Question

1. You are planning to do some experiments to demonstrate the dynamic nature of microtubules. How you would fluorescently label microtubules in order to use for FRAP experiments inside a cell?
2. Once you have labeled microtubules, you decide to treat the cells with taxol and then perform FRAP What would be your control for this experiment? What are your predictions for the experimental and control experiment results?

Answer 1

Ans. -

The quantification of the immobile fraction of molecules is only possible by FRAP. This parameter is of great interest to avoid epiphenomenal observations by svFCS. While the two methods are inversely sensitive to the concentration of fluorescent probes, there exists a range for which measurements are compatible. Implementing FRAP and FCS on the same setup is easy to do with an acousto-optic modulator (AOM) to precisely adjust the laser power. A small region of a surface or volume containing mobile fluorescent molecules (or fluorescently labeled macromolecules) is exposed to a brief intense pulse of light thereby causing irreversible photochemical bleaching of the fluorophore in that region.

Fluorescence Recovery After Photobleaching (FRAP) has been considered the most widely applied method for observing translational diffusion processes of macromolecules. The resulting information can be used to determine kinetic properties, like the diffusion coefficient, mobile fraction, and transport rate of the fluorescently labeled molecules. FRAP employs irradiation of a fluorophore with a short laser pulse. For very fast kinetics the FlyMode can be applied. In this mode, reading out the signal during the x-fly back of the scanner provides a time resolution between lines, instead of frames, for the FRAP experiment.

Microtubules are intrinsically dynamic polymers, and their dynamics play a crucial role in mitotic spindle assembly, the mitotic checkpoint, and chromosome movement. In living cells, suppression of microtubule dynamics is responsible for the ability of taxol to inhibit mitotic progression and cell proliferation. Using quantitative fluorescence video microscopy, examined the effects of taxol on the dynamics of individual microtubules in two living human tumor cell lines. At equivalent intracellular taxol concentrations, dynamic instability was inhibited similarly in the two cell lines. Taxol blocked cell cycle progress at the metaphase/anaphase transition and inhibited cell proliferation.

A scientific control is an experiment or observation designed to minimize the effects of variables other than the independent variable. This increases the reliability of the results, often through a comparison between control measurements and the other measurements. If the initial hypothesis is not supported, you can go back to the drawing board and hypothesize a new answer to the question and a new way to test it. If your hypothesis is supported, you might think of ways to refine your hypothesis and test those. The action of predicting future events; an instance of this, a prophecy, a forecast. Hypothesis in science: A tentative, testable, and falsifiable explanation for an observed phenomenon in nature.