高速ビジョンを用いたリアルタイムビデオモザイキングと安定化に関する研究

広島大学(Hiroshima University)博士(工学)Doctor of Engineeringdoctora

Studies of Single-Molecule Dynamics in Microorganisms

Fluorescence microscopy is one of the most extensively used techniques in the life sciences. Considering the non-invasive sample preparation, enabling live-cell compliant imaging, and the specific fluorescence labeling, allowing for a specific visualization of virtually any cellular compound, it is possible to localize even a single molecule in living cells. This makes modern fluorescence microscopy a powerful toolbox. In the recent decades, the development of new, "super-resolution" fluorescence microscopy techniques, which surpass the diffraction limit, revolutionized the field. Single-Molecule Localization Microscopy (SMLM) is a class of super-resolution microscopy methods and it enables resolution of down to tens of nanometers. SMLM methods like Photoactivated Localization Microscopy (PALM), (direct) Stochastic Optical Reconstruction Microscopy ((d)STORM), Ground-State Depletion followed by Individual Molecule Return (GSDIM) and Point Accumulation for Imaging in Nanoscale Topography (PAINT) have allowed to investigate both, the intracellular spatial organization of proteins and to observe their real-time dynamics at the single-molecule level in live cells. The focus of this thesis was the development of novel tools and strategies for live-cell SingleParticle Tracking PALM (sptPALM) imaging and implementing them for biological research. In the first part of this thesis, I describe the development of new Photoconvertible Fluorescent Proteins (pcFPs) which are optimized for sptPALM lowering the phototoxic damage caused by the imaging procedure. Furthermore, we show that we can utilize them together with Photoactivatable Fluorescent Proteins (paFPs) to enable multi-target labeling and read-out in a single color channel, which significantly simplifies the sample preparation and imaging routines as well as data analysis of multi-color PALM imaging of live cells. In parallel to developing new fluorescent proteins, I developed a high throughput data analysis pipeline. I have implemented this pipeline in my second project, described in the second part of this thesis, where I have investigated the protein organization and dynamics of the CRISPR-Cas antiviral defense mechanism of bacteria in vivo at a high spatiotemporal level with the sptPALM approach. I was successful to show the differences in the target search dynamics of the CRISPR effector complexes as well as of single Cas proteins for different target complementarities. I have also first data describing longer-lasting bound-times between effector complex and their potential targets in vivo, for which only in vitro data has been available till today. In summary, this thesis is a significant contribution for both, the advances of current sptPALM imaging methods, as well as for the understanding of the native behavior of CRISPR-Cas systems in vivo

Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations

Despite significant progress, high-speed live-cell super-resolution studies remain limited to specialized optical setups, generally requiring intense phototoxic illumination. Here, we describe a new analytical approach, super-resolution radial fluctuations (SRRF), provided as a fast graphics processing unit-enabled ImageJ plugin. In the most challenging data sets for super-resolution, such as those obtained in low-illumination live-cell imaging with GFP, we show that SRRF is generally capable of achieving resolutions better than 150 nm. Meanwhile, for data sets similar to those obtained in PALM or STORM imaging, SRRF achieves resolutions approaching those of standard single-molecule localization analysis. The broad applicability of SRRF and its performance at low signal-to-noise ratios allows super-resolution using modern widefield, confocal or TIRF microscopes with illumination orders of magnitude lower than methods such as PALM, STORM or STED. We demonstrate this by super-resolution live-cell imaging over timescales ranging from minutes to hours

