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Measuring the Effects of Microtubule-Associated Proteins on Microtubule Dynamics In Vitro


Zanic MMarija . Methods in molecular biology (Clifton, N.J.). 2016 1 1; 1413(). 47-61


Microtubule dynamic instability, the process by which individual microtubules switch between phases of growth and shrinkage, is essential for establishing the architecture of cellular microtubule structures, such as the mitotic spindle. This switching process is regulated by a complex network of microtubule-associated proteins (MAPs), which modulate different aspects of microtubule dynamic behavior. To elucidate the effects of MAPs and their molecular mechanisms of action, in vitro reconstitution approaches with purified components are used. Here, I present methods for measuring individual and combined effects of MAPs on microtubule dynamics, using purified protein components and total-internal-reflection fluorescence (TIRF) microscopy. Particular focus is given to the experimental design, proper parameterization, and data analysis.