Fluorescence Probing of Fluctuating Microtubule using a Covalent Fluorescent Probe: Effect of Taxol

摘要

abstract_textpFluctuation in the local microenvironment of a dynamic microtubule, undergoing continuous growth and shrinkage, is monitored by time resolved confocal microscopy. For this, we have attached a fluorescent probe (CPM, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin), covalently to tubulin dimers. In this experiment, the laser is focused on a small microtubule seed immobilized on a glass slide by neutravidin and biotin interaction. Tubulin monomers and other agents present in the solution cause growth and shrinkage. We have monitored emission maximum and solvation dynamics of the probe, CPM. Emission maximum (lambda(em)(max)) of CPM labeled tubulin dimer indicates a highly exposed medium similar to that in human serum albumin, HSA denatured by 6 M Guanidinium Hydrochloride (GdnHCl). However, solvation dynamics of CPM in tubulin dimer is more than 10 times slower than that in denatured HSA. lambda(em)(max) and solvation dynamics of CPM in tubulin dimer is similar to those in fluctuating microtubules and taxol (paclitaxel) stabilized microtubules. The fluorescence intensity of CPM exhibits intermittent oscillation in 1-2 seconds time scale in dynamic microtubules. No such fluctuation is observed in the case of unpolymerized tubulin dimer or when microtubules are stabilized by taxol. This strategy offers a new physicochemical approach of monitoring polymerization (growth) and depolymerization (shrinkage) of tubulin./p/abstract_text