Synthetic noise control in eukaryotic gene expression and signal transduction

A certain level of randomness is inherent to every biological process, causing individual cells in a clonally identical population to vary in the number of protein molecules. This variation that was termed gene expression noise arises from stochastic fluctuations and the variability of numbers and states of the involved expression machinery. Noise causes suboptimal protein concentrations, which can negatively affect biological processes. Nature has selected for noise-reducing mechanisms when they benefit cellular fitness. Most synthetic genetic circuits and signaling pathways, however, lack systems that control gene expression noise, which can reduce their functionality. Here, we report the construction of a synthetic noise tuning system in Saccharomyces cerevisiae. We present data acquired by flow cytometry, using a measurement setup that we optimized for minimal nonspecific biological and technical variations. The system we developed allows the tuning of expression noise of a target gene using externally added small molecules to control the transcription rate via inducible promoters and the mRNA degradation rate via inducible ribozyme sequences. We demonstrate the functionality of the noise tuner by achieving up to 3-fold noise differences in the expression of a fluorescence reporter gene. We benchmarked the performance of the noise tuner by comparing it to semi-synthetic systems with fixed mRNA degradation rates, mediated by native yeast terminators. Stochastic simulations of an analytical model that links gene expression to population-level distributions of protein numbers were used to reproduce the experimental findings and revealed the mechanisms underlying the observations: In the given parameter space, noise was mainly affected by the transcription rate, whereas the mean expression was governed by both, the transcription rate and the translational burst size defined by the mRNA degradation rate. The objective of the development of the noise tuner was twofold: the first goal was to reduce gene expression noise in contexts where it proves to be detrimental. The second goal was to establish the noise tuner as a tool to investigate the influence of noise in complex networks. We applied the noise tuner to different genes in the yeast mating pathway, a model signal transduction pathway and the basis for numerous studies in the field of synthetic biology. We determined that the noise tuner, when applied to different genes of the pathway resulted in detectable changes in pathway noise. Detailed analysis of the negative pathway regulator gene SST2 set to either high or low noise resulted in up to 50 % difference in pathway noise between the two settings. We demonstrated that the low noise setting of SST2 expression lead to improved information transmission through the pathway. Categorization of cell morphologies during stimulation with mating pheromone suggested a more precise, switch-like response in the low-noise SST2 cells. To investigate whether noise tuning principle we describe here was also applicable to native genes, we selected five yeast genes with reportedly extreme mRNA production and degradation rates. We used the corresponding promoters and terminators to drive the expression of a reporter gene to observe a general trend towards low noise for genes with high transcription and mRNA degradation rates and vice versa. The results of a gene ontology analysis of the two most extreme cases supported a hypothesis that noise levels are linked to protein function. In this thesis we report the design, construction and successful application of a synthetic noise tuner. Our results illustrate the impact of gene expression noise of individual components on pathway performance – but we also show that this can be controlled. We suggest that design principles for low-noise gene expression, such as those presented in this thesis, should be taken into account for the synthetic modification and de novo design of signal transduction pathways and other networks

Interlaced biophysical methods to unveil membrane receptor organization

From the introduction: "In my thesis I set out to address these issues by a toolbox of fluorescence imaging techniques such as F\uf6rster Resonance Energy Transfer, Fluorescence Anisotropy Imaging, and Spatio-temporal image correlation spectroscopy. I investigated receptor properties such as membrane mobility, microtubule and caveolin-1 binding, and TRPV1 oligomerization status. My experimental strategies benefited from the use of genetically-encodable fluorescent reporters belonging to the green fluorescent protein family.

Genetically encodable fluorescent protein markers in advanced optical imaging

Optical fluorescence microscopy plays a pivotal role in the exploration of biological structure and dynamics, especially on live specimens. Progress in the field relies, on the one hand, on technical advances in imaging and data processing and, on the other hand, on progress in fluorescent marker technologies. Among these, genetically encodable fluorescent proteins (FPs) are invaluable tools, as they allow facile labeling of live cells, tissues or organisms, as these produce the FP markers all by themselves after introduction of a suitable gene. Here we cover FP markers from the GFP family of proteins as well as tetrapyrrole-binding proteins, which further complement the FP toolbox in important ways. A broad range of FP variants have been endowed, by using protein engineering, with photophysical properties that are essential for specific fluorescence microscopy techniques, notably those offering nanoscale image resolution. We briefly introduce various advanced imaging methods and show how they utilize the distinct properties of the FP markers in exciting imaging applications, with the aim to guide researchers toward the design of powerful imaging experiments that are optimally suited to address their biological questions