Nucleocytoplasmic transport of large cargos: theory and single-molecule microscopy : a dissertation /
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| Format: | Thesis Book |
| Language: | English |
| Published: |
[College Station, Tex.] :
[Texas A&M University System Health Science Center],
[2013]
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| Subjects: |
| Abstract: | ABSTRACT: In eukaryotic cells, the nuclear envelope (NE) surrounds the genome in the nucleus. The macromolecular traffic between the two resultant compartments, the cytoplasm and the nucleus, is critical to cell function. The transport machinery, the nuclear pore complex (NPC), is located in the NE, spanning both membranes. Within the NPC central channel, natively disorderd polypeptides, which contain many glycine-phenylalanine repeats (FG-repeats), form a semi-permeable barrier and regulate bidirectional nuclear transport. The barrier allows free diffusion of smaller molecules but nuclear transport receptors (NTRs) are required for the translocation of larger molecules. The arrangement and characteristics of the FG-polypeptides are key to understanding nuclear transport. Transport through the FG-barrier can be modeled mathematically based on a few assumptions that are consistent with known NPC properties. Since NTRs interact with the FG-repeat motifs and transit through the NPC with cargos, the movement of a cargo complex within the NPC can be considered as diffusion in an effective potential facilitated by the NTR-FG interaction energy. In this model, the transport rate, as well as the transport efficiency, can be estimated by measurements from bulk transport experiments. Parameters that affect the transport rate and efficiency are discussed in this simple model. Real-time single molecule fluorescence methods were used to examine the nuclear import of a large model cargo, M9-[beta]Gal-8c (~500 kD). Due to its tetrameric structure, M9-[beta]Gal-8C has four M9 signal sequences, each of which binds an NTR. We showed that efficient transport of this large model cargo requires multiple NTRs. Large cargo carried by single NTR was capable of binding to the FG-polypeptides but not to penetrate the barrier. Avidity calculations suggested that the interactions between the NTR and the FG repeats are weak and the density of accessible FG binding motifs is low. We propose a model in which the central FG-polypeptides are densely packed and the peripheral FG-polypeptides are sparsely distributed. Interaction energies are complicated by the entropic cost of displacing FG-polypeptides, and, possibly, the enthalpic cost of breaking FG-FG interactions. |
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| Item Description: | Vita. "Major Subject: Biomedical Sciences". "Submitted to The Office of Research and Graduate Studies of The Texas A&M University System Health Science Center In partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2013." Approved as to style and content by: Siegfried M. Musser, Kathryn J. Ryan, Van G. Wilson, Kayla J. Bayless, Geoffrey M. Kapler. |
| Physical Description: | xiv, 114 leaves : illustrations (mostly color) ; 28 cm. |
| Bibliography: | Includes bibliographical references (leaves 24-33,47-48,78-85,109-111). |