Studying neuronal microtubule organization and microtubule-associated proteins using single molecule localization...

The formation and maintenance of highly polarized neurons critically depends on the proper organization of the microtubule (MT) cytoskeleton. In axons, MTs are uniformly oriented with their plus-end pointing outward whereas in mature dendrites MTs have mixed orientations. MT organization and dynamics can be regulated by MT-associated proteins (MAPs). Plus-end tracking proteins are specialized MAPs that decorate plus-ends of growing MTs and regulate neuronal polarity, neurite extension, and dendritic spine morphology. Conventional fluorescence microscopy enables observation of specific cellular components through molecule-specific labeling but provides limited resolution (∼250 nm). Therefore, electron microscopy has until now provided most of our knowledge about the precise MT organization in neurons. In the past decade, super-resolution fluorescence microscopy techniques have emerged that circumvent the diffraction limit of light and enable high-resolution reconstruction of the MT network combined with selective protein labeling. However, preserving MT ultrastructure, MAP binding, high labeling density, and antibody specificity after fixation protocols is still quite challenging. In this chapter, we provide an optimized protocol for two-color direct stochastic optical reconstruction microscopy imaging of neuronal MTs together with their growing plus-ends to probe MT architecture and polarity.