On the structural response of eukaryotic cells

textThe actin, microtubule and intermediate filament cytoskeletal polymer assemblies, along with their accessory proteins, govern the mechanical or structural response of an eukaryotic cell to an external stress. Using statistical mechanics tools, this dissertation investigates the molecular properties, such as mesh size, persistence length and filament length, that determine the structural strength of the in vivo polymer networks, with an emphasis on the actin network or the cortex of cells. Our study of actin shows how the wide range of shear moduli from 1 Pa to 1 kPa that spans the viscous sol-like state to the elastic gel-like state witnessed in eukaryotic cells can be achieved through transient crosslinking and the spatial distribution of actin and actin crosslinking proteins alone. Thus, this gel-sol transition is achieved without the action of any severing or capping proteins that depolymerize the actin network. In order to understand how the microscopic quantities controlling the structural properties of these in vivo polymers are related to the deformation of a cell observed experimentally, a cell model is created by us. It starts with modeling the actin cortex as a thick shell and increases in complexity to include microtubules and the nucleus. Our cell model predicts that the structural response of the cell amplifies changes in molecular properties such as the in vivo actin concentration. Hence, the sensitivity of the structural response to cytoskeletal changes can be used to distinguish between different cells such as normal and cancer cells and can serve as an indicator of disease.Physic

Bidirectional Mechanical Response Between Cells and Their Microenvironment

Cell migration and invasion play a role in many physiological and pathological processes and are therefore subject of intensive research efforts. Despite of the intensively investigated biochemical processes associated with the migration and invasion of cells, such as cancer cells, the contribution ofmechanobiological processes to themigratory capacity of cells as well as the role of physical polymeric phase transitions is not yet clearly understood. Unfortunately, these experiments are not very informative because they completely disregard the influence of the three-dimensional cell environment. Despite this data situation, it was possible to adequately demonstrate that there exists a direct mechanical interplay between cells and theirmicroenvironment in both directions, where both elements can bemechanically altered by one another. In line with these results, it has turned out that the mechanobiological molecular processes through which cells interact with each other and additionally sense their nearby microenvironment have an impact on cellular functions such as cellular motility. The mechanotransduction processes have become the major focus of biophysical research and thereby, diverse biophysical approaches have been developed and improved to analyze the mechanical properties of individual cells and extracellular matrix environments. Both, the cell mechanics and matrix environmentmechanics regulate the cellmigration types in confined microenvironments and hence it seems to be suitable to identify and subsequently present a common bidirectional interplay between cells and their matrix environment. Moreover, hallmarks of the mechanophenotype of invasive cells and extracellular matrices can be defined. This review will point out how on the one hand the intracellular cytoskeletal architecture and on the other hand the matrix architecture contribute to cellular stiffness or contractility and thereby determines the migratory phenotype and subsequently the emergence of a distinct migration mode. Finally, in this review it is discussed whether universal hallmarks of the migratory phenotype can be defined

Filament Nucleation Tunes Mechanical Memory in Active Polymer Networks

Incorporating growth into contemporary material functionality presents a grand challenge in materials design. The F‐actin cytoskeleton is an active polymer network that serves as the mechanical scaffolding for eukaryotic cells, growing and remodeling in order to determine changes in cell shape. Nucleated from the membrane, filaments polymerize and grow into a dense network whose dynamics of assembly and disassembly, or “turnover,” coordinates both fluidity and rigidity. Here, the extent of F‐actin nucleation is varied from a membrane surface in a biomimetic model of the cytoskeleton constructed from purified protein. It is found that nucleation of F‐actin mediates the accumulation and dissipation of polymerization‐induced F‐actin bending energy. At high and low nucleation, bending energies are low and easily relaxed yielding an isotropic material. However, at an intermediate critical nucleation, stresses are not relaxed by turnover and the internal energy accumulates 100‐fold. In this case, high filament curvatures template further assembly of F‐actin, driving the formation and stabilization of vortex‐like topological defects. Thus, nucleation coordinates mechanical and chemical timescales to encode shape memory into active materials

