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Characterization of moving neurofilaments in cultured neurons

Yan, Yanping
http://rave.ohiolink.edu/etdc/view?acc_num=osu1134349238

Abstract Details

2006, Doctor of Philosophy, Ohio State University, Neuroscience.
Neurofilament proteins are the major cytoskeletal component of most neurons and they are transported by slow axonal transport, whose mechanism is still controversial. Live cell imaging has revealed that GFP-tagged neurofilament proteins move rapidly along axons in predominantly filamentous form but the overall rate is slow because they spend most of the time pausing. In this thesis I test the hypotheses that these moving filamentous structures represent single neurofilament polymers and that moving and pausing neurofilaments might differ in their polypeptide composition or phosphorylation state. To test these hypotheses, I have developed a novel perfusion technique for capturing fluorescent filaments as they move through naturally occurring gaps in the axonal neurofilament array of cultured sympathetic neurons. Using quantitative immunofluorescence microscopy and correlative light and electron microscopy, I demonstrate that the captured structures are single continuous neurofilament polymers. To analyze their polypeptide composition, I processed the captured filaments and filaments in detergent-splayed cytoskeletons for immunofluorescence microscopy using antibodies specific for various neurofilament proteins. All neurofilaments contained four different neurofilament proteins (NFL, NFM, á-internexin and peripherin) and on average each was present along >95% of the neurofilament length. Since there was no difference between the polypeptide composition of the moving filaments and the overall neurofilament population, most of which are pausing at any point in time, these data indicate that moving and pausing neurofilaments do not differ in their polypeptide composition. Immunostaining with antibody RT97, which recognizes phosphoepitopes on NFM and NFH that have been correlated with slowing of neurofilament transport, revealed no evidence for hypophosphorylation of moving neurofilaments. All moving neurofilaments had the epitope distributed along almost their entire length and quantitative comparison of RT97 immunofluorescence intensity along moving and splayed neurofilaments further confirms that moving neurofilaments are not hypophosphorylated at this epitope relative to the overall population. I conclude (1) that neurofilament proteins move in the form of single assembled polymers, (2) that moving and pausing neurofilaments are complex heteropolymers comprised of at least four different subunit proteins, and (3) that phosphorylation at the RT97 epitope is not a key regulator of neurofilament movement in cultured sympathetic neurons.
Anthony Brown (Advisor)
Biology, Neuroscience
neurofilament; axonal transport; live cell imaging; immunofluorescence microscopy; electron microscopy;

Recommended Citations

Citations

  • Yan, Y. (2006). Characterization of moving neurofilaments in cultured neurons [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1134349238

    APA Style (7th edition)

  • Yan, Yanping. Characterization of moving neurofilaments in cultured neurons. 2006. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1134349238.

    MLA Style (8th edition)

  • Yan, Yanping. "Characterization of moving neurofilaments in cultured neurons." Doctoral dissertation, Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=osu1134349238

    Chicago Manual of Style (17th edition)

osu1134349238
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© 2005, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.