INVESTIGATING THE MECHANISM OF BACTERIAL CELL DIVISION WITH SUPERRESOLUTION MICROSCOPY

The molecular mechanisms that drive bacterial cytokinesis are attractive antibiotic targets that remain poorly understood. The machinery that performs cytokinesis in bacteria has been termed the 'divisome' (see Chapter 1 for description). The most widely-conserved divisome protein, FtsZ, is an essential tubulin homolog that polymerizes into protofilaments in a nucleotide-dependent manner. These protofilaments assemble at midcell to form the ‘Z-ring’, which has been the prevailing candidate for constrictive force generation during cell division. However, it has been difficult to experimentally test proposed Z-ring force generation models in vivo due to the small size of bacteria (< 1 μm diameter for E. coli) compared to the diffraction-limited resolution of light (~ 0.3 μm). In this work, quantitative superresolution and time-lapse microscopy were applied to examine whether Z-ring structure and function indeed play limiting roles in driving E. coli cell constriction (Chapter 2). Surprisingly, these studies revealed that the rate of septum closure during constriction is robust to substantial changes in many Z-ring properties, including the GTPase activity of FtsZ, molecular density of the Z-ring, the timing of Z-ring disassembly, and the absence of Z-ring assembly regulators. Further investigation revealed that septum closure rate is instead highly coupled to the rate of cell wall growth and elongation, and can be modulated by coordination with chromosome segregation. Taken together, these results challenge the Z-ring centric view of constriction force generation, and suggest that cell wall synthesis and chromosome segregation likely drive the rate and progress of cell constriction in bacteria. These investigations were made possible by advancements in quantitative superresolution microscopy techniques (see Chapter 3 for overview). One major obstacle encountered during the course of this work, and shared by those utilizing localization-based superresolution microscopy techniques, was the overestimation of molecule numbers caused by fluorophore photoblinking. Thus, Chapter 4 describes a systematic characterization of the effects of photoblinking on the accurate construction and analysis of superresolution images. These characterizations enabled the development of a simple method to identify the optimal clustering thresholds and an empirical criterion to evaluate whether an imaging condition is appropriate for accurate superresolution image reconstruction. Both the threshold selection method and imaging condition criterion are easy to implement within existing PALM clustering algorithms and experimental conditions

Super-resolution mapping of receptor engagement during HIV entry

The plasma membrane (PM) serves as a major interface between the cell and extracellular stimuli. Studies indicate that the spatial organisation and dynamics of receptors correlate with the regulation of cellular responses. However, the nanoscale spatial organisation of specific receptor molecules on the surface of cells is not well understood primarily because these spatial events are beyond the resolving power of available tools. With the development in super-resolution microscopy and quantitative analysis approaches, it optimally poises me to address some of these questions. The human immunodeficiency virus type-1 (HIV-1) entry process is an ideal model for studying the functional correlation of the spatial organisation of receptors. The molecular interactions between HIV envelope glycoprotein (Env) and key receptors, CD4 and co-receptor CCR5/CXCR4, on the PM of target cells have been well characterised. However, the spatial organisation that receptors undergo upon HIV-1 binding remains unclear. In this project, I established a Single Molecule Localisation Microscopy (SMLM) based visualisation and quantitative analysis pipeline to characterise CD4 membrane organisation in CD4+ T cells, the main host cell target for HIV-1 infection. I found that prior to HIV engagement, CD4 and CCR5 molecules are organised in small distinct clusters across the PM. Upon HIV-1 engagement, I observed dynamic congregation and subsequent dispersal of virus-associated CD4 clusters within 10min. I further incorporated statistical modelling to show that this reorganisation is not random. This thesis provides one of the first nanoscale imaging and quantitative pipelines for visualising and quantifying membrane receptors. I showed that this quantitative approach provides a robust methodology for understanding the recruitment of HIV-1 receptors before the formation of a fusion pore. This methodology can be applied to the analyses of the nanoscale organisation of PM receptors to link the spatial organisation to function

