / Fall Semester 2016. / November 18, 2016. / Includes bibliographical references. / Kenneth A. Taylor, Professor Directing Thesis; M. Elizabeth Stroupe, Committee Member; P. Bryant
Chase, Committee Member; Wu-Min Deng, Committee Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_405689 |
| Contributors | Summerill, Corinne Alethea Oriana (authoraut), Taylor, Kenneth A. (professor directing thesis), Stroupe, Margaret Elizabeth (committee member), Chase, P. Bryant (committee member), Deng, Wu-Min (committee member), Florida State University (degree granting institution), College of Arts and Sciences (degree granting college), Department of Biological Science (degree granting departmentdgg) |
| 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 (104 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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