The actin cytoskeleton plays a vital role in various biological processes such as cell migration, morphogenesis, and intracellular trafficking. The polymerization of actin filaments at membranes provides the force for generating dynamic actin structures such as protrusions and invaginations that drive these processes. In filopodia, which are finger-like protrusions comprised of bundled actin filaments, actin regulatory proteins are believed to assemble a distal 'tip complex' which stimulates actin nucleation at the membrane. However how these regulators collectively behave in a macromolecular complex still remains poorly understood. To understand the macromolecular nature of these complexes, I investigated the dynamic properties and spatial organization of actin regulatory factors, using an in vitro reconstitution assay for filopodia-like structures (FLS) utilizing artificial lipid bilayers and Xenopus laevis egg extracts. FRAP analysis of seven actin regulatory factors (Toca-1, N-WASP, GTPase-binding domain, Ena, VASP, Diaph3, Fascin) revealed that the FLS tip complex has both dynamic and stable properties, with different proteins displaying distinct dynamics. Further analyses on the membrane-binding protein Toca-1 showed that its dynamic turnover is controlled by interactions with actin and exchange of molecules with solution. Single-molecule localization microscopy resolved the nanoscale organization of Toca-1, showing its arrangement into flat plaque-like and narrowly elevated tubular substructures. Plaque-like structures showed similarities to phase-transition patterns, while tubule-like structures closely resembled those previously found to decorate membrane tubules in vitro, which are thought to be involved in endocytic membrane remodeling. Endocytic accessory proteins such as SNX9 and Dynamin2 were also found to localize to FLS tips. This work provides new insights into the dynamics and organization of protein ensembles at actin nucleation sites, and proposes a novel link between endocytosis and filopodia formation, which is relevant to understanding how cells decide when and where to assemble actin at the membrane.

Human Natural Killer (NK) cells are regulated by a variety of germ-line encoded activating and inhibitory receptors. Broadly, activating receptors detect ligands that are expressed or up-regulated on cancerous or infected cells, while inhibitory receptors bind self-molecules to induce tolerance against healthy cells. Highly homologous pairs of activating and inhibitory receptors are also expressed on NK cells, including Killer Ig-like Receptors KIR2DL1 and KIR2DS1, which bind the same ligands, class I MHC proteins from the C2 group. Here, two super-resolution microscopy techniques, stimulated emission depletion (STED) and ground state depletion microscopy followed by individual molecule return (GSDIM) were used to examine the nanometre-scale organization of KIR2DL1 and KIR2DS1, as well as molecules engaged in their signalling. Both receptors were observed to constitutively assemble in nanometre-scale clusters at the surface of NK cells but displayed differential patterns of clustering - the activating receptor KIR2DS1 formed nanoclusters 2.3-fold larger than its inhibitory counterpart KIR2DL1. Site-directed mutagenesis established that the size of nanoclusters was controlled by transmembrane amino-acid 233, a lysine in KIR2DS1. Mutated variant of KIR2DS1 in which lysine 233 was substituted with alanine formed significantly smaller clusters than the wild-type KIR2DS1. Reciprocally, substitution of isoleucine found at position 233 in KIR2DL1 sequence with lysine resulted in the receptor assembling into larger clusters. Super-resolution microscopy also revealed two ways in which KIR nanoclusters impact signalling. First, KIR2DS1 and DAP12 nanoclusters were juxtaposed in the resting-cell state but coalesced upon receptor ligation. Second, quantitative super-resolution microscopy revealed that membrane-proximal clusters of the kinase ZAP-70 or phosphatase SHP-1, as well as their phosphorylated active forms, were more often found in contact with larger KIR nanoclusters. Together, this work has established that size of KIR nanoclusters depends on the transmembrane sequence and impacts downstream signalling.

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Natural Killer (NK) cells are cytotoxic lymphocytes that can recognize and kill virally infected or tumour transformed cells by the secretion of cytolytic granules containing perforin. An individual NK cell can kill several target cells sequentially. Each target cell can trigger NK cell activation via different activating ligands and here we report that the order in which ligands are encountered affects the NK cell response. When NK cells are repeatedly activated via their Fc receptor CD16, with the therapeutic antibody rituximab, perforin secretion decreases with each stimulation. However, perforin secretion is restored to its initial level upon subsequent activation by MICA, which ligates NKG2D. Repeated stimulation of NK cells via MICA also decreases the degranulation capacity of NK cells but, strikingly, this effect cannot be rescued by a subsequent stimulation with rituximab. The strength of perforin secretion is also translated to killing of Daudi target cells, expressing different ligands. When Daudi, opsonised with rituximab is the first target NK cell encounters, the sequential killing of another opsonised rituximab or Daudi, expressing MICA will not be affected. But, when Daudi-MICA is met first, the consecutive killing of Daudi-MICA as well as Daudi-rituximab will be impaired. We found that the mechanism underlying these differential outcomes involves shedding of CD16, which occurs upon NK cell activation through both, CD16 and NKG2D. Shedding of CD16 renders the cells insensitive to further activation via that receptor but they remain competent for further activation through NKG2D. Interestingly, however, we also identified the beneficial role of CD16 shedding for NK cell serial killing. NK cells are more motile on rituximab-coated surfaces than on MICA-coated surfaces and their migration speed decreases upon inhibition of CD16 shedding. Moreover, the inhibition of CD16 shedding also prevents the NK cell detachment from rituximab opsonised Daudi cells. Thus, the shedding of the receptor can serve to augment NK cell motility to move between target cells. Efficient NK cell detachment also correlated with their increased survival. Finally, we report that CD16 is constitutively organised in small, dense nanoclusters and that the ligation with rituximab does not affect their spatial distribution. Despite the shedding of the receptor, leading to less protein molecules at the surface, the area of these clusters remains the same. Together these data suggest that CD16 shedding hinders NK cell cytotoxicity against opsonised targets, but promotes their movements between different targets. Thus, receptor shedding is important for efficient NK cell serial killing. Manipulation of CD16 shedding, perhaps by boosting its recovery, might therefore represent an important target for NK cell-based therapies including treatments with therapeutic antibodies.