Dynamics of the plasma membrane transporter GLUT4

Glucose homeostasis in the human body is maintained by hormones of the pancreas, mostly glucagon and insulin. Insulin is secreted when blood glucose levels are high and triggers a signalling cascade that results in glucose uptake via the glucose transporter GLUT4 in peripheral tissues. GLUT4 is the only glucose transporter that responds to insulin stimulation and it slowly recycles between intracellular storage compartments and the plasma membrane. In the basal state, the majority of GLUT4 is intracellularly localised. Insulin stimulation results in movement (“translocation”) of GLUT4 from these intracellular stores to the plasma membrane. The signalling cascade from insulin binding to its receptor to translocation of GLUT4 is comparatively well understood. Less is known about the dynamics of GLUT4 within the plasma membrane itself. Advances in light microscopy techniques, such as Total Internal Reflection Fluorescence and super-resolution microscopy, have allowed new insights into the events in the membrane. It has recently been proposed that GLUT4 is located in plasma membrane clusters and that another effect of insulin is the dispersal of these GLUT4 clusters. The main objective of this work was to develop a microscopy-based assay to visualise and quantify these clusters and to investigate the molecular mechanisms behind clustering and dispersal of the glucose transporter in response to insulin. The majority of this work has been carried out in 3T3 L1 adipocytes, a widely used cell model for the study of GLUT4. However, this cell line is difficult to maintain, and its genetic manipulation is very challenging. For this reason, we investigated HeLa cells as a suitable substitute cell model for preliminary screenings. Using Total Internal Reflection Fluorescence Microscopy and Spatial Intensity Distribution Analysis, we gained new insight into the dynamics of plasma membrane GLUT4 in both 3T3 L1 adipocytes and HeLa cells. We found that the transporter forms an oligomer of high order in the plasma membrane in both cell types. Further, we compared the dynamics of GLUT4 mobilisation in response to insulin and found similar results. Based on these findings, we carried out an siRNA knock-down screening to determine proteins involved in intracellular GLUT4 trafficking and found that GOSR1 and Ykt6 are promising targets for further examination. Single molecule localisation microscopy allowed us to accomplish our aim to assay GLUT4 clustering and dispersal. Using dSTORM and Ripley’s K-function, as well as Bayesian cluster analysis methods, we showed that GLUT4 is indeed located in clusters in the plasma membrane and that insulin stimulation leads to its dispersal. We found that treatment with Galectin-3, a drug that inhibits glucose uptake, impedes the dispersal. Building upon previous research in our group that identified EFR3a as a membrane-localised protein involved in glucose uptake, we knocked-down EFR3a in 3T3 L1 adipocytes and found that this also disrupts GLUT4 dispersal, which we hypothesise could be a potential drug target for type 2 diabetes. Taken together, the findings presented in this thesis suggest HeLa cells as a suitable cell model for initial assessments of research questions related to GLUT4 trafficking. Furthermore, a robust assay to measure GLUT4 dispersal was established

