Abstract
The neuronal microtubule cytoskeleton underlies the polarization and proper functioning of neurons, amongst others by providing tracks for motor proteins that drive intracellular transport. Different subsets of neuronal microtubules, varying in composition, stability, and motor preference, are known to exist, but the high density of microtubules has so far precluded mapping their relative abundance and three-dimensional organization. Here, we use different super-resolution techniques (STED, Expansion Microscopy) to explore the nanoscale organization of the neuronal microtubule network in rat hippocampal neurons. This revealed that in dendrites acetylated microtubules are enriched in the core of the dendritic shaft, while tyrosinated microtubules are enriched near the plasma membrane, thus forming a shell around the acetylated microtubules. Moreover, using a novel analysis pipeline we quantified the absolute number of acetylated and tyrosinated microtubules within dendrites and found that they account for 65-75% and ~20-30% of all microtubules, respectively, leaving only few microtubules that do not fall in either category. Because these different microtubule subtypes facilitate different motor proteins, these novel insights help to understand the spatial regulation of intracellular transport.
Highlights
The extended and polarized morphology of neurons is established and maintained by the cytoskeleton (Stiess and Bradke, 2011; Bentley and Banker, 2016)
We started by mapping the spatial distribution of acetylated and tyrosinated microtubules throughout the dendrite using both 2D and 3D stimulated emission depletion (STED) microscopy
Consistent with our earlier observations, this revealed that acetylated microtubules in DIV9 neurons tend to be distributed closer to the central axis of the dendrite, while the tyrosinated microtubules seem to be enriched at the outer surface, close to the membrane (Figure 1A,B)
Summary
The extended and polarized morphology of neurons is established and maintained by the cytoskeleton (Stiess and Bradke, 2011; Bentley and Banker, 2016). One of the functions of the microtubule cytoskeleton is to provide a transport network inside the neurons long axon and branched dendrites (Kapitein and Hoogenraad, 2015; Burute and Kapitein, 2019). Directional transport is enabled by the structural polarity of microtubules, which is recognized by motor proteins that drive transport to either the minus end (dynein) or plus end (most kinesins). Using this network, intracellular cargos attached to microtubule-based motor proteins (kinesins and dynein) can be shipped between different neuronal compartments (Hirokawa et al, 2010; Kapitein and Hoogenraad, 2011). An important component is the composition and spatial distribution of microtubule tracks, which is the main subject of this study
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