The new field of protein-based nano-technology takes advantage of the complex interactions between proteins to form unique structures with properties that cannot be achieved with traditional components. Microtubules (MTs), self assembled proteinaceous hollow filaments, offer promise in the development of MT-based nano-systems. The compelling need for the controlled assembly of 3D MT arrays is the fundamental motivation for the first part of this research. We report on the morphology of MTs grown in a crowded environment in the form of high viscosity fluids containing agarose and a novel process that enables the assembly of MTs supported by gel-based 3D scaffolds. Our research on MTs and their interaction with other molecules lead us to discover extraordinary spherulitic structures that changed the course of the project. The novel subject situate us into a complicated dilemma that question the nature of MT asters reported in experiments carried out in cells. The second part of this research is focused in the crystallization ofTaxol, a MT stabilizing molecule used as anti-cancer drug. It was confirmed via fluorescent and differential interference contrast microscopy that Taxol crystals can be decorated with fluorescent proteins and fluorochromes without perturbing their morphology. We used theoretical calculations to further investigate Taxol-fluorescent agent interactions. Furthermore, the crystallization of Taxol was studied in pure water, aqueous solutions containing tubulin proteins and tubulin-containing agarose gels. We demonstrated that tubulin is able to heterogeneously nucleate Taxol spherulites. To explain the formation of tubulin-Taxol nuclei a new, secondary Taxol-binding site within the tubulin heterodimer is suggested. Results presented in this work are important for in vivo and in vitro microtubule studies due to the possibility of mistaking these Taxol spherulites for microtubule asters. Thus, we are confirming the need for careful interpretation of fluorescence microscopy observations of MT structures when large concentrations of Taxol are used as stabilizing agent in cells.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/195413 |
| Date | January 2009 |
| Creators | Castro Carmona, Javier Servando |
| Contributors | Deymier, Pierre A., Deymier, Pierre A., Seraphin, Supapan, Loy, Douglas A, Yoon, Jeong-Yeol |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | English |
| Detected Language | English |
| Type | text, Electronic Dissertation |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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