Establishing super-resolution imaging of biosilica-embedded proteins in diatoms

Kieselalgen – auch Diatomeen genannt – verfügen über die einzigartige Fähigkeit, nanostrukturierte, hierarchisch aufgebaute Zellwände aus Siliziumdioxid – auch als Biosilica bekannt – mit beispielloser Genauigkeit und Reproduzierbarkeit zu bilden. Ein tieferes Verständnis für diesen Prozess, der als “Biomineralisation“ bekannt ist, ist nicht nur auf dem Gebiet der Grundlagenforschung zu Kieselalgen sehr bedeutsam, sondern auch für die Nutzung dieser Nanostrukturierung in den Materialwissenschaften oder der Nanobiotechnologie. Nach dem derzeitigem Stand der Wissenschaft wird diese Strukturierung durch die Selbstorganisation von Proteinmustern, an denen sich das Siliziumdioxid bildet, erreicht. Um die Funktion und das Zusammenspiel einzelner Proteine, die an diesem Biomineralisationsprozess beteiligt sind, entschlüsseln zu können, ist es essentiell ihre strukturelle Organisation aufzuklären und diese mit den morphologischen Zellwandmerkmalen zu korrelieren. Die Größenordnung dieser Merkmale ist im Bereich von Nanometern angesiedelt. Mit Hilfe der Elektronenmikroskopie können diese Biosilicastrukturen aufgelöst werden, jedoch ist keine proteinspezifische Information verfügbar. Ziel dieser Arbeit war es daher, eine Technik zu etablieren, die in der Lage ist, einzelne Biosilica-assozierte Proteine mit Nanometer-Präzision zu lokalisieren. Um dieses Ziel zu erreichen, wurde Einzelmoleküllokalisationsmikroskopie (single-molecule localization microscopy, kurz: SMLM) beispielhaft in der Kieselalge Thalassiosira pseudonana etabliert. Die Position verschiedener Biosilica-assoziierte Proteine innerhalb des Biosilicas und nach dessen chemischer Auflösung wurde mit einer hohen räumlichen Auflösung bestimmt. Um quantitative Ergebnisse zu erhalten, wurde ein Analyse-Workflow entwickelt, der grafische Benutzeroberflächen und Skripte für die Visualisierung, das Clustering und die Kolokalisation von SMLM Daten beinhaltet. Um optimale Markierungen für SMLM an Biosilica-eingebetteten Proteinen zu finden, wurde ein umfassendes Screening von photo-schaltbaren fluoreszierenden Proteinen durchgeführt. Diese wurden als Fusionsproteine mit Silaffin3, einem Protein, welches eng mit der Biosilica-Zellwand assoziiert ist, exprimiert. Es konnte gezeigt werden, dass nur drei von sechs Kandidaten funktional sind, wenn sie in Biosilica eingebettet sind. Silaffin3 konnte indirekt mittels SMLM mit einer Lokalisationsgenauigkeit von 25 nm detektiert werden. Dies erlaubte es, seine strukturelle Organisation aufzulösen und Silaffin3 als eine Hauptkomponente in der Basalkammer der Fultoportulae zu identifizieren.:1 INTRODUCTION 1 1.1 Diatoms – a model system for biomineralization 3 1.2 Imaging of biosilica and associated organic components 8 1.3 Single-molecule localization microscopy (SMLM) 10 2 METHODS & METHOD DEVELOPMENT FOR SMLM DATASETS 17 2.1 Super-resolution reconstruction 19 2.2 Tools for SMLM resolution estimates 21 2.3 Voronoi tessellation for noise-removal and cluster estimation 25 2.4 Tools for SMLM cluster analysis 27 2.5 Coordinate-based co-localization 32 2.6 PairRice – A novel algorithm to extract distances between cluster pairs 33 2.7 SiMoNa – A new GUI for exploring SMLM datasets 35 3 RESOLUTION OF THE SMLM SETUP TESTED WITH DNA ORIGAMI NANOSTRUCTURES 41 3.1 DNA origami as a length standard 42 3.2 Global resolution estimates 44 3.3 Local resolution estimates 47 3.4 Conclusion 53 4 EVALUATION OF PHOTO-CONTROLLABLE FLUORESCENT PROTEINS FOR PALM IN DIATOMS 55 4.1 Selecting PCFPs to minimize interference with the diatom autofluorescence 56 4.2 Screening results for cytosolic and biosilica-embedded PCFPs 58 4.3 The underlying conversion mechanism 61 4.4 Conclusion 63 5 IMAGING THE SIL3 MESHWORK 65 5.1 Analyzing protein layer thickness using tpSil3-Dendra2 65 5.2 Imaging the valve region using tpSil3 68 5.3 Resolution and localization parameters of tpSil3 70 5.4 Conclusion 72 6 DECIPHERING CINGULIN PATTERNS WITH CO LOCALIZATION STUDIES 73 6.1 A two-color cingulin construct for PALM-STORM 73 6.2 Steps towards PALM-STORM: screening, alignment, and imaging routine 76 6.3 Co-localization studies: quantification, clustering, and correlations 83 6.4 Conclusion 91 7 OUTLOOK 93 8 MATERIALS & METHODS 97 8.1 Microscope specifications 97 8.2 DNA origami annealing and AFM measurements 99 8.3 Diatom sample preparations 100 8.4 Fluorescence imaging conditions 102 8.5 Buffer systems 103 9 APPENDICES 105 9.1 Tables and Protocols 105 9.2 Satellite projects 112 9.2.1 Quantitative fluorescence intensity analysis of 3D time-lapse confocal microscopy data in diatoms 112 9.2.2 Applying neural networks to filter SMLM localizations 118 9.2.3 In vivo imaging at super-resolution conditions using SOFI 121 9.2.4 Quantifying chromatic aberrations in the microscope using fiducials 123 10 REFERENCES 