Tracing Actin Filament Bundles in Three-Dimensional Electron Tomography Density Maps of Hair Cell Stereocilia

Cryo-electron tomography (cryo-ET) is a powerful method of visualizing the three-dimensional organization of supramolecular complexes, such as the cytoskeleton, in their native cell and tissue contexts. Due to its minimal electron dose and reconstruction artifacts arising from the missing wedge during data collection, cryo-ET typically results in noisy density maps that display anisotropic XY versus Z resolution. Molecular crowding further exacerbates the challenge of automatically detecting supramolecular complexes, such as the actin bundle in hair cell stereocilia. Stereocilia are pivotal to the mechanoelectrical transduction process in inner ear sensory epithelial hair cells. Given the complexity and dense arrangement of actin bundles, traditional approaches to filament detection and tracing have failed in these cases. In this study, we introduce BundleTrac, an effective method to trace hundreds of filaments in a bundle. A comparison between BundleTrac and manually tracing the actin filaments in a stereocilium showed that BundleTrac accurately built 326 of 330 filaments (98.8%), with an overall cross-distance of 1.3 voxels for the 330 filaments. BundleTrac is an effective semi-automatic modeling approach in which a seed point is provided for each filament and the rest of the filament is computationally identified. We also demonstrate the potential of a denoising method that uses a polynomial regression to address the resolution and high-noise anisotropic environment of the density map

From the mechanical properties of single cells to those of simple tissues

As interest in biophysics and biophysical modelling has grown in the cell and developmental biology communities, a variety of techniques have been developed to measure the mechanical properties of single cells. Atomic Force Microscopy (AFM) has become one of the preferred methods for these measurements primarily due to its ease of operation and commercial availability. However, measurements on soft cells with a variable surface topography require an additional level of care so that the predicted contact area with the cell surface is accurately estimated. Using combined AFM and confocal microscopy I have shown that with pyramidal tipped cantilevers the cell body can easily deform to the shape of the tip but can also touch the underside of the AFM cantilever beam causing an overestimation of elasticity. Such artefactual increases in contact area could be avoided by using spherical tipped cantilevers or tips with a high aspect ratio. I examined the role of the cytoskeleton and cell contractility in setting single cell stiffness with AFM. With techniques such as AFM, the rheology of single cells is becoming increasingly well characterised. The next logical step in furthering our understanding of organ and embryo mechanics is to scale up investigations to simple tissues such as on cell thick monolayers. I have developed methods to measure the mechanical properties of MDCK epithelial cell monolayers under AFM indentation or planar extension. Using deep indentation of monolayers cultured on soft gels I have measured the evolution of mechanical properties upon the establishment of cell-cell junctions. The relative mechanical stiffnesses of monolayer-gel composites evolve as cell contacts are established and required the formation of mature contractile adherens junctions. To measure the planar mechanical properties of cell monolayers I designed a system to create monolayers freely suspended from their susbstrate between two test rods. Cell monolayers have a higher stiffness than their cellular constituents due to the organisation of the cell cytoskeleton upon the formation of matured intercellular junctions

Molecular Control of Actin Cortex Architecture During Cell Division

Animal cell shape is controlled by gradients in contractile tension of the actin cortex. The cortex is a thin actomyosin network supporting the plasma membrane. At the molecular level, contractile tension is generated by myosin motors pulling on actin filaments. Along- side myosin, actin connectivity has been shown to be key to cortical tension regulation. Understanding molecular organisation of the actin cortex is thus key to understanding cortical tension. To understand how cortical composition changes when tension changes, and to identify potential molecular regulators of cortical tension, I firstly compared protein composition of interphase and mitotic cortices. Indeed, interphase and mitotic cells were previously shown to di↵er in cortical tension. I isolated cortical fractions from cells in these stages of cell cycle, by isolating cortex-enriched blebs. Using mass spectrometry, we detected over 922 proteins in blebs isolated from synchronised cells. Among 238 actin-related proteins, we showed a role for septins in the regulation of the mitotic cell shape. Overall, we created a comprehensive dataset of potential regulators of cortex mechanics. In the second part of my PhD, I focused on the role of actin crosslinkers in cortex tension regulation. In particular, I focused on the role of actin crosslinker size for their localisation and in tension regulation. To this aim, we created artificial crosslinkers, for which I was able to modulate size independently of other features. We created artificial crosslinkers between 5 and 35 nm long, which successfully localised to actin structures. I investigated the role of artificial crosslinkers in the control of cortical thickness, tension and cell division. Together, in this thesis, I investigate new levels of regulation of cortical organisation and tension at the molecular level

