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

Methods in Cell Biology
Anaël ChazeauLukas C Kapitein

Abstract

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 protoco...Continue Reading

Citations

Aug 28, 2019·Nature Methods·A DesclouxA Radenovic
Mar 7, 2020·Biomedical Optics Express·Marijn SiemonsLukas C Kapitein
Sep 13, 2017·Frontiers in Cellular Neuroscience·Anne F J JanssenLukas C Kapitein
Jun 17, 2020·Nature Communications·K S GrußmayerT Lasser
May 13, 2019·Methods : a Companion to Methods in Enzymology·Angélique JimenezChristophe Leterrier
Jun 9, 2021·Nature Communications·Marijn E SiemonsLukas C Kapitein

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