Optical cell manipulation allows precise and non-invasive exploration of mammalian cell function and physiology for medical applications. Plants, however, represent a vital component of the Earth's ecosystem and the knowledge gained from using optical tools to study plant cells can help to understand and manipulate useful agricultural and ecological traits. This thesis explores the potential of several biophotonic techniques in plant cells and tissue. Laser-mediated introduction of nucleic acids and other membrane impermeable molecules into mammalian cells is an important biophotonic technique. Optical injection presents a tool to deliver dyes and drugs for diagnostics and therapy of single cells in a sterile and interactive manner. Using femtosecond laser pulses increases the tunability of multiphoton effects and confines the damage volume, providing sub-cellular precision and high viability. Extending current femtosecond photoporation knowledge to plant cells could have sociological and environmental benefits, but presents different challenges to mammalian cells. The effects of varying optical and biological parameters on optical injection of a model plant cell line were investigated. A reconfigurable optical system was designed to allow easy switching between different spatial modes and pulse durations. Varying the medium osmolarity and optoinjectant size and type affected optoinjection efficacy, allowing optimisation of optical delivery of relevant biomolecules into plant cells. Advanced optical microscopy techniques that allow imaging beyond the diffraction limit have transformed biological studies. An ultimate goal is to merge several biophotonic techniques, creating a plant cell workstation. A step towards this was demonstrated by incorporating a fibre-based optical trap into a commercial super-resolution microscope for manipulation of cells and organelles under super-resolution. As proof-of-concept, the system was used to optically induce and quantify an immunosynapse. The capacity of the super-resolution microscope to resolve structure in plant organelles in aberrating plant tissue was critically evaluated.

Over the past few decades, fluorescence microscopy has proven to become the most widely used imaging technique in the field of life sciences. Unfortunately, all classical optical microscopy techniques have one thing in common: their resolution is limited by the diffraction. Thankfully, due to the very strong interest, development of fluorescent microscopy techniques is very intense, with novel solutions surfacing repeatedly. The major breakthrough came with the appearance of super-resolution microscopy techniques, enabling imaging well below the diffraction barrier and opening the new era of nanoscopy. Among the fluorescent super-resolution techniques, Stimulated Emission Depletion (STED) microscopy has been particularly interesting, as it is a purely optical technique which does not require post image processing. STED microscopy has proven to resolve structures down to the molecular resolution. However, super-resolution microscopy is not a cure to all the problems and it also has its limits. What has shown to be particularly challenging, was the super-resolution imaging of thick samples. With increased thickness of biological structures, the aberrations increase and signal-to-noise (SNR) decreases. This becomes even more evident in the super-resolution imaging, as the nanoscopic techniques are especially sensitive to aberrations and low SNR. The aim of this work is to propose and develop a 3D STED microscope that can successfully image thick biological samples with nanoscopic resolution. In order to achieve that, adaptive optics (AO) has been employed for correcting the aberrations, using the indirect wavefront sensing approach. This thesis presents a custom built 3D STED microscope with the AO correction and the resulting images of thick samples with resolution beyond diffraction barrier. The developed STED microscope achieved the resolution of 60nm in lateral and 160nm in axial direction. What is more, it enabled super-resolution imaging of thick, aberrating samples. HeLa, RPE-1 cells and dopaminergic neuron differentiated from human IPS cells were imaged using the microscope. The results shown in this thesis present 3D STED imaging of thick biological samples and, what is particularly worth to highlight, 3D STED imaging at the 80μm depth, where the excitation and depletion beams have to propagate through the thick layer of tissue. 3D STED images at such depth has not been reported up to date.

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Fluorescence microscopy offers the opportunity to image biological samples noninvasively in real time and has become an essential analytical tool in the biomedical laboratory. Nonetheless, the phenomenon of diffraction imposes stringent limitations on the resolving power of conventional microscopes, preventing the spatial resolution of fluorescent species co-localized within areas of nanoscaled dimensions. Time, however, can be exploited to distinguish fluorophores within the same subdiffraction area, if their fluorescence can be switched independently, and reconstruct sequentially their spatial distribution. In this context, photolytic reactions and photochromic transformations can be invoked to switch fluorescence under optical control. Fluorescent units, such as inorganic semiconductor nanoparticles and organic dyes, and photoactive components can be operated within a common supramolecular matrix or integrated within the same molecular construct to produce photoswitchable fluorescent assemblies. In the resulting systems, electronic communication between the components can be designed in order to photoactivate or photodeactivate fluorescence respectively. Both mechanisms can be exploited to overcome diffraction, and ultimately permit the reconstruction of images with resolution down to the nanometer level, in combination with appropriate illumination protocols.