127Diatoms feature the unique ability to form nanopatterned hierarchical silica cell walls with unprecedented accuracy and reproducibility. Gathering a deeper understanding of this process that is known as “biomineralization” is vitally important not only in the field of diatom research. In fact, the nanopatterning can also be exploited in the fields of material sciences or nanobiotechnology. According to the current understanding, the self-assembly of protein patterns along which biosilica is formed is key to this nanopatterning. Thus, in order to unravel the function of individual proteins that are involved in this biomineralization process, their structural organization has to be deciphered and correlated to morphological cell wall features that are in the order of tens of nanometer. Electron microscopy is able to resolve these features but does not provide protein-specific information. Therefore, a technique has to be established that is able to localize individual biosilica-associated proteins with nanometer precision. To achieve this objective, single-molecule localization microscopy (SMLM) for the diatom Thalassiosira pseudonana has been pioneered and exploited to localize different biosilica associated proteins inside silica and after silica removal. To obtain quantitative data, an analysis workflow was developed including graphical user interfaces and scripts for SMLM visualization, clustering, and co-localization. In order to find optimal labels for SMLM to target biosilica-embedded proteins, a comprehensive screening of photo-controllable fluorescent proteins has been carried out. Only three of six candidates were functional when embedded inside biosilica and fused to Silaffin3 – a protein that is tightly associated with the biosilica cell wall. Silaffin3 could be localized using SMLM with a localization precision of 25 nm. This allowed to resolve its structural organization and therefore identified Silaffin3 as a major component in the basal chamber of the fultoportulae. Additionally, co-localization studies on cingulins – a protein family hypothesized to be involved in silica formation – have been performed to decipher their pattern-function relationship. Towards this end, novel imaging strategies, co-localization calculations and pattern quantifications have been established. With the help of these results, the spatial arrangement of cingulins W2 and Y2 could be compared with unprecedented resolution. In summary, this work has laid ground for quantitative SMLM studies of proteins in diatoms in general and contributed insights into the spatial organization of proteins involved in biomineralization in the diatom T. pseudonana.:1 INTRODUCTION 1 1.1 Diatoms – a model system for biomineralization 3 1.2 Imaging of biosilica and associated organic components 8 1.3 Single-molecule localization microscopy (SMLM) 10 2 METHODS & METHOD DEVELOPMENT FOR SMLM DATASETS 17 2.1 Super-resolution reconstruction 19 2.2 Tools for SMLM resolution estimates 21 2.3 Voronoi tessellation for noise-removal and cluster estimation 25 2.4 Tools for SMLM cluster analysis 27 2.5 Coordinate-based co-localization 32 2.6 PairRice – A novel algorithm to extract distances between cluster pairs 33 2.7 SiMoNa – A new GUI for exploring SMLM datasets 35 3 RESOLUTION OF THE SMLM SETUP TESTED WITH DNA ORIGAMI NANOSTRUCTURES 41 3.1 DNA origami as a length standard 42 3.2 Global resolution estimates 44 3.3 Local resolution estimates 47 3.4 Conclusion 53 4 EVALUATION OF PHOTO-CONTROLLABLE FLUORESCENT PROTEINS FOR PALM IN DIATOMS 55 4.1 Selecting PCFPs to minimize interference with the diatom autofluorescence 56 4.2 Screening results for cytosolic and biosilica-embedded PCFPs 58 4.3 The underlying conversion mechanism 61 4.4 Conclusion 63 5 IMAGING THE SIL3 MESHWORK 65 5.1 Analyzing protein layer thickness using tpSil3-Dendra2 65 5.2 Imaging the valve region using tpSil3 68 5.3 Resolution and localization parameters of tpSil3 70 5.4 Conclusion 72 6 DECIPHERING CINGULIN PATTERNS WITH CO LOCALIZATION STUDIES 73 6.1 A two-color cingulin construct for PALM-STORM 73 6.2 Steps towards PALM-STORM: screening, alignment, and imaging routine 76 6.3 Co-localization studies: quantification, clustering, and correlations 83 6.4 Conclusion 91 7 OUTLOOK 93 8 MATERIALS & METHODS 97 8.1 Microscope specifications 97 8.2 DNA origami annealing and AFM measurements 99 8.3 Diatom sample preparations 100 8.4 Fluorescence imaging conditions 102 8.5 Buffer systems 103 9 APPENDICES 105 9.1 Tables and Protocols 105 9.2 Satellite projects 112 9.2.1 Quantitative fluorescence intensity analysis of 3D time-lapse confocal microscopy data in diatoms 112 9.2.2 Applying neural networks to filter SMLM localizations 118 9.2.3 In vivo imaging at super-resolution conditions using SOFI 121 9.2.4 Quantifying chromatic aberrations in the microscope using fiducials 123 10 REFERENCES 12

