Neurons, which heavily utilize microtubule-dependent vesicle transport, may be especially vulnerable to the presence of Taxol crystals. Our hypothesis is supported by the discovery that Taxol-treated cultured bovine aortic endothelial cells are defective in microtubule-dependent vesicle transport [26].
Again, intracellular crystallization could provide a mechanism by which transport is impaired. Neutropenia characterized by abnormally low neutrophil counts is another serious side effect of Taxol chemotherapy [27]. Neutrophils and platelets are derived from a common myeloid progenitor, and both circulate in the blood.
Their appearance indicates that Taxol may crystallize within platelets. It could be interesting to examine the microtubule configuration in Taxol-treated neutrophils. Furthermore, in a clinical study, Jiko et al. Neutrophils are similarly affected by high concentrations of Taxol [29]. Neutrophils and platelets in Taxol-containing serum are in some ways analogous to cultured cells in Taxol-containing medium.
Caov-3 ovarian carcinoma cells attain a high intracellular Taxol concentration Thus, it is not unreasonable to think that blood cells may also attain saturation at clinically applicable concentrations, potentially resulting in neutropenia or other hematological aberrations.
This observation substantiates the well-established mechanism by which low concentrations of Taxol [30] are sufficient to disrupt cell division, by stabilizing spindle microtubules. It is also consistent with our proposed mechanism by which somewhat higher but still clinically relevant concentrations are cytotoxic.
Given that Taxol may crystallize intracellularly, it could be worth determining whether or how other anti-mitotic drugs crystallize intracellularly or form non-microtubule polymers that may be decorated by tubulin. For example, colchicine has been reported to induce a variety of tubulin-containing arrays that start out as transitory cortical strands.
Discodermolide has been reported to increase the microtubule polymer mass and induce bundling of microtubules [32] , and laulimalide enhances microtubule polymerization, based on turbidity [33]. We hope our data will provide a steppingstone for others to determine whether Taxol or similar drugs can crystallize intracellularly, and if so, under what conditions.
For each treatment, a 0. Otherwise, each 0. The small sample volume results in flatter droplets, thus improving image clarity. Samples were visualized at room temperature using a digital-enhanced DIC microscope Zeiss Axiovert equipped with a 1. Crystals were removed from the tube and pipetted under Halocarbon oil into a chamber slide. We confirmed that the crystals were actually present in larger volumes of solution e. Using both plastic tips and glass micropipettes, we pipetted crystals from tube to slide while observing the transfer under the microscope.
The crystals emerged from the tips preformed, and could be seen immediately at multiple focal planes, not just at the focal plane adjacent to the glass.
To promote microtubule assembly, 1. For concentration-dependence of aster formation, 0. For decoration of the crystals with tubulin, 2. A Narishige microinjector was used to control tubulin flow. The tip opening was wide enough that intact microtubules could pass through as seen in Figure 2 B. Conceived and designed the experiments: DZ. Wrote the paper: MF. Critical review of manuscript: DZ BW. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field.
Abstract Taxol is a potent anti-mitotic drug used in chemotherapy, angioplastic stents, and cell biology research. Introduction In , Taxol paclitaxel was isolated from the Pacific yew tree, Taxus brevifolia , and shown experimentally to have antitumor activity [1].
Results and Discussion Taxol is known to function by binding the inner surface of microtubules and stabilizing them in their polymerized state [2] , [5]. Download: PPT. Figure 1. Taxol crystals resemble asters and bundles formed in the presence of Taxol-stabilized microtubules. Figure 2.
Taxol crystals bind fluorescently labeled tubulin subunits. Figure 3. Concentration- and time-dependence of aster formation. Generation of Taxol-stabilized microtubules To promote microtubule assembly, 1. Author Contributions Conceived and designed the experiments: DZ.
References 1. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J Am Chem Soc — View Article Google Scholar 2. View Article Google Scholar 3. View Article Google Scholar 4. Biochemistry — View Article Google Scholar 5. Cell 79— Nanoscale superstructures are the sources of inspiration for the design of complex materials, i. By modifying the diameter of microtubules, one can control the nanotubes geometric parameters important in biomaterials applications.
The basic geometry is the shape of nanoparticles, which influences these parameters tremendously with different aspect ratios. For example, nanoscale cylindrical particles with higher aspect ratios diameter of nm and height of nm internalized rapidly 5. The cellular uptake depends on a shape of a nanoparticle and ultimately the designing of the nanoparticle for effective nanomedicine delivery Chun, ; Brazil, Nogales et al.
The ring curvature is due to a kink between dimers and a kink at monomer—monomer interface of similar or decreased magnitude. These unique properties of microtubules can be applied in nanoscience to create supramolecular systems. A monomer is a single nanoparticle with self-assembled tubular structures mimicking microtubules. Materials scientists are motivated by technological advancements to alter or tailor the material of interest. This process has lead to discovery of a new class of functional materials and superior functionality as evidenced by a novel concept in the field of nanotechnology and microtubule dynamics.
This commentary is based on the work published in Nature Materials in by Ojeda-Lopez et al. The transition from bundles of Taxol-stabilized microtubules 26 nm to inverted tubulin tubules bundled phase These compact polymeric assemblies resembled like a stack of rings with the inside-out model system, wherein the outer surface of inverted tubulin tubules corresponds to the inner lumen of microtubules Figure 1.
The different aspects of hierarchically ordered nanostructures, electrostatic interactions, conformational transition, and nanoparticle assemblies have been elucidated.
Experimental results have shown that the microtubules lateral and vertical surfaces cause the transition to form inverted tubulin tubule structures. SAXS data have shown microtubules bundled at 15 mM spermine concentration, whereas, at 30 mM, it was completely in the inverted tubulin tubule bundled phase at room temperature after 10 days. This suggested that the ring formation leading to such inverted structures are induced by highly charged spermine localized in small volume between the lateral contacts in microtubule.
Spermine lowers the straight-to-curved energy barrier with increasing concentrations. In addition, at 2. The structural properties of microtubules are crucial for nanotechnological applications. Needleman et al. Whereas, in the presence of small divalent cations, highly anisotropic living necklace bundles were formed due to the large surface area.
Thus, microtubule bundling architectures of controlled dimensions as templates can be tailored for miniaturized materials. From the technological viewpoint, these nanomaterials find interesting applications in microtubule-binding drug, MAP-tau protein, dynamin protein, tubulin GTPase enzyme Safinya et al. They also find applicability in circuitry components such as carbon nanotubes and conducting polymers, thermoresponsive polymer Thess et al.
Figure 1. Curves ahead: transformation from straight tubes to stacked rings via nanometer scale microtubule assembly and disassembly phenomenon for delivering nanomedicine Taxol. Add Taxol Stepwise Add taxol to the polymerized microtubules in the following stepwise manner. Incubate for 10 minutes. Incubate for 15 minutes.
NOTE : Taxol must be added stepwise in order to avoid precipitation. The final taxol concentration should match or be in equimolar excess to the tubulin protein concentration i. Taxol must be included in all subsequent buffers. STEP 6. STEP 7. Resuspend Pipette off and discard the supernatant. Pipette off and discard the cushion. Use a cut pipette tip to avoid shearing the microtubules. NOTE : After polymerizing, taxol-stabilized microtubules are to be handled at room temperature.
The taxol-stabilized microtubules are now ready for experimental use and can be kept at room temperature for several days. Recommended Products. Unlabeled Tubulin Protein.
Quick View. Cycled Tubulin. Lyophilized Tubulin. Labeled Tubulin Protein.
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