1,721,615 research outputs found
Spatial Cell Biology: Dissecting and directing intracellular transport mechanisms
No full textCellular compartmentalization and intracellular transport mechanisms are important to establish and maintain the spatial organisation of proteins and organelles needed to ensure proper cellular functioning. Especially in polarized cells like neurons, the proper distribution of proteins into the right cellular compartment is crucial for correct functioning. In the first part of this thesis, we studied the transport of glutamate receptors into dendrites and synapses. Active long-range transport of recycling endosomes containing these glutamate receptors is mediated by the microtubule cytoskeleton and by motor proteins of the kinesin and dynein families. Myosin-V motor proteins can subsequently transport these endosomes into dendritic spines. Once recycling endosomes fuse with the plasma membrane either in the dendrite or spine, glutamate receptors are released onto the plasma membrane in which they can diffuse laterally. We showed that recycling endosomes in dendritic spines are required to maintain glutamate receptor levels at synapses and synapse architecture. We also found that spine morphology has an impact on how much glutamate receptors can be retained at synapses and thereby contributes to compartmentalization of the receptor. We then focused on the development of a novel optogenetic methods to manipulate organelle transport and positioning in living cells. Using light-sensitive dimerization domains of the LOV and phytochrome classes, we could recruit motor proteins of choice to a specific type of organelle. This led to rapid but highly controllable relocalization of these organelles in the illuminated area of interest allowing us to observe the effect of acute mislocalizations. Moreover, combining these two orthogonal optogenetic systems, we demonstrate simultaneous control of two different organelles. Using these assays we show that recycling endosomes have a local function in axonal outgrowth. A better understanding of the local functions of organelle positioning and the underlying mechanisms that regulate these processes may lead to novel therapeutic targets in diseases such as microvillus-inclusion disease (MVID), neurodegeneration or other developmental defects
Cellulaire dynamica – beweging in ons brein
No full textIn deze publieke les zal ik u het komende half uur de achtergronden van mijn onderzoeksgebied ‘celbiologie van zenuwcellen’ en mijn visie op de relatie tussen fundamenteel onderzoek en onze maatschappij met u bespreken. De celbiologie en neurobiologie zijn onderzoeksgebieden die de geheimen van het leven willen begrijpen en gebruiken. Ik zal u kennis laten maken met moleculen en celbiologische processen in onze hersenen, die bepalen of wij gezond zijn of niet. Ik wil met name de dynamiek – de beweging - van zenuwcellen met u bespreken
Strengthening Microtubules by Cuts that Heal
No full textMicrotubule-severing enzymes, which can remove tubulin dimers from microtubule lattices, participate in cytoskeletal remodeling in various contexts. A recent study showed that partially damaged microtubule shafts and new microtubule ends generated by these enzymes can incorporate GTP-tubulin and serve as sites of microtubule rescue and re-growth, explaining how severing enzymes can amplify microtubule arrays
Cellulaire dynamica – beweging van begin tot het eind
No full textDe bouwstenen van het leven zijn ons door grote recente ontwikkelingen in de biologie bekend geworden. We kennen de volledige sequentie van ons genoom en krijgen een steeds beter overzicht van de moleculen waaruit onze cellen zijn opgebouwd. De volgende vraag is hoe deze bouwstenen samenwerken om zo het leven vormen. Om deze vraag te kunnen beantwoorden moet men de dynamiek en werking van de cellulaire componenten in levende cellen zichtbaar maken. Moderne microscopische technieken maken dit mogelijk. Zo kan men de continue bewegingen van de filamenten van het celskelet en zelfs individuele moleculen nauwkeurig volgen. Deze dynamiek kan men vervolgens reconstrueren met gezuiverde componenten en hierdoor de moleculaire mechanismen die verantwoordelijk zijn voor deze continue beweging van het leven ontrafelen. Deze kennis is nodig om betere medicijnen tegen kanker, infecties en ouderdomsgerelateerde ziektes te ontwikkelen
Fishing in the ocean: Use of mass spectrometry to study protein interactions involved in cytoskeletal organization and cell division