Spatial point pattern analysis with application to confocal microscopy data

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Probing the formation of mammalian cell lipid microdomains with nanometer-scale imaging secondary ion mass spectrometry

Over forty years ago, Singer and Nicholson began studying the organization of the plasma membranes on mammalian cells. Since then, our understanding of this membrane has shifted from that of a homogeneous to a more heterogenous environment of proteins and lipids. The complexity of living cells have made for difficulties in obtaining a complete understanding of the plasma membrane, especially that of the sphingolipids’ organization. The recent application of imaging mass spectrometry to biological samples has enabled for the chemical analysis of the plasma membrane. Through the use of a high resolution imaging secondary ion mass spectrometer (NanoSIMS 50, Cameca), we have been able to chemically map the distributions of sphingolipids on the plasma membrane of murine fibroblast cells with an astonishing sub-100 nm resolution. This required us to metabolically incorporate a rare, stable label into the molecules of interest and chemically preserving the cells in an effort to maintain close to a native organization of the membrane. The labeled lipid species could then be visualized with the NanoSIMS, providing us with a closer understanding of lipids within the plasma membrane. These experiments have revealed that sphingolipid-enriched domains exist in the plasma membrane on a length scale of ~200 nm, and they form non-random clustering on the cell surface. Disruption of the cellular cytoskeleton resulted in a loss of detectable statistically significant domains. In addition, the rapid transport of sphingolipids to the plasma membrane has made for complex interpretation of the role that vesicle transport plays in domain formation. Further studies using this methodology of chemically imaging mammalian cells, most importantly disruption of vesicle trafficking, may help to definitively identify the mechanisms behind the formation of these lipid domains as well as elucidate the full purpose they serve in the cell

NEW TECHNOLOGIES FOR LIVE CELL FLUORESCENCE IMAGING OF POST-TRANSLATIONAL MODIFICATIONS

Post translational modifications (PTMs) of proteins serve critical roles in both signal transduction as well as gene regulation. Phosphorylation is an important means by which signals are transmitted, processed, and amplified within a cell, while methylation is a critical regulator of chromatin organization mediated by histone proteins. The spatial distribution and temporal dynamics of these PTMs must be tightly controlled to allow for proper functioning. In signaling pathways, distinct spatiotemporal patterning of signaling activities is a critical means by which cells use the same molecular components to regulate diverse functionalities. In gene regulation, precise spatial organization of histone methylation allows for the proper distributions of heterochromatin and euchromatin, controlling which parts of the genome are transcriptionally active or repressed. In this dissertation, we discuss three new genetically encodable fluorescence technologies to expand the information we can obtain about the spatiotemporal dynamics of PTMs in real time in live cells. First, we discuss a new suite of genetically encodable fluorescent biosensors that are both single-color and ratiometric. These FLuorescent Anisotropy Reporters (FLARE) are based on homo-FRET, and they allow for multiparameter imaging of cell signaling activities, such as kinase activity and second-messengers. We then present a panel of biosensors called Fluorescent fLuctuation INcrease by Contact (FLINC) sensors that allow for the creation of sub-diffraction limit resolution maps of kinase activity. They were then used to map highly localized signaling microdomains of the Protein Kinase A (PKA) pathway in the plasma membrane. Finally, we describe a new fluorescent probe for superresolution mapping of tri-methylated histone 3 lysine 9 (H3K9me3), a critical histone modification for regulating the formation of heterochromatin in the nucleus. By engineering a mutant of the chromobox homolog protein 1 (CBX1) with high affinity for H3K9me3 and fusing it with a photoactivatable fluorescent protein, we demonstrate the ability to create sub-diffraction limit resolution maps of H3K9me3 in live cells.

Microscopy of defects in semiconductors

In this chapter, the authors discuss microscopy techniques that can be useful in addressing defects in semiconductors. They focus on three main families: scanning probe microscopy, scanning electron microscopy and transmission electron microscopy. They first address the basic principles of the selected microscopy techniques In discussions of image formation, they elucidate the mechanisms by which defects are typically imaged in each technique. Then, in the latter part of the chapter, they describe some key examples of the application of microscopy to semiconductor materials, addressing both point and extended defects and both two-dimensional (2D) and three-dimensional (3D) materials