Microtubule, fluorescent

A EXPERIMENTAL FIGURE 20-10 Fluorescence microscopy reveals in vivo growth and shrinkage of individual microtubules. Fluorescently-labeled tubulin was microinjected into cultured human fibroblasts. The cells were chilled to depolymerize preexisting microtubules into tubulin dimers and were then incubated at 37 °C to allow repolymerization. 

A) Microtubules, fluorescent dye FITC B) Actin filament, fluorescent dye telramethylrhodamine-B-isothiocyanate (TRITC)... 

Mitchison, T.J. (1989). Polewards microtubule flux in the mitotic spindle Evidence from photoactivation of fluorescence. J. Cell Biol. 109, 637-652. 

Gonczy This is very difficult to quantitate. As you have seen from the immunofluorescence images, there are many microtubules on either side in wild-type moreover, anaphase B takes places within a couple of minutes. Therefore, it will be difficult to uncover potential transient changes in microtubule numbers using fixed specimens. However, we have generated a green fluorescent protein... 

Drummond, D. R., Carter, N. and Cross, R. A. (2002). Multiphoton versus confocal high resolution z-sectioning of enhanced green fluorescent microtubules Increased multiphoton photobleaching within the focal plane can be compensated using a Pockels cell and dual widefield detectors. J. Microsc. 206, 161-9. 

The staining of germinated pollen of Hippeastrum hybridum with colchicine demonstrates green-yellow emission of microtubules (better vision in black-white image) around nuclei of pollen grain (threads at the division of the nucleus) and spermium on the tip of the pollen tube, where spermium moves, as well as in some bridge sites of the tube (Fig. 10). The similar fluorescent allelochemicals may be also used as fluorescent dyes at the cellular diagnostics (Roshchina, 2005 b). 

Scherson, T., Kreis, T.E., Schlessinger, J., Littauer, U., Borisy, G.G., and Geiger, B. (1984) Dynamic interactions of fluorescently labeled microtubule-associated proteins in living cells./. Cell Biol. 99, 425-434. 

Vigers, G.P.A., Cone, J.R., and Mcintosh, 1. (1988) Fluorescent microtubules break up under illumination./. Cell Biol. 107, 1011. 

Until the past decade, the cytoplasm was widely considered to be structurally unorganized with the main division of labor at the organellar level. Certainly, relatively little was known about the nature of the cyto-skeleton (with the notable exception of the mitotic apparatus and striated muscle), and the dynamics of cytoplasmic behavior were conceptualized vaguely in terms of sol-gel transitions without a sound molecular foundation. Substantial improvements in electron, light, and fluorescence microscopy, as well as the isolation of discrete protein components of the cytoskeleton, have led the way to a much better appreciation of the structural organization of the cytoplasm. Indeed, the lacelike network of thin filaments, intermediate filaments, and microtubules in nonmuscle cells is as familiar today as the organelles identified... 

Fluorescein-labeled proteins are also used to measure the translational mobility of proteins and lipids by the Fluorescence Recovery After Photo-bleaching technique [54-59]. The uniformly labeled fluorescent sample is flashed with an intense light source to bleach a spot, thus producing a concentration gradient. The rate of recovery of fluorescence in that bleached area is measured and used to calculate the diffusion coefficient of the probe dye into the bleached zone. Such diffusion coefficient measurements have been used to determine the association constants of proteins in cells [60], to measure the exchange of tubulin between the cytoplasm and the microtubules [61,62], to study the polymerization-depolymerization process of actin [63-65] and to monitor the changes that occur upon cell maturation [66,67]. 

Figure 7-32 Micrograph of a mouse embryo fibroblast was obtained using indirect immunofluorescence techniques.313 The cells were fixed with formaldehyde, dehydrated, and treated with antibodies (formed in a rabbit) to microtubule protein. The cells were then treated with fluorescent goat antibodies to rabbit /-globulins (see Chapter 31) and the photograph was taken by fluorescent light emission. Courtesy of Klaus Weber.

Motion of kinesin heads has been observed by movement of microtubules over biotinylated kinesin fixed to a steptavidin-coated surface,209 by direct observation of fluorescent kinesin moving along microtubules,171 and by optical trap interferometry.210 Kinesin heads move by 8-nm steps, evidently the exact length... 

Tagging individual proteins with fluorescent molecules allows them to be monitored in live cells, enabling rapid discovery of many intricate details about the cellular chemistry. For example, fluorescent tags on molecules that have been packaged into small vesicles have been monitored as they travel along microtubules within the axons via complex protein machines. 

Motion analysis and particle tracking methods enable users to follow the movement over time of tagged particles, such as fluorescently labeled cell surface molecules, microtubules, nucleic acids, lipids, and other objects with subpixel resolution.287 These methods allow scientists to measure jc and y coordinates, velocity, mean displacement, mean vector length, and more. 

Fluorescent CPMV [93] particles were used as a reporter tag in a standard microtubule gliding assay... 

Fig. 2 Left cytoplasmic microtubules in interphase kidney epithelial cells imaged with the fluorescent paclitaxel derivative Flutax-2 (green) and nuclear DNA stained with Hoescht 33342 (blue). Right mitotic spindle from a dividing metaphase cell with similarly imaged microtubules and chromosomes. Bars indicate 10 pm (micrographs courtesy of Isabel Barasoain)...

The problem was overcome with the use of mildly fixed microtubules [10] in which the paclitaxel binding site is unaltered, while protected from cold and dilution depolymerisation and a paclitaxel molecule bound to a fluorescent probe (either fluorescein or difluorofluorescein) [8], The fluorescent derivatives of paclitaxel were then tested to check that they compete with taxoids for the same site, with the same 1 1 stoichiometry producing the same celular effects as paclitaxel and docetaxel, thus it can be assumed that they bind for the same site [9], Using these stabilized microtubules, it is possible to determine precisely the binding constant of the fluorescent taxoid which was found to be of the order of 108 M at 25°C. 

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