Keratin Dynamics: Modeling the Interplay between Turnover and Transport

Keratin are among the most abundant proteins in epithelial cells. Functions of the keratin network in cells are shaped by their dynamical organization. Using a collection of experimentally-driven mathematical models, different hypotheses for the turnover and transport of the keratin material in epithelial cells are tested. The interplay between turnover and transport and their effects on the keratin organization in cells are hence investigated by combining mathematical modeling and experimental data. Amongst the collection of mathematical models considered, a best model strongly supported by experimental data is identified. Fundamental to this approach is the fact that optimal parameter values associated with the best fit for each model are established. The best candidate among the best fits is characterized by the disassembly of the assembled keratin material in the perinuclear region and an active transport of the assembled keratin. Our study shows that an active transport of the assembled keratin is required to explain the experimentally observed keratin organization.Comment: 27 pages, 11 Figure

Model-based Curvilinear Network Extraction and Tracking toward Quantitative Analysis of Biopolymer Networks

Curvilinear biopolymer networks pervade living systems. They are routinely imaged by fluorescence microscopy to gain insight into their structural, mechanical, and dynamic properties. Image analysis can facilitate understanding the mechanisms of their formation and their biological functions from a quantitative viewpoint. Due to the variability in network geometry, topology and dynamics as well as often low resolution and low signal-to-noise ratio in images, segmentation and tracking networks from these images is challenging. In this dissertation, we propose a complete framework for extracting the geometry and topology of curvilinear biopolymer networks, and also tracking their dynamics from multi-dimensional images. The proposed multiple Stretching Open Active Contours (SOACs) can identify network centerlines and junctions, and infer plausible network topology. Combined with a $k$-partite matching algorithm, temporal correspondences among all the detected filaments can be established. This work enables statistical analysis of structural parameters of biopolymer networks as well as their dynamics. Quantitative evaluation using simulated and experimental images demonstrate its effectiveness and efficiency. Moreover, a principled method of optimizing key parameters without ground truth is proposed for attaining the best extraction result for any type of images. The proposed methods are implemented into a usable open source software ``SOAX\u27\u27. Besides network extraction and tracking, SOAX provides a user-friendly cross-platform GUI for interactive visualization, manual editing and quantitative analysis. Using SOAX to analyze several types of biopolymer networks demonstrates the potential of the proposed methods to help answer key questions in cell biology and biophysics from a quantitative viewpoint

KRAP tethers IP3 receptors to actin and licenses them to evoke cytosolic Ca2+ signals

Regulation of IP3 receptors (IP3Rs) by IP3 and Ca2+ allows regenerative Ca2+ signals, the smallest being Ca2+ puffs, which arise from coordinated openings of a few clustered IP3Rs. Cells express thousands of mostly mobile IP3Rs, yet Ca2+ puffs occur at a few immobile IP3R clusters. By imaging cells with endogenous IP3Rs tagged with EGFP, we show that KRas-induced actin-interacting protein (KRAP) tethers IP3Rs to actin beneath the plasma membrane. Loss of KRAP abolishes Ca2+ puffs and the global increases in cytosolic Ca2+ concentration evoked by more intense stimulation. Over-expressing KRAP immobilizes additional IP3R clusters and results in more Ca2+ puffs and larger global Ca2+ signals. Endogenous KRAP determines which IP3Rs will respond: it tethers IP3R clusters to actin alongside sites where store-operated Ca2+ entry occurs, licenses IP3Rs to evoke Ca2+ puffs and global cytosolic Ca2+ signals, implicates the actin cytoskeleton in IP3R regulation and may allow local activation of Ca2+ entry. 2021, The Author(s).The authors thank Martyn Reynolds and Stephen Tovey (Cairn, UK) for help with super-resolution confocal microscopy. Supported by a Wellcome Senior Investigator Award (grant no. 101844), and by a grant (grant no. BB/T012986/1) and studentship (to H.A.S) from the Biotechnology and Biological Sciences Research Council UK. P.A.-A. is a research fellow of Emmanuel College, Cambridge.Scopu

