A practical review on the measurement tools for cellular adhesion force

Cell cell and cell matrix adhesions are fundamental in all multicellular organisms. They play a key role in cellular growth, differentiation, pattern formation and migration. Cell-cell adhesion is substantial in the immune response, pathogen host interactions, and tumor development. The success of tissue engineering and stem cell implantations strongly depends on the fine control of live cell adhesion on the surface of natural or biomimetic scaffolds. Therefore, the quantitative and precise measurement of the adhesion strength of living cells is critical, not only in basic research but in modern technologies, too. Several techniques have been developed or are under development to quantify cell adhesion. All of them have their pros and cons, which has to be carefully considered before the experiments and interpretation of the recorded data. Current review provides a guide to choose the appropriate technique to answer a specific biological question or to complete a biomedical test by measuring cell adhesion

Binding Kinetics of Proteins at Immune-Cell Contacts

Protein-protein interactions are crucial in numerous cellular functions and biological processes that take place inside our body. It is therefore not surprising that these interactions also govern the response of our body´s defence mechanism, the so-called immune system, towards an infection. Understanding how proteins interact entails studying the binding affinity (strength) and the lifetime (duration) of the protein-protein interaction to better decompose how an immune response is initiated and how we can explore this knowledge to treat diseases. In this thesis, total internal fluorescence microscopy (TIRF) and single-molecule imaging were used to observe and characterize protein-functionalized supported lipid bilayers (SLBs) interacting with immune cells to obtain the binding kinetics of various protein-protein pairs.In the first part of this thesis, the interaction between the rat CD2 (rCD2) adhesion protein and its ligand rat CD48T92A (rCD48T92A), a high-affinity mutant of the wild type rat CD48, was used to establish a new method of obtaining single-cell binding affinities of T cells interacting with SLBs using imidazole titrations. The results showed a relatively small spread in the rCD2-rCD48T92A binding affinity values despite the considerable spread of receptor densities within the cell population. The lifetime of the rCD2/rCD48T92A interaction was also investigated using single-molecule imaging and tracking displaying a similarly small lifetime spread within the cell population. Using both these methods, the single-cell binding affinity and lifetime of the cell population can be investigated and their spread can provide information concealed withpopulation-average techniques.The second part of the thesis focused on the CD4 co-receptor whose role in initiating an immune response is ambiguous. Even though the CD4 co-receptor increases the sensitivity of T cell signalling manyfold, it binds to its ligand, peptide major histocompatibility complex II (pMHCII), with the lowest binding affinity known to this day. The CD4-MHC II interaction is so weak that adhesion molecules are needed to ensure a successful CD4-MHC II contact formation. For this reason, the influence of an adhesion molecule, rat CD2, on the obtained binding kinetics of the human CD4 co-receptor was initially examined showing that the accumulation of CD4 was influenced when having a high concentration of bound CD2 inside the cell-SLB contacts. Later, the studies focused on the CD4-TCR-MHC II ternary complex where it was demonstrated that the presence of L3-12 TCR strongly supported the CD4-MHC II interaction by increasing the local density of MHC II inside the cell-SLB contacts. However, the presence of TCR did not seem to significantly influence the specific affinity for CD4 to MHC II. Lastly, CD4 binding studies showed that the co-receptor did not noticeably affect the TCR-MHC II binding at physiological levels of hCD4 in the SLB

Selective and wash‐resistant fluorescent dihydrocodeinone derivatives allow single‐molecule imaging of μ‐opioid receptor dimerization

μ‐Opioid receptors (μ‐ORs) play a critical role in the modulation of pain and mediate the effects of the most powerful analgesic drugs. Despite extensive efforts, it remains insufficiently understood how μ‐ORs produce specific effects in living cells. We developed new fluorescent ligands based on the μ‐OR antagonist E‐p‐nitrocinnamoylamino‐dihydrocodeinone (CACO), that display high affinity, long residence time and pronounced selectivity. Using these ligands, we achieved single‐molecule imaging of μ‐ORs on the surface of living cells at physiological expression levels. Our results reveal a high heterogeneity in the diffusion of μ‐ORs, with a relevant immobile fraction. Using a pair of fluorescent ligands of different color, we provide evidence that μ‐ORs interact with each other to form short‐lived homodimers on the plasma membrane. This approach provides a new strategy to investigate μ‐OR pharmacology at single‐molecule level

Real-time visualization of clustering and intracellular transport of gold nanoparticles by correlative imaging.

