Painted Roads: A super-resolution study of the neuronal cytoskeleton

The correct distribution of molecules to different compartments is important for the assembly of all cells in our body. Active transport of these molecular building blocks is especially important in large polarized cells, such as neurons, to develop and maintain their form and function. Neurons consist of two types of long protrusions, axons and dendrites, that differ in function and molecular composition. Upon excitation of a neuron, the axon releases a chemical signal into contact zones with the dendrites of the next neuron in the network that can subsequently propagate the signal through its own axon. The form of the axon and dendrites is determined by the cytoskeleton, a network of biopolymers and auxiliary proteins that provide mechanical stability and plasticity. Additionally, molecular motors use cytoskeletal components as the highways of the cell for active transport. Motor proteins distribute cargoes into the axon and dendrites by walking along the actin and microtubule filaments. These are structurally polarized biopolymers with a plus- and minus-end and different motor proteins exclusively walk either to the plus- or minus-end of a microtubule or actin filament. The work in this thesis addresses how the exact organization of the neuronal cytoskeleton affects molecular transport. To map the architecture of the cytoskeleton as accurately as possible, we have used different advanced microscopy techniques, predominantly single-molecule localization microscopy (SMLM). One of the main challenges of SMLM is combining different strategies to visualize the cytoskeleton components at the same time. To better visualize actin filaments, we optimized the purification and labelling of a small protein fragment, called lifeAct. This led to an easy to use actin labelling strategy that is compatible with other existing SMLM methods. This resulted in more in depth insights into the organization of actin clusters and cables at the initial segment of the axon and the spines of dendrites. Additionally, we have studied transport in living cells to identify the function of these structures. To better understand the traffic rules along the cellular microtubule highways in neurons, it is important to known their orientation with high precision. In order to achieve this, we have developed a new method to reconstruct a high resolution map of the neuronal microtubule network while directly imposing their orientations. We termed this method motor-PAINT. We focused on the dendritic microtubule organization in order to better understand how kinesins selectively navigate along the mixed microtubules of the dendrites. Our experiments showed that microtubules with the same orientation are bundled into unidirectional highways. Subsequent correlative and pharmacological measurements showed that microtubules that are oriented with their minus-end away from the cell body, are in general more stable. Because kinesins prefer different groups of microtubules, we could then propose a new model for selective transport. The research in this thesis provides us with deeper understanding in the architecture of the neuronal cytoskeleton and the traffic rules ine neurons. These fundamental insights are important to better understand how neurons are build and where it goes wrong in developmental disorders or during neurodegenerative diseases.