Fluorescence Methods for Investigation of Living Cells and Microorganisms

Fluorescence methods play a leading role in the investigation of biological objects. They are the only non-destructive methods for investigating living cells and microorganisms in vivo. Using intrinsic and artificial fluorescence methods provides deep insight into mechanisms underlying physiological and biochemical processes. This book covers a wide range of modern methods involved in experimental biology. It illustrates the use of fluorescence microscopy and spectroscopy, confocal laser scanning microscopy, flow cytometry, delayed fluorescence, pulse-amplitude-modulation fluorometry, and fluorescent dye staining protocols. This book provides an overview of practical and theoretical aspects of fluorescence methods and their successful application in the investigation of static and dynamic processes in living cells and microorganisms

Studying Large Multi-Protein Complexes Using Single Molecule Localization Microscopy

Biology would not be where it is today without fluorescence microscopy. It is arguably one of the most commonly used tools in the biologists toolbox and it has helped scientists study the localization of cellular proteins and other small things for decades, but it is not without its limitations. Due to the diffraction limit, conventional fluorescence microscopy is limited to micrometer-range structures. Science has long relied upon electron microscopy and X-ray crystallography to study phenomena that occur below this limit. However, many of lifes processes occur between these two spatial domains. Super-resolution microscopy, the next stage of evolution of fluorescence microscopy, has the potential to bridge this gap between micro and nano. It combines superior resolutions of down to a few nanometers with the ability to view objects in their natural environments. It is the ideal tool for studying the large, multi-protein complexes that carry out most of lifes functions, but are too complex and fragile to put on an electron microscope or into a synchrotron. A form of super-resolution microscopy called SMLM Microscopy shows especially high promise in this regard. With its ability to detect individual molecules, it combines the high resolution needed for structural studies with the quantitative readout required for obtaining data on the stoichiometry of multi-protein complexes. This thesis describes new tools which expand the toolbox of SMLM with the specific aim of studying multi-protein complexes. First, the development of a novel fluorescent tagging system that is a mix of genetic tagging and immuno-staining. The system, termed BC2, consists of a short, genetically encodable peptide that is targeted by a nanobody (BC2 nanobody). The system brings several advantages. The small tag is not disruptive to the protein it is attached to and the small nanobody can get into tight spaces, making it an excellent tag for dense multi-protein structures. Next, several new variants of some commonly used green-to-red fluorescent proteins. The novel variants, which can be converted with a combination of blue and infrared light are especially useful for live-cell imaging. The developed fluorescent proteins can also be combined with photo-activatable fluorescent proteins to enable imaging of several targets with the same color protein. Finally, an application of the latter technique to study the multi-protein kinetochore complex and gain first glimpses into its spatial organization and the stoichiometry of its subunits

Multi-Scale Force Transmission to and Within the Nucleus.

PhD Theses.The mechanical state of cells, controlled primarily by cytoskeletal (CSK) networks (actin, microtubules and intermediate filaments) is a critical component of maintaining healthy function. Forces transmitted through the cytoskeleton influence the organisation and state of nuclear material, leading to changes in gene expression. This thesis aims to increase our understanding of the role of the CSK networks, specifically the intermediate filament keratin, and their interplay in integrating mechanical forces. We primarily use immunofluorescence imaging of the CSK networks and the nucleus, supported by Atomic Force Microscopy. We work in human epidermal keratinocytes (HEKs), as they are rich in keratin, whose role in cytoskeletal force transmission is under-studied. Since drugs to disrupt keratin are scarce, we first established that Withaferin-A, a compound previously used to disrupt vimentin intermediate filaments, can disrupt keratin at non cyto-toxic doses; impacting cell mechanics and migration. Following from this, Withaferin-A was used alongside established cyto-modulatory drugs to disrupt CSK networks, quantifying a range of properties describing their organisation. These data were fitted to nuclear parameters that described opposing functions on the nuclear state of HEKs for keratin and tubulin, with keratin protecting the nucleus from mechanical force. Finally, machine and deep learning techniques were used to expand the mathematical modelling of data. By training networks to predict nuclear location from only CSK images, a causative relationship between CSK organisation and nuclear location can be derived. In addition, we develop new models to rapidly analyse Atomic Force Microscopy curves and generate synthetic cell images. These results demonstrate the important role of keratin in protecting the nucleus from mechanical force and that deep learning techniques can be used in the study of cell mechanics to gain new insights

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