Mechanistic understanding of the endocytosis and intracellular trafficking of nanoparticles is essential for designing smart theranostic carriers. Physico-chemical properties, including size, clustering and surface chemistry of nanoparticles regulate their cellular uptake and transport. Significantly, even single nanoparticles could cluster intracellularly, yet their clustering state and subsequent trafficking are not well understood. Here, we used DNA-decorated gold (fPlas-gold) nanoparticles as a dually emissive fluorescent and plasmonic probe to examine their clustering states and intracellular transport. Evidence from correlative fluorescence and plasmonic imaging shows that endocytosis of fPlas-gold follows multiple pathways. In the early stages of endocytosis, fPlas-gold nanoparticles appear mostly as single particles and they cluster during the vesicular transport and maturation. The speed of encapsulated fPlas-gold transport was critically dependent on the size of clusters but not on the types of organelle such as endosomes and lysosomes. Our results provide key strategies for engineering theranostic nanocarriers for efficient health management

Selective and Wash‐Resistant Fluorescent Dihydrocodeinone Derivatives Allow Single‐Molecule Imaging of μ‐Opioid Receptor Dimerization

μ‐Opioid receptors (μ‐ORs) play a critical role in the modulation of pain and mediate the effects of the most powerful analgesic drugs. Despite extensive efforts, it remains insufficiently understood how μ‐ORs produce specific effects in living cells. We developed new fluorescent ligands based on the μ‐OR antagonist E‐p‐nitrocinnamoylamino‐dihydrocodeinone (CACO), that display high affinity, long residence time and pronounced selectivity. Using these ligands, we achieved single‐molecule imaging of μ‐ORs on the surface of living cells at physiological expression levels. Our results reveal a high heterogeneity in the diffusion of μ‐ORs, with a relevant immobile fraction. Using a pair of fluorescent ligands of different color, we provide evidence that μ‐ORs interact with each other to form short‐lived homodimers on the plasma membrane. This approach provides a new strategy to investigate μ‐OR pharmacology at single‐molecule level

Fast Neuronal Imaging using Objective Coupled Planar Illumination Microscopy

Complex computations performed by the brain are produced by activities of neuronal populations. There is a large diversity in the functions of each individual neuron, and neuronal activities occur in the time scale of milliseconds. In order to gain a fundamental understanding of the neuronal populations, one has to measure activity of each neuron at high temporal resolution, while investigating enough neurons to encapsulate the neuronal diversity. Traditional neurotechniques such as electrophysiology and optical imaging are constrained by the number of neurons whose activities can be simultaneously measured or the speed of measuring such activities. We have developed a novel light-sheet based technique called Objective Coupled Planar Illumination: OCPI) microscopy which is capable of measuring simultaneous activities of thousands of neurons at high speeds. In this thesis I pursue the following two aims: * Improve OCPI microscopy by enhancing the spatial resolution deeper in tissue. Tissue inhomogeneity and refractive index mismatch at the surface of the tissue lead to optical aberrations. We have compensated for such aberrations by: 1) miniaturizing the OCPI illumination optics, so as to enable more vertical imaging of the tissue,: 2) correcting for the angular defocus caused by the refraction at the immersion fluid/tissue interface, and: 3) applying adaptive optics to correct for higher order optical aberrations. The improvement in the depth at which one can image tissue will enable the measurement of activities of neuronal populations in cortical areas. * Measure the diversity in the expression pattern of VSNs responsive to sulfated steroids. Nodari et al. have identified sulfated steroids as a novel family of ligands which activate vomeronasal sensory neurons: VSNs). Due to the experimental constraints, it has not been possible to obtain a comprehensive understanding of the number, location and functional characteristics of the sulfated steroid responsive VSNs. Applying OCPI microscopy and calcium imaging to simultaneously image thousands of VSNs, we show that the sulfated steroid responsive neurons: 1) have unique ligand preferences,: 2) are predominantly present in the apical regions of the VNO, and: 3) that the choice of expression of a receptor type is not purely stochastic

Ligand-induced dynamics of neurotrophin receptors investigated by single-molecule imaging approaches

Neurotrophins are secreted proteins that regulate neuronal development and survival, as well as maintenance and plasticity of the adult nervous system. The biological activity of neurotrophins stems from their binding to two membrane receptor types, the tropomyosin receptor kinase and the p75 neurotrophin receptors (NRs). The intracellular signalling cascades thereby activated have been extensively investigated. Nevertheless, a comprehensive description of the ligand-induced nanoscale details of NRs dynamics and interactions spanning from the initial lateral movements triggered at the plasma membrane to the internalization and transport processes is still missing. Recent advances in high spatio-temporal resolution imaging techniques have yielded new insight on the dynamics of NRs upon ligand binding. Here we discuss requirements, potential and practical implementation of these novel approaches for the study of neurotrophin trafficking and signalling, in the framework of current knowledge available also for other ligand-receptor systems. We shall especially highlight the correlation between the receptor dynamics activated by different neurotrophins and the respective signalling outcome, as recently revealed by single-molecule tracking of NRs in living neuronal cells