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Ca2+ levels in the cell play an important role in both animals and plants. Abnormalities in these Ca2+ levels are related to several cancers. Therefore, many studies focus on Ca2+ concentrations in the cell. Often, proteins that light up when binding to Ca2+ are used to observe the presence of Ca2+. This light is then detected for imaging. This technique works fine for superficial imaging, but not if you want to image processes that happen deeper into the tissue. Photoacoustic (PA) imaging gets around this problem by detecting ultrasonic waves. To create such a signal, these Ca2+ binding proteins should radiate warmth instead of emitting light. To achieve this we introduced mutations into existing Ca2+ sensors. The main aim of these mutations was to stop this sensor from emitting light and make it radiate warmth instead. Due to Covid19-measures, we miss essential data to conclude if the created mutants are better candidates than the original senor for use in PA. We propose possible mutations for further research that might dim the original sensor. The second part of the project focuses on monitoring Ca2+ levels in cells that are induced by ErbB receptors on the cell membrane. ErbB receptors can give the cell signals to increase cell survival, growth or to start processes that will lead to the cell’s death. Abnormalities in these receptors are related to many cancers. These receptors need to be activated before the signal can travel further into the cell. Activation starts after a small molecule (ligand) binds to the receptor. This changes the shape of the receptor and makes it more likely to form pairs with other receptors or with itself (dimers). One receptor of this family, ErbB3, is not very active. For this reason, it is assumed that ErbB3 always needs to pair up with another family member before it is able to send signals down the cell. Some mutations in ErbB3 are found regularly in cancer. In this work, we wanted to observe if these mutations in ErbB3 have an influence on Ca2+ levels in cells. We found that some cells containing the normal ErbB3 receptor enhance Ca2+ levels, in contrast with cells containing the mutant receptor. This indicates that ErbB3 mutants do not the receptor more active. We then monitored Ca2+ concentrations in cells containing the normal ErbB3 receptor and other ErbB family members. We found that these kinds of cells could influence signals more than cells containing only the mutant ErbB3 receptor. Because of the Covid-19 outbreak, planned experiments involving cells containing the mutant ErbB3 receptor and normal other receptors were not carried out.

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Caenorhabditis elegans is a round worm living in free soil and is an often used model organism in laboratory environment. As its whole genome is known, it is very easy to make it transgenic. This transparent worm provides us with a convenient way to study neurobiology by labeling the proteins of interest with fluorescent proteins, so they can be seen by fluorescence microscopy. Optical microscopy however has a resolution limit of around 200 nanometer, so if we want to see objects smaller than this we need to resort to super-resolution microscopy techniques. The aim of this master thesis is to identify single synapses (i.e. connection point between 2 neurons through which information travels) in the neurons of C. elegans, namely between AIY and RIA interneurons, which belongs to the thermo-sensing circuit. These synapses we expected are smaller than 200nm and should be visualized by a super-resolution technique, called photoactivated localization microscopy (PALM). More specifically we are interested in the glutamate receptor subunit 3 (GLR-3), one component of these synapses. The neurons themselves are bigger and can be visualized using confocal laser scanning microscopy (CLSM). Transgenic DNA-constructs (plasmids) that can be used to highlight the target neurons were made and micro-injected into the worms as to make them fluorescent. After successful selection, we could observe AIY interneuron with enhanced cyan fluorescent protein (eCFP), and RIA interneuron with enhanced green fluorescent protein (eGFP), while we can see the glutamate receptors with the orange mEos2 fluorescent protein. By employing CLSM, the neurites of these two neurons were localized by fast 3D scanning, and the cross-point between the axons and the dendrite was identified. Subsequently, the sample was subjected to PALM, to visualize the expression pattern of the glutamate receptors at the identified crosspoint region in super-resolution (a resolution of 40nm could be achieved). In total, 2 axon cross-points were identified by CLSM. Therefore, this master thesis confirmed the advancement of the technique combining CLSM with PALM in the field of neurobiology research. Based on the present work, we aim to extend the study to elucidating the molecular mechanisms behind learning behavior and memory formation in the near future.