No full textProtein functions often depend on physical interactions with other proteins. Sketching a map of protein-protein interactions is an important step to understand cellular processes. Over past decades, mass spectrometry based proteomics has increasingly become the method of choice for the identification and characterization of protein interactions. In this thesis, we have combined various mass spectrometry-based methods, diverse biochemical and molecular genetics approaches and advanced imaging techniques to get a better insight in protein interactions involved in cytoskeletal organization and cell division. Chemical cross-linking mass spectrometry provides a novel way to analyse protein complexes. In this thesis, we examine an integrated workflow that combines chemical cross-linking mass spectrometry with genetic fragmentation technique to accurately locate the minimal binding domains responsible for protein interactions. We show that chemical cross-linking mass spectrometry can efficiently guide the design of truncation or deletion mutants, however, not all the cross-links we found are within the minimal binding domain. Furthermore, our data indicate that chemical cross-linking mass spectrometry-based prediction of protein interaction domains might work better for folded protein domains than for protein regions predicted to be intrinsically disordered. We apply chemical cross-linking mass spectrometry in studying the interaction between the flavoprotein monooxygenase MICAL3 and the centralspindlin component MKLP1 during cytokinesis. We show that MKLP1 interacts with and recruits MICAL3 to the midbody. We propose that during cytokinesis, MICAL3 acts as a protein-binding hub, which promotes maturation of the intercellular bridge and abscission. We also use mass spectrometry-based technique to identify new binding partners for the microtubule minus-end-interacting proteins. We further investigate the role of these proteins in the regulation of microtubule minus end organization in interphase and in mitosis
Focus on the axon: from neuronal development to dynamic synapses
No full textNeurons acquire an optimized structure for the reception and processing of information, with long protrusions extending from the neuronal cell body. These protrusions are morphologically and functionally different from each other, and are crucial for the establishment of a dynamic and intricate neuronal network. The axon is a long protrusion responsible for the transmission of electrochemical signals to other neurons, while dendrites receive these signals. The propagation of information from the axon of one neuron to the dendrite of another happens at specialized regions called synapses. When a signal travels along the axon and reaches the presynapse, it induces the fusion of synaptic vesicles with the plasma membrane in the presynaptic terminal, releasing neurotransmitters. These neurotransmitters can bind to receptors present at the postsynaptic membrane of the receiving neuron, generating a signal that can be transmitted to the next neuron. To ensure the accurate communication between neurons, synapses must be correctly assembled during development and supplied with specific proteins to the pre- and postsynaptic compartments. As most of the proteins are synthesized in the cell body, they need to be transported to the proper location by motor proteins, which use the underlying actin and microtubule cytoskeleton as rails. The axon extends considerably from the soma to reach the appropriate synaptic partners, and the molecular mechanisms underlying this process have intrigued many neuroscientists. We can study axon development in a controlled environment by culturing neurons from rodent brains. In this system, the axon is formed within the first 24 hours after plating, and the factors that support axon development and outgrowth have to be delivered by motor proteins to the proper location. We have addressed the role of motor proteins in the extension of the axon in hippocampal neurons. Besides their role in axonal outgrowth, we showed that motor proteins are also required for the maintenance of synapses along the dendrites of hippocampal neurons. The presynaptic terminals are very dynamic structures, appearing, disappearing and reappearing at the same location along the axon. Inhibitory presynaptic boutons have been shown to be particularly dynamic, but the molecular mechanisms underlying these dynamics are not understood. In this thesis, we studied a new regulatory pathway of inhibitory presynaptic bouton dynamics triggered by the guidance protein Semaphorin4D
VHH Activators and Inhibitors for Protein Kinase C Epsilon
No full textProtein kinase C epsilon (PKCε), which is one of the novel PKC isozymes, is widely expressed throughout the body and has important roles in the function of the nervous, cardiovascular and immune systems. In order to better understand PKCε regulated pathways, isozyme specific activity modulators are desperately needed. Such compounds could also be developed into drugs for diseases such as diabetes, cancer and Alzheimer’s disease, where PKCε dysregulation is implicated. This thesis describes the selection and characterization of PKCε specific llama single chain antibodies, known as VHHs. VHHs have several advantages compared to the conventional antibodies consisting of two heavy and two light chains. VHHs are small, easy to clone, convenient to genetically modify, remarkably