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Subcellular compartmentalisation is necessary for eukaryotic cell function. Spatial and temporal regulation of kinesin activity is essential for building these local environments via control of intracellular cargo distribution. Kinesin binding protein (KBP) interacts with a subset of kinesins via their motor domains, inhibits their microtubule (MT) attachment and blocks their cellular function. However, its mechanisms of inhibition and selectivity have been unclear. Here we use cryo-electron microscopy to reveal the structure of KBP and of a KBP-kinesin motor domain complex. KBP is a TPR-containing, right-handed α-solenoid that sequesters the kinesin motor domain’s tubulin-binding surface, structurally distorting the motor domain and sterically blocking its MT attachment. KBP uses its α-solenoid concave face and edge loops to bind the kinesin motor domain, and selected structure-guided mutations disrupt KBP inhibition of kinesin transport in cells. The KBP-interacting motor domain surface contains motifs exclusively conserved in KBP-interacting kinesins, suggesting a basis for kinesin selectivity.

Because of the low stoichiometry of protein phosphorylation, targeted enrichment prior to LC–MS/MS analysis is still essential. The trend in phosphoproteome analysis is shifting toward an increasing number of biological replicates per experiment, ideally starting from very low sample amounts, placing new demands on enrichment protocols to make them less labor-intensive, more sensitive, and less prone to variability. Here we assessed an automated enrichment protocol using Fe(III)-IMAC cartridges on an AssayMAP Bravo platform to meet these demands. The automated Fe(III)-IMAC-based enrichment workflow proved to be more effective when compared to a TiO2-based enrichment using the same platform and a manual Ti(IV)-IMAC-based enrichment workflow. As initial samples, a dilution series of both human HeLa cell and primary rat hippocampal neuron lysates was used, going down to 0.1 μg of peptide starting material. The optimized workflow proved to be efficient, sensitive, and reproducible, identifying, localizing, and quantifying thousands of phosphosites from just micrograms of starting material. To further test the automated workflow in genuine biological applications, we monitored EGF-induced signaling in hippocampal neurons, starting with only 200 000 primary cells, resulting in ∼50 μg of protein material. This revealed a comprehensive phosphoproteome, showing regulation of multiple members of the MAPK pathway and reduced phosphorylation status of two glutamate receptors involved in synaptic plasticity.

Because of the low stoichiometry of protein phosphorylation, targeted enrichment prior to LC–MS/MS analysis is still essential. The trend in phosphoproteome analysis is shifting toward an increasing number of biological replicates per experiment, ideally starting from very low sample amounts, placing new demands on enrichment protocols to make them less labor-intensive, more sensitive, and less prone to variability. Here we assessed an automated enrichment protocol using Fe(III)-IMAC cartridges on an AssayMAP Bravo platform to meet these demands. The automated Fe(III)-IMAC-based enrichment workflow proved to be more effective when compared to a TiO2-based enrichment using the same platform and a manual Ti(IV)-IMAC-based enrichment workflow. As initial samples, a dilution series of both human HeLa cell and primary rat hippocampal neuron lysates was used, going down to 0.1 μg of peptide starting material. The optimized workflow proved to be efficient, sensitive, and reproducible, identifying, localizing, and quantifying thousands of phosphosites from just micrograms of starting material. To further test the automated workflow in genuine biological applications, we monitored EGF-induced signaling in hippocampal neurons, starting with only 200 000 primary cells, resulting in ∼50 μg of protein material. This revealed a comprehensive phosphoproteome, showing regulation of multiple members of the MAPK pathway and reduced phosphorylation status of two glutamate receptors involved in synaptic plasticity.

Dendrites have a unique microtubule organization. In vertebrates, dendritic microtubules are organized in antiparallel bundles, oriented with their plus ends either pointing away or toward the soma. The mixed microtubule arrays control intracellular trafficking and local signaling pathways, and are essential for dendrite development and function. The organization of microtubule arrays largely depends on the combined function of different microtubule regulatory factors or generally named microtubule-associated proteins (MAPs). Classical MAPs, also called structural MAPs, were identified more than 20 years ago based on their ability to bind to and copurify with microtubules. Most classical MAPs bind along the microtubule lattice and regulate microtubule polymerization, bundling, and stabilization. Recent evidences suggest that classical MAPs also guide motor protein transport, interact with the actin cytoskeleton, and act in various neuronal signaling networks. Here, we give an overview of microtubule organization in dendrites and the role of classical MAPs in dendrite development, dendritic spine formation, and synaptic plasticity.

Dendrites have a unique microtubule organization. In vertebrates, dendritic microtubules are organized in antiparallel bundles, oriented with their plus ends either pointing away or toward the soma. The mixed microtubule arrays control intracellular trafficking and local signaling pathways, and are essential for dendrite development and function. The organization of microtubule arrays largely depends on the combined function of different microtubule regulatory factors or generally named microtubule-associated proteins (MAPs). Classical MAPs, also called structural MAPs, were identified more than 20 years ago based on their ability to bind to and copurify with microtubules. Most classical MAPs bind along the microtubule lattice and regulate microtubule polymerization, bundling, and stabilization. Recent evidences suggest that classical MAPs also guide motor protein transport, interact with the actin cytoskeleton, and act in various neuronal signaling networks. Here, we give an overview of microtubule organization in dendrites and the role of classical MAPs in dendrite development, dendritic spine formation, and synaptic plasticity.

