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The brain consists of billions of brain cells, also called neurons, which form a complex network. To ensure proper signal transduction from cell to cell, it is of great importance that each neuron has the right building blocks at the right moment at the right place. This transport is mediated by so called motor proteins. The research in this thesis focusses on a family of motor proteins, the kinesins. There are more than 40 different kinesins in neurons and there are even more different types of building blocks. Therefore, each neuron has a complex transport network, in which each kinesin knows exactly what to transport where. This thesis describes the development of a new method that can be used to study kinesins. This method is combined with other biochemical techniques to study the regulation of the kinesin KIF1A. First, the interaction of KIF1A with the inhibiting protein KBP is elucidated, which regulates the activity of the motor. Next, different building blocks of KIF1A are identified and it is shown that specificity for each building block is regulated by different adaptor proteins. Furthermore, it is shown that KIF1A interacts with KIF5 in the transport of the TrkB receptor. These insights into the regulation of KIF1A contribute to our fundamental knowledge about the transport mechanisms in neurons. This is an important step in understanding the neuronal transport network and ultimately to solve brain diseases caused by disruptions in this network.

The brain consists of billions of brain cells, also called neurons, which form a complex network. To ensure proper signal transduction from cell to cell, it is of great importance that each neuron has the right building blocks at the right moment at the right place. This transport is mediated by so called motor proteins. The research in this thesis focusses on a family of motor proteins, the kinesins. There are more than 40 different kinesins in neurons and there are even more different types of building blocks. Therefore, each neuron has a complex transport network, in which each kinesin knows exactly what to transport where. This thesis describes the development of a new method that can be used to study kinesins. This method is combined with other biochemical techniques to study the regulation of the kinesin KIF1A. First, the interaction of KIF1A with the inhibiting protein KBP is elucidated, which regulates the activity of the motor. Next, different building blocks of KIF1A are identified and it is shown that specificity for each building block is regulated by different adaptor proteins. Furthermore, it is shown that KIF1A interacts with KIF5 in the transport of the TrkB receptor. These insights into the regulation of KIF1A contribute to our fundamental knowledge about the transport mechanisms in neurons. This is an important step in understanding the neuronal transport network and ultimately to solve brain diseases caused by disruptions in this network.

The use of super-resolution microscopy in recent years has revealed that proteins often form small assemblies inside cells and are organized in nanoclusters. However, determining the copy number of proteins within these nanoclusters constitutes a major challenge because of unknown labeling stoichiometries and complex fluorophore photophysics. We previously developed a DNA-origami-based calibration approach to extract protein copy number from super-resolution images. However, the applicability of this approach is limited by the fact that the calibration is dependent on the specific labeling and imaging conditions used in each experiment. Hence, the calibration must be repeated for each experimental condition, which is a formidable task. Here, using cells stably expressing dynein intermediate chain fused to green fluorescent protein (HeLa IC74 cells) as a reference sample, we demonstrate that the DNA-origami-based calibration data we previously generated can be extended to super-resolution images taken under different experimental conditions, enabling the quantification of any green-fluorescent-protein-fused protein of interest. To do so, we first quantified the copy number of dynein motors within nanoclusters in the cytosol and along the microtubules. Interestingly, this quantification showed that dynein motors form assemblies consisting of more than one motor, especially along microtubules. This quantification enabled us to use the HeLa IC74 cells as a reference sample to calibrate and quantify protein copy number independently of labeling and imaging conditions, dramatically improving the versatility and applicability of our approach.

Atkins et al. identify a new role for Fidgetin-like 1 in motor axon navigation via its regulation of bidirectional axonal transport. They show that Fidgetin-like 1 binds Kif1bβ and the opposed polarity-directed motor dynein/dynactin in a molecular complex and controls circuit wiring by reducing dynein velocity in developing motor axons., Neuronal connectivity relies on molecular motor-based axonal transport of diverse cargoes. Yet the precise players and regulatory mechanisms orchestrating such trafficking events remain largely unknown. We here report the ATPase Fignl1 as a novel regulator of bidirectional transport during axon navigation. Using a yeast two-hybrid screen and coimmunoprecipitation assays, we showed that Fignl1 binds the kinesin Kif1bβ and the dynein/dynactin adaptor Bicaudal D-1 (Bicd1) in a molecular complex including the dynactin subunit dynactin 1. Fignl1 colocalized with Kif1bβ and showed bidirectional mobility in zebrafish axons. Notably, Kif1bβ and Fignl1 loss of function similarly altered zebrafish motor axon pathfinding and increased dynein-based transport velocity of Rab3 vesicles in these navigating axons, pinpointing Fignl1/Kif1bβ as a dynein speed limiter complex. Accordingly, disrupting dynein/dynactin activity or Bicd1/Fignl1 interaction induced motor axon pathfinding defects characteristic of Fignl1 gain or loss of function, respectively. Finally, pharmacological inhibition of dynein activity partially rescued the axon pathfinding defects of Fignl1-depleted larvae. Together, our results identify Fignl1 as a key dynein regulator required for motor circuit wiring.

Kinesin-1 (conventional kinesin) is a molecular motor that transports various cargo such as endoplasmic reticulum and mitochondria in cells. Its two head domains walk along microtubule by hydrolyzing ATP, while the tail domains at the end of the long stalk bind to the cargo. When a kinesin is not carrying cargo, its motility and ATPase activity is inhibited by direct interactions between the tail and head. However, the mechanism of this tail regulation is not well understood. Here, we apply single molecule fluorescence resonance energy transfer (smFRET) to observe this interaction in stalk-truncated kinesin. We found that kinesin with two tails forms a folding conformation and dissociates from microtubules, whereas kinesin with one tail remains bound to the micro-tubule and is immobile even in the presence of ATP. We further investigated the head-tail interaction as well as head-head coordination on the microtubule at various nucleotide conditions. From these results, we propose a two-step inhibition model for kinesin motility.

We studied molecular motors in long-range motility of early endosomes (EEs) in a fungal model system that contains a bipolar, dendrite-like microtubule (MT) array. Dynein moves retrograde EEs over ∼10 μm, before kinesin-3 takes over for a further ∼80 μm along antipolar MT bundles. Thus kinesin-3 is the major motor for retrograde EE motility., The polarity of microtubules (MTs) determines the motors for intracellular motility, with kinesins moving to plus ends and dynein to minus ends. In elongated cells of Ustilago maydis, dynein is thought to move early endosomes (EEs) toward the septum (retrograde), whereas kinesin-3 transports them to the growing cell tip (anterograde). Occasionally, EEs run up to 90 μm in one direction. The underlying MT array consists of unipolar MTs at both cell ends and antipolar bundles in the middle region of the cell. Cytoplasmic MT-organizing centers, labeled with a γ-tubulin ring complex protein, are distributed along the antipolar MTs but are absent from the unipolar regions. Dynein colocalizes with EEs for 10–20 μm after they have left the cell tip. Inactivation of temperature-sensitive dynein abolishes EE motility within the unipolar MT array, whereas long-range motility is not impaired. In contrast, kinesin-3 is continuously present, and its inactivation stops long-range EE motility. This indicates that both motors participate in EE motility, with dynein transporting the organelles through the unipolar MT array near the cell ends, and kinesin-3 taking over at the beginning of the medial antipolar MT array. The cooperation of both motors mediates EE movements over the length of the entire cell.