stable, and tend to be highly specific for the target antigen. PKCε specific VHHs were selected in one round from an immune phage-VHH library using phage display. The VHHs that showed the strongest binding to human PKCε were produced in E. coli as monovalent proteins and tested for their ability to influence PKCε activity in in vitro kinase activity assays. Three VHHs (A10, C1 and D1) were found to increase the activity of PKCε, whereas three other VHHs (C7, E6 and G8) inhibited PKCε kinase activity. These effects were PKCε specific, since the VHHs had no effect on the activity of PKCδ, PKCθ or protein kinase A. A more detailed kinetic analysis revealed that the activators increased PKCε activity by increasing the maximum rate of the reaction and/or by speeding up the reaction. Furthermore, VHHs E6 and G8 were found to be non-competitive enzyme inhibitors. Interestingly, the binding site of all of the tested VHHs was in the catalytic domain of PKCε. Moreover, the binding of the VHHs was found to be conformation dependent and species specific. In order to study whether the VHHs could also influence PKCε activity in a cellular context, the VHHs were cloned into a mammalian expression vector with a C-terminal mCherry tag, and the VHH-mCherry proteins were transiently expressed in HeLa cells. Several cellular effects, such as changes in cell morphology, increases in cytotoxicity, and inhibition of PKCε downregulation, were observed for some of the VHH-mCherry constructs. The most direct indication that the VHHs can also influence PKCε activity in cells came from PKCε-EGFP translocation studies, where the activator A10-mCherry increased the rate of PKCε-EGFP translocation to the membranes in response to phorbol ester stimulation. In contrast, the inhibitors C7-mCherry and G8-mCherry decreased the rate of translocation. The results presented in this thesis demonstrate that highly specific VHHs against an intracellular antigen can be selected in one round from an immune phage-VHH library. Furthermore, it was shown that the selected VHHs can act as activators or inhibitors of PKCε activity in vitro and in cells. In addition to the potential therapeutic applications of the VHHs for diseases with aberrant PKCε signalling, these PKCε specific activity modulators will be valuable tools in research into PKCε regulated pathway
Connecting the neuronal proteome: Unraveling protein dynamics in neurons using Mass Spectrometry-based proteomics
No full textSimilarly to the amazon rainforest which is formed by a multitude of different trees, many of them growing around and on top of each other with their branches and roots tightly interconnected, the human brain is composed by approximately 100 billion neurons organized into a complex matrix of connections and packed into highly specialized layers. In order to establish and maintain neuronal connections, neurons must be able to sort and deliver proteins to specific compartments with extreme precision. In fact, the protein composition of each single neuronal compartment not only determines its unique architectural morphology and function but has also a more profound effect on the formation of the neuronal network which is the essence of our brain. In this perspective, it is essential to study the primary building blocks of life, the proteins, alongside with their interactions and the molecular mechanisms regulating their functions. A key concept of modern biology is that proteins participate in complex, interconnected networks, rather than linear pathways. In the field of neurobiology, proteomics has recently started to showcase its full potential as a powerful methodology able to outperform classical biochemical approaches by allowing a more global understanding of protein dynamics and a more detailed characterization of intracellular protein networks and complexes. This thesis describes our efforts in advancing the understanding of selected mechanisms in neurons by using Mass Spectrometry (MS)-based proteomics applications. Latest MS techniques have been widely used to investigate neuronal differentiation, kinesin-mediated neuronal transport and protein-interaction networks in physiological or pathological condition
Masters of the tips: End Binding proteins orchestrate microtubule plus- and minus-end dynamics