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Mammalian cells have the ability to respond to their environment and change their behavior correspondingly. The cells recognize the environmental signals, which can be chemical or physical, with the help of special signal transducing molecules. One of the molecules is epidermal growth factor receptor (EGFR). EGFR recognizes epidermal growth factor (EGF) outside the cell, forms dimer, and autophosphorylates. The phosphorylated EGFR has a lot of binding partners inside the cell. Some of them transduce the signal and lead to a change in the behavior of the cell. Some others are responsible for positive or negative feedback regulation of the signal. These molecules are working in a concerted way for proper response to the environmental cues. EGFR regulates various cell fates such as growth, division, death, and differentiation. Therefore, EGFR and its binding partners are key molecules to our health, and many types of cancer are caused by improper regulation of them. In this thesis, we aimed to study how the binding partners of EGFR are recruited from the inside of the cell to the border where EGFR is located when EGFR gets the EGF signal. For that, we created plasmid DNAs to express EGFR labeled with a red fluorescent protein and its binding partners, Grb2, MIG6 and PLCg,labeled with a green fluorescent protein in mammalian cells. By introducing these plasmids, cells produce labeled EGFR and binding partners. We took a series of pictures of the labeled molecules by scanning the surface of the cells. These scanned images hold a lot of information about the dynamics of molecules, which can be obtained by analyzing using an appropriate image-processing tool. I used the tool called RICS and analyzed the speed of the molecules as well as their tendency to bind. By assuming that all the molecules move at the same average speed, we could show that the binding partners of EGFR slowed down upon stimulation with EGF. We interpreted that more of the binding partners of EGFR would bind to it when it got stimulated, since EGFR is a lot slower than its binding partners. We inspected this interpretation in two different ways. First, we assumed that the molecules were moving at two possible speeds: one where they are moving freely and one where they are moving together with EGFR. We could show that the amount of freely moving molecules decreased and the amount of molecules moving together with EGFR increased when EGFR got its signal. Secondly, by analyzing pictures of EGFR and the binding partner together, we can directly see when two molecules are moving together and therefore when they bind to each other. We showed that more of the binding partners bind to EGFR when EGFR gets the EGF signal. We only scratched the surface with these image processing techniques. In principle, it is possible to analyze when and where the binding event is happening at the cell surface. This information has been shown to be very important for the decision making in the cell. There were some technical issues regarding the red label and the stability of the microscope to keep the focus on the cell surface. We suggested a number of solutions for these issues in this thesis. We believe that these image processing techniques will become more important for studying these decision making mechanisms in cells and that the knowledge from these techniques can eventually translate in better medicines to fight cancer and other diseases.

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Een stap in de goede richting voor Alzheimer onderzoek Alzheimer, de meeste mensen kennen wel iemand die met deze toetakelende ziekte te kampen heeft. Daarenboven zal het aantal getroffen mensen alleen maar toenemen, aangezien we steeds ouder worden. Aan de andere kant zullen we dan kunnen rekenen op minder (financiële) steun, vanwege de vergrijzing. Zo ziet de situatie er nu alleszins uit, maar wat als er medicatie zou kunnen worden ontwikkeld die Alzheimer kan voorkomen, genezen, of een halt toeroepen? Alzheimer wordt veroorzaakt door het afsterven van neuronen (zenuwcellen) in de hersenen. Daarenboven wordt het gekenmerkt door de aanwezigheid van zogenaamde neurofibrillaire ‘klitten’ die een proteïne genaamd Tau bevatten. Er worden dus als het ware kluwens van samengeklit Tau gevormd, waardoor deze proteïne zijn functie niet meer kan uitoefenen of misschien zelfs toxisch wordt. Dit is een probleem, aangezien Tau een noodzakelijke rol speelt in het functioneren van neuronen. Tau kan gemakkelijk van structuur veranderen en kan daardoor fase scheiding ondergaan, waarbij verschillende Tau proteïnen groeperen in vloeistof-achtige druppeltjes. Hoewel deze druppeltjes een normale functie zouden kunnen hebben, zouden ze ook een overgang kunnen representeren van ‘gezond’ Tau naar pathologisch Tau. Daarnaast blijkt ook de fosforylatie van Tau, met andere woorden, het toevoegen van fosfaatgroepen, geassocieerd te zijn met pathologische functies. Wij trachtten te onderzoeken of de fosforylatie van Tau op bepaalde locaties een effect heeft op de structuur en het fase scheiding gedrag van deze proteïne. Dit werd gedaan door middel van het nabootsen van fosforylatie. Aangezien Tau vaak van structuur verandert, is het moeilijk om deze te bestuderen met klassieke technieken. Daarom gebruikten wij een techniek waarbij we de afstand tussen bepaalde locaties van de moleculen kunnen meten. Aangezien de afstand tussen slechts twee verschillende locatie-paren werd getest, konden er weinig conclusies getrokken worden betreffende de structuur van de proteïne. Er waren echter wel aanwijzingen dat fosforylatie zorgt voor meer dynamische proteïnen. Deze zouden meer switchen tussen verschillende structuren. Daarnaast zagen we dat er grotere druppeltjes gevormd werden bij Tau waar fosforylatie bij werd nagebootst. Dit zou het gevolg kunnen zijn van de extra negatieve ladingen die aanwezig zijn in deze proteïne, waardoor de interacties binnen en tussen moleculen zouden kunnen veranderen. Deze bevindingen dragen bij aan het beter begrijpen van hoe Alzheimer tot stand komt, wat op zijn beurt kan bijdragen aan de ontwikkeling van medicatie.

