Mapping the Neuronal Cytoskeleton with Expansion Microscopy

Abstract

Neuronal cells possess a highly polarized morphology which is crucial for its functioning and development. A key determinant in establishing and maintaining this functional polarization is the microtubule cytoskeleton, a filamentous biopolymer network. Although this microtubule network has been studied extensively, the exact spatial organization remains unknown due to subdiffraction size of filaments and dense bundle packing in neurites. In this paper we describe optimization of a recently developed super-resolution technique, Expansion Microscopy, for mapping the microtubule organization in hippocampal neuron cultures. By combining Expansion Microscopy with Stimulated Emission Depletion Microscopy we were able to reach a resolution of 44 nm (as measured by Fourier Ring Correlation). We found that acetylated- and tyrosinatedmicrotubules show a clear difference in spatial distribution in the dendrite. Acetylated microtubules are concentrated around the central axes of the dendrite while tyrosinated-microtubules are located closer to the membrane of the dendrite.