No full textMicrotubules are dynamic polymers built of dimers of α- and β-tubulin, which attach to each other in a head-to-tail fashion. Microtubules can rapidly switch between phases of growth and shortening, a behavior known as “dynamic instability”. In cells, microtubule dynamics are also tightly controlled by numerous factors including microtubule plus end tracking proteins (+TIPs). End Binding proteins (EBs) are highly conserved +TIPs. In mammalian cells, the EB family includes three members, EB1, EB2 and EB3. EBs form the core of the +TIP network by recruiting to microtubule ends many structurally and functionally diverse +TIPs. EBs can also alter the properties of microtubule plus ends by promoting GTP hydrolysis by β-tubulin and possibly by modifying microtubule end structure. In Chapter 2, we applied CRISPR/Cas9 technology to generate a series of EB1, EB2 and EB3 mutant cell lines. Surprisingly, we found that EBs strongly affect microtubule minus-end organization: microtubule minus ends stabilized by CAMSAP2 were detached from Golgi membranes and the Golgi apparatus became more compact. Further studies showed that co-organization of microtubules and Golgi membranes depends on the EB1/EB3-myomegalin-AKAP450 complex, which tethers microtubules to the Golgi and counteract compaction of Golgi stacks. In Chapter 3, we investigated how EBs and their partners affect the properties of growing microtubule plus ends. We generated new EB1, EB2 and EB3 triple knockout HeLa cell lines. We compare the length of EB comets in vivo and in vitro. We found a stable, depolymerization-resistant microtubule zone in the tip-proximal region. We also found that EBs inhibit mobility of microtubule tips and make their growth more persistent. Experiments with EB deletion mutants showed that these properties depend on the recruitment of EB binding partners. In Chapter 4, we explored the possibility that liquid-liquid phase separation (LLPS) plays a role in microtubule tip regulation. We found that SLAIN2 can form droplet in cells, particularly in EB1, EB2 and EB3 triple knockout cells. We also found that EBs and chTOG were recruited to SLAIN2 droplets, whereas CLASP and CLIP170 were not. We also found that SLAIN2 and chTOG, but not EB3, could efficiently form droplets in vitro, and the droplets containing chTOG could nucleate microtubule asters. In Chapter 5, we investigated how different microtubule binding domains could affect microtubule dynamics in vitro when targeted to microtubule tips by the N-terminal part of EB3. We found that fusions of SLAIN2 and spectraplakin caused no obvious changes, fusion of the TOG domains of CLASP2 suppressed catastrophes or induced rescues. Fusion of different TOG domain of chTOG increased catastrophe frequency. Fusion of the GAR domain of spectraplakin and the CAP-Gly domains of CLIP170 induced rescues, fusion of the microtubule binding domain of MAP7 reduced microtubule depolymerization rate. The same fusion proteins did not affect microtubule dynamics in EB1, EB2 and EB3 triple knockout cells as they did in vitro. In conclusion, we have obtained new insights into how EBs control microtubule networks and generated new tools that will help in future to unravel cellular mechanisms controlling microtubule polymerization
Cellular cartography: mapping the neuronal microtubule network using super-resolution microscopy
No full textDescribed in this thesis are the development and use of novel single molecule localization microscopy technologies to gain new insights into (neuronal) microtubule organization. The image quality of single molecule localization microscopy (SMLM) depends on a sound optical setup. Aberrations introduced by optical components result in optical distortions, lower photon counts, and a decreased localization precision. Avoiding aberrations or correcting them is therefore of great importance. We show that SMLM deep in biological samples is aided by the use of a deformable mirror to decrease aberrations. At the same time the deformable mirror can be used to encode information about the z-position of molecules allowing them to be localized with high accuracy in three dimensions. Optimized fixation protocols and fluorescent probes are just as important for high quality images. The immunostaining protocol described in this thesis allows SMLM of microtubules and microtubule associated proteins with low background signal and high labeling density. This protocol was used to show that microtubule organization drastically changes during neuronal development, and that the microtubule associated protein CAMSAP2 decorates MT minus-ends in neurons. Optimization of fluorescent probes in terms of brightness, return percentage, and size also leads to higher quality SMLM imaging. The commonly used bright monomeric red fluorescent protein mCherry turned out to be a high quality SMLM probe when exploiting a purely chemical caging mechanism with a return percentage up to 80%. Other novel probes very small in size, called tubulin nanobodies, were shown to enable investigation of individual microtubules in dense bundles, which is not possible with conventional antibodies. A property of microtubules that cannot be determined with immunostaining is their orientation. We showed, using a processive kinesin motor protein as a probe for SMLM. that the orientation of MTs can be determined in fixed cells and, surprisingly, that there is local orientational order in the dendritic MT array. In conclusion, described in this thesis are the development of novel SMLM methods and their use to find new insights in the neuronal microtubule organization
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