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Microtubule dynamics reconstituted in vitro and imaged by single-molecule fluorescence microscopy


Gell CChristopher , Bormuth VVolker , Brouhard GJGary J , Cohen DNDaniel N , Diez SStefan , Friel CTClaire T , Helenius JJonne , Nitzsche BBert , Petzold HHeike , Ribbe JJan , Schäffer EErik , Stear JHJeffrey H , Trushko AAnastasiya , Varga VVladimir , Widlund POPer O , Zanic MMarija , Howard JJonathon . Methods in cell biology. 2010 1 1; 95(). 221-45


In vitro assays that reconstitute the dynamic behavior of microtubules provide insight into the roles of microtubule-associated proteins (MAPs) in regulating the growth, shrinkage, and catastrophe of microtubules. The use of total internal reflection fluorescence microscopy with fluorescently labeled tubulin and MAPs has allowed us to study microtubule dynamics at the resolution of single molecules. In this chapter we present a practical overview of how these assays are performed in our laboratory: fluorescent labeling methods, strategies to prolong the time to photo-bleaching, preparation of stabilized microtubules, flow-cells, microtubule immobilization, and finally an overview of the workflow that we follow when performing the experiments. At all stages, we focus on practical tips and highlight potential stumbling blocks.