Item does not contain fulltext Identification of genes involved in hereditary vestibular disease is growing at a remarkable pace. Mutant mouse technology can be an important tool for understanding the biological mechanism of human vestibular diseases.

The accumulation of cellular damage, including DNA damage, is hypothesized to contribute to aging-related neurodegenerative changes. DNA excision repair cross-complementing group 1 (Ercc1) knock-out mice represent an accepted model of neuronal aging, showing gradual neurodegenerative changes, including loss of synaptic contacts and cell body shrinkage. Here, we used the Purkinje cell-specific Ercc1 DNA-repair knock-out mouse model to study aging in the mouse cerebellum. We performed an in-depth quantitative proteomics analysis, using stable isotope dimethyl labeling, to decipher changes in protein expression between the early (8 weeks), intermediate (16 weeks), and late (26 weeks) stages of the phenotypically aging Ercc1 knock-out and healthy littermate control mice. The expression of over 5,200 proteins from the cerebellum was compared quantitatively, whereby 79 proteins (i.e. 1.5%) were found to be substantially regulated during aging. Nearly all of these molecular markers of the early aging onset belonged to a strongly interconnected network involved in excitatory synaptic signaling. Using immunohistological staining, we obtained temporal and spatial profiles of these markers confirming not only the proteomics data but in addition revealed how the change in protein expression correlates to synaptic changes in the cerebellum. In summary, this study provides a highly comprehensive spatial and temporal view of the dynamic changes in the cerebellum and Purkinje cell signaling in particular, indicating that synapse signaling is one of the first processes to be affected in this premature aging model, leading to neuron morphological changes, neuron degeneration, inflammation, and ultimately behavior disorders.

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 human brain is the most complicated organ, that processes the information from the sensory organs, forms memories and controls motor functions. Neurons are the fundamental units of the brain. These highly specialized cells are connected via synapses. There are 100 billion neurons in the human brain that form the enormous complexity. To understand how the brain works, it is essential to study the fundamental units, the neurons. Neurons are highly polarized cells and critically rely on their cytoskeletal filaments to acquire and maintain their specialized morphology and functions. Microtubules are the major cytoskeletal components of neurons and they are important for neurodevelopment from differentiation, migration and development to synapses connection. They serve as tracks for long distance transport, support neuronal morphology, and control local signaling events. In neurons, microtubules are organized into a specialized architecture. Neurons express abundant microtubule-associated proteins (MAPs). It is becoming clear that MAPs are critical for the establishment and maintenance of the microtubule architecture. To date, our understanding on MAPs and their function is still quite limited, and it is what we aim to explore in this thesis.

The microtubule cytoskeleton is essential for nearly every stage of neurodevelopment, fromdifferentiation,migration, polarization, axon and dendrite development to synapse connections.During these processes, microtubules serves as ‘roads’ for motor proteins mediated cargotransport over long distances. As the neuron from human PNS can have axons with a totallength ranging from 10 mm to 100 m, the microtubule based motors transport life sustainingmaterials such as organelles, proteins or mRNAs from the soma to the axon terminal. Neurons express abundant MAPs to regulate the microtubule cytoskeleton. MAPs were initially defined as proteins that bind to and stabilize microtubules. More recently it is becoming clear that MAPs can regulate the microtubule network in many other ways, which will be explored in this thesis. In chapter 2 we describe the emerging concept that regionally confined MAPs act as a “MAP code” regulating motor activity. We found that MAP7 family members MAP7/7D1 are specifically enriched in somatodendritic compartment of the neuron, whereas MAP7D2 localizes in the proximal axon and promotes Kinesin-1 mediated cargo transport into axons. In chapter 3 we discussed how the E3 ubiquitin ligase family members MID1 and MID2 associate with microtubule cytoskeleton and control axon and dendrite development. In chapter 4 we show how MAPs act as microtubule crosslinkers to form the proximal axon specific microtubule fascicles. In chapter 5 we discuss the cross-talk between TRIM46 induced parallel microtubules and the AnkG mediated submembrane domain coordinate to drive theAIS assembly to establish neuronal polarity. In chapter 6 we discuss the mechanisms that underlie the specific microtubule organization in axons; the “MAP code” that guide Kinesin-1 with cargos out of dendrites and into axons; the mechanisms of how MAPs are confined to different compartments of neurons and how MAPs regulate axon branching.

Neurons are highly polarized cells with two distinct processes called axons and dendrites. To establish and maintain their specialized morphology and function, neurons use molecular motors: kinesins, myosins and dynein to steer cargo transport along the cytoskeleton into axons and dendrites. However, little is known about how specific cargos are directed into the distinct compartments of the neuronal cell. The work presented in this thesis focuses on investigating the molecular mechanisms of regulation of the intracellular transport in neurons. Here, we identify novel regulatory mechanisms for cargo-motor, motor-cytoskeleton and motor-motor interaction necessary for proper neuronal sorting, development and function.