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In the Alzheimer's disease (AD) field, the amyloid hypothesis remains the dominant hypothesis wherein an overproduction and/or clearance defect of toxic, aggregation-prone amyloid peptides is considered a major perpetrator of neurodegeneration and cognitive decline. Amyloid-β peptides are the result of a sequential cleavage of the amyloid precursor protein (APP).This amyloidogenic process starts when APP encounters Bace1, which sheds the extracellular domain of APP giving rise to membrane-bound APP C-terminal fragment (CTF), a direct substrate for γ-secretase proteolysis. Alternatively, APP can also undergo a non-amyloidogenic processing by ADAM10 and γ-secretase, releasing shorter non-amyloidogenic peptides. Recently, it was suggested that γ-secretase forms pre-existing complexes with the respective sheddases. However, these were biochemical studies, so whether they actually exist in cells is unclear.γ-secretase consists of four transmembrane proteins including nicastrin (NCT), PEN2, APH1 and presenilin (PSEN), the last being the catalytic subunit. Active γ-secretase complexes reside in post-Golgi compartments with PSEN1-complexes being distributed at the cell surface and endosomal compartments while PSEN2-complexes are restricted to late endosomes/lysosomes.Interestingly, mutations in PSENs resulting in familial early onset AD (FAD), appear to affect this processivity increasing the production of relatively longer/more toxic Aβ peptides.In the past years, a 3.5Å atomic resolution of γ-secretase structure was resolved revealing three conformational states and tight association of phosphatidylcholine molecules. More recently, the 3D structure of the complex including its substrates APP and Notch provided insight in how and where γ-secretase processing occurs. Based on this, computational modelling studies predicted conformational changes dependent on the lipid microenvironment, including cholesterol.Moreover, lipidomic studies on γ-secretase showed high sensitivity of the complex to lipid changes. Overall, γ-secretase structure is very dynamic and association to cholesterol or other lipid molecules determine changes in structure. Therefore, alterations to lipid environment or mutations that change affinity to certain lipids could therefore contribute as well to amyloidogenesis.Amyloidogenic processing has been proposed to occur preferentially in cholesterol and sphingomyelin rich nanodomains or lipid rafts. These are considered signaling platforms that include or exclude proteins based on their biophysical characteristics. Historically, lipid rafts were defined by biochemical fractionation with detergents, however, this approach is prone to induce association artifacts. As lipid rafts are also too small to be resolved by confocal microscopy, advanced microscopy techniques including single molecule microscopy have madepossible to analyze in situ lipid rafts.In parallel, microscopy studies on actin gave rise to an alternative mechanism to explain the heterogeneous distribution of proteins and lipids, the 'picket-fence' hypothesis. Here, actin forms an intracellular mesh that can hinder the movement of proteins and direct their movement to specific locations. To be effective to all proteins and lipids across the bilayer, this actin 'fence' would require 'pickets' or transmembrane proteins associated to actin that help in the movement of proteins and lipids bound only to the exoplasmic leaflet of the plasma membrane (PM). For long time no such pickets have being found, until recently thanks to single particle tracking (SPT) on living cells CD44 was shown to act as a picket, helping in organizing proteins and lipids at the PM. Therefore, evidence suggests that if existing, lipid rafts would be controlled by the actin cytoskeleton. To our knowledge, the use of super-resolution microscopy has not been exploited to study γ-secretase at the PM, including its dynamics and relationships to the lipid microenvironment. So far, most of our knowledge on γ-secretase is gathered essentially using biochemical approaches.Thus, a major objective of my research has been to setup advanced microscopy techniques, including structured illumination microscopy (SIM), photoactivated localization microscopy (PALM)/stochastic reconstruction microscopy (STORM) to study the biology of γ-secretase incellular lipid bilayers. First we addressed the distribution of PSEN1/γ-secretase complexes with respect to cluster formation, size and density. We found that strikingly instead of finding clusterization claimed by lipid rafts, we mostly found monomeric or dimeric associations of the complex. Moreover, by tagging two subunits of the same complex, we could visualize and confirm the stochiometry of the complex subunits. Next we addressed its association with a substrate or sheddase, were we found no significant associations for γ-secretase and its sheddases as recent biochemical studies have proposed. However, we did found association of γ-secretase with its substrate APP, which was expected. Finally, we went to living cells to address PSEN1/γ-secretase lateral diffusion (single particle tracking or SPT-PALM) and how this is influenced by internal (e.g. FAD mutations) and external factors such as cholesterol and actin. Although we found, as expected, an increase in mobility after actin depletion, cholesterol depletion turned out to immobilize, even at short time-points, the complex. This result indicate that cholesterol is indeed associated to γ-secretase and plays a critical role in its mobility, most likely affecting its conformation and thus, its conformation. But the precise mechanism is still under study.