LineRESOLFT microscopy

Recent advances in RESOLFT (reversible saturable optical fluorescence transitions) microscopy have enabled the non-invasive three-dimensional visualization of numerous structures in living cells at high spatial resolution. This technique, which utilizes low light intensities, has manifold potential applications in the life sciences. The work presented here envisions further broadening the applications of RESOLFT microscopy by implementing a scheme for fast image acquisition of large fields of view, applicable to thick specimen. With a pattern consisting of line-shaped intensity minima, this novel technique, called lineRESOLFT, permits fast imaging of living cells at ~40nm lateral resolution while offering strong optical sectioning. The full potential of this method is further illustrated by the achievement of continuous three-dimensional imaging of neurons in living brain slices with high spatio-temporal resolution, enabling the observation of rapid spine motility for large fields of view on the second time scale

Intermediate Filament Mechanics Across Scales – From Single Filaments to Single Interactions and Networks in Cells

The mechanical properties of cells are largely determined by the cytoskeleton. The cytoskeleton is an intricate and complex structure formed by protein filaments, motor proteins, and crosslinkers. The three main types of protein filaments are microtubules, actin filaments, and intermediate filaments ( IFs ). Whereas the proteins that form microtubules and actin filaments are exceptionally conserved throughout cell types and organisms, the family of IFs is diverse. For example, the IF protein vimentin is expressed in relatively motile fibroblasts, and keratin IFs are found in epithelial cells. This variety of IF proteins might therefore be linked to the various mechanical properties of different cell types. In the scope of this thesis, I combine studies of IF mechanics on different time scales and in systems of increasing complexity, from single filaments to networks in cells. This multiscale approach allows for the simplification necessary to interpret observations while adding increasing physiological context in subsequent experiments. We especially focus on the tunability of the IF mechanics by environmental cues in these increasingly complex systems. In a series of experiments, including single filament elongation studies, single filament stretching measurements with optical tweezers, filament-filament interaction measurements with four optical tweezers, microrheology, and isotropic cell stretching, we characterize how electrostatic (pH and ion concentration) and hydrophobic interactions (detergent) provide various mechanisms by which the mechanics of the IF cytoskeleton can be tuned. These studies reveal how small changes, such as charge shifts, influence IF mechanics on multiple scales. In combination with simulations, we determine the mechanisms by which charge shifts alter single vimentin filament mechanics and we extract energy landscapes for interactions between single filaments. Such insights will provide a deeper understanding of the mechanisms by which cells can maintain their integrity and adapt to the mechanical requirements set by their environment

Novel insights into hair structure and the effects of chemical stressors on hair and skin using label-free advanced light microscopy

There is a need for a better method to image hair as the current methods involve embedding the hair in resin, which may produce artefacts, or using dyes which are limited in their depth of penetration into the hair. The research performed in this thesis endeavours to characterise the cellular structure of human hair with label-free imaging using autofluorescence and fluorescence lifetime imaging. Wavelengths were shown to selectively excite the hair cuticle, cortex and medulla, and subcellular compartments. Development of an optical transverse imaging method enabled discoveries including different fluorescence lifetimes across the cuticle cell layers and suggests the cuticle layers possess differing chemical environments. A new method was developed to distinguish between eumelanin and pheomelanin using 405nm and 633nm wavelengths. The newly developed methods were additionally used in the characterisation of an unidentified hair and skin disorder, which found poorly differentiated cuticle cells and showed differences in the fluorescence lifetimes of the hair compared to control hairs. The hair care industry needs more efficacious chemical depilatories and information into their action. This was elucidated using the developed methods and a new dynamic imaging method. Potassium thioglycolate was shown to cause drastic expansion of the hair which was amplified by the addition of guanidine carbonate, creating fissures through the cuticle and into the cortex. Other experimental depilatory formulations were tested and were found to have varying effects upon the structure of the hair. New chemical depilatories require development because existing depilatories can cause irritation in the skin. Potassium thioglycolate and guanidine carbonate were tested on HaCaT cells, isolated cornified envelopes, and HEKn cells in a 3D epidermal model. An investigation into the differentiation, proliferation and acute stress response of the cells showed that the treatments had no significant effect on these markers. However, the chemicals negatively affected HaCaT cell viability and damaged the cornified envelopes. Despite this, the viability and structural integrity of the living cells of the epidermal model were maintained through the protection provided by the stratum corneum

Mathematical Methods for the Quantification of Actin-Filaments in Microscopic Images

In cell biology confocal laser scanning microscopic images of the actin filament of human osteoblasts are produced to assess the cell development. This thesis aims at an advanced approach for accurate quantitative measurements about the morphology of the bright-ridge set of these microscopic images and thus about the actin filament. Therefore automatic preprocessing, tagging and quantification interplay to approximate the capabilities of the human observer to intuitively recognize the filaments correctly. Numerical experiments with random models confirm the accuracy of this approach

Periderm invasion contributes to epithelial formation in the teleost pharynx

The gnathostome pharyngeal cavity functions in food transport and respiration. In amniotes the mouth and nares are the only channels allowing direct contact between internal and external epithelia. In teleost fish, gill slits arise through opening of endodermal pouches and connect the pharynx to the exterior. Using transgenic zebrafish lines, cell tracing, live imaging and different markers, we investigated if pharyngeal openings enable epithelial invasion and how this modifies the pharyngeal epithelium. We conclude that in zebrafish the pharyngeal endoderm becomes overlain by cells with a peridermal phenotype. In a wave starting from pouch 2, peridermal cells from the outer skin layer invade the successive pouches until halfway their depth. Here the peridermal cells connect to a population of cells inside the pharyngeal cavity that express periderm markers, yet do not invade from outside. The latter population expands along the midline from anterior to posterior until the esophagus-gut boundary. Together, our results show a novel role for the periderm as an internal epithelium becomes adapted to function as an external surface.Agência financiadora Ghent University Research Fund - BOF24J2015001401 Cancer Prevention Research Institute of Texas - RR140077info:eu-repo/semantics/publishedVersio

Dynamics of non-muscle myosin II organization into contractile networks and fibers at medial cell cortex

The cellular morphology of adhered cells depends crucially on the formation of a contractile meshwork of parallel and cross-linked stress fibers along the contacting surface. The motor activity and mini-filament assembly of non-muscle myosin II is an important component of cell-level cytoskeletal remodeling during mechanosensing. To monitor the dynamics of non-muscle myosin II, we used confocal microscopy to image cultured HeLa cells that stably express myosin regulatory light chain tagged with GFP (MRLC-GFP). MRLC-GFP was monitored in time-lapse movies at steady state and during the response of cells to varying concentrations of blebbistatin (which disrupts actomyosin stress fibers). Using image correlation spectroscopy analysis, we quantified the kinetics of disassembly and reassembly of actomyosin networks and compared to studies by other groups. This analysis suggested the following processes: myosin minifilament assembly and disassembly; aligning and contraction; myosin filament stabilization upon increasing contractile tension. Numerical simulations that include those processes capture some of the main features observed in the experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness. We discuss methods to monitor myosin reorganization using non-linear imaging methods

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