COMPOSITE NETWORK OF ACTIN AND MICROTUBULE FILAMENTS, SELF-ORGANIZATION AND STEADY-STATE DYNAMICS

Actin and microtubule filaments, with their auxiliary proteins, enable the cytoskeleton to perform vital processes in the cell by tuning the organizational, mechanical properties and dynamics of the network. Despite their critical importance and interactions in cells, we are only beginning to uncover information about the composite network. Here, I use florescence microscopy to explore the role of filaments characteristics, interactions and activities in the self-organization and steady-state dynamics of the composite network of filaments. First, I discuss active self-organization of semiflexible actin and rigid microtubule filaments in the 2D composite network while myosin II and kinesin-1 motor proteins propel actin and microtubule filaments, respectively. Second, I studied the steady-state mobility of the 3D composite network is studied when the interactions of filaments are regulated by the varying amount of crosslinkers. In a composite network where only actin filaments crosslinked using biotin-NeutrAvidin molecules, microtubule mobility is tuned by actin crosslinking and displays non-monotonic dependence on the amount of actin crosslinkers. Third, I included antiparallel microtubule crosslinkers, MAP65, as well as biotin-NeutrAvidin actin crosslinkers to reveal the different roles of these crosslinkers in the structure and mobility of the composite network. While actin crosslinkers dictated the mobility, microtubule crosslinkers control the co-localization of filaments. Finally, I worked on an active composite network of actin, microtubule, and myosin II motor proteins. The structural changes in the contractile composite network is characterized using correlation length measurements. These results provide a valuable insight into the cytoskeletal filaments interactions and their vital roles in various biological processes in cells. Furthermore, this knowledge could enable us to design autonomous bioinspired materials with tunable mechanical properties

Dynamic nanostructured scaffolds as advanced biomaterials

Growing replacement tissues and organs in the laboratory will revolutionise healthcare; however, the maturation of cells into functional tissue constructs requires the controlled presentation of biochemical factors within a mechanically suitable scaffold. In nature, the presentation of such signals is provided through factors and structures existent within the nanoarchitecture of the extracellular matrix (ECM); therefore, in tissue engineering there is significant need to develop dynamic advanced artificial tissue constructs capable of mimicking the complexities of the native ECM. The requirement for bioactive, innervated constructs that contain biologically relevant signals delivered through tuneable mechanisms has yet to be achieved. One approach to address this key-challenge is offered through bioprinting, which allows for the controlled spatial distribution of bioinks containing cells, structures and signals within a single printed construct. However, currently bioprinting applications are severely limited by bioink function - with the majority of bioinks either lacking sufficient mechanical properties or biochemical signalling. Therefore, there is a key need to develop bioinks which adequately mimic the native ECM on a nanostructured, chemical level - particularly in establishing effective control over cell fate and tissue innervation. Tissue composition and extracellular signalling varies substantially between tissue-types, and therefore, advanced approaches that allow for ease of mechanical and biological tuneability through modular mechanisms would provide a practical avenue for bioink development. Self-assembling peptides (SAPs) are a unique class of biomaterials capable of spontaneously forming simple biomimetic structures which entangle to form highly hydrated, bioactive networks with favourable conditions for cell maturation. These biomaterials are easily tuned through modification of amino acid sequence, enabling tailored control over biochemical signalling between cells and scaffold. This provides the ability to artificially replicate natural signalling in a controlled manner - bringing about desired cell behaviour. Using these peptides, a variety of synergistic ECM-protein analogues have been developed, including Fmoc-FRGDF containing fibronectin's attachment motif RGD, and Fmoc-DIKAV, containing laminin's attachment motif IKVAV. Fmoc-SAPs possess the ability to be further functionalised through macromolecule addition, allowing for the presentation of charged, developmentally or structurally-important macromolecules on the surface of peptide fibrils. These macromolecules can integrate with the peptide networks, facilitating additional signalling and allowing for mechanical tunability. Here, we take advantage of these properties to develop an advanced and dynamic bioink for bioprinting applications. Initially, material enhancement is investigated through development of multi-sequence scaffolds. Specifically, Fmoc-FRGDF is combined with a synergistic cell attachment motif PHSRN, either through sequence engineering (Fmoc-FRGSFPHSRN) or through control over assembly properties (Fmoc-FRGDF/Fmoc-PHSRN coassembly). Here, the coassembled (Fmoc-FRGDF/Fmoc-PHSRN) system forms a synergistic network which promotes the attachment, proliferation and migration of muscle cells in vitro. The potential of Fmoc-SAP multi-sequence scaffolds is further investigated through the development of an artificial tumour microenvironment for cancer-cell studies. Here, Fmoc-FRGDF is combined with Fmoc-DIKVAV and used as a spheroid (LLC, NOR-10, LLC + NOR-10) micro-environment. The coassembled Fmoc-FRGDF/Fmoc-DIKVAV microenvironment enhances cancer-cell growth and progression compared to 2D cultures, non-encapsulate spheroids, and spheroids encapsulated in agarose. Agarose was selected as a control owing to the similar physical properties yet lack of biofunctionalisation. Results from this study reinforce the potential of Fmoc-SAPs as advanced microenvironments, and further support the ease of biological functionalisation inherent with this material. Further scaffold functionalisation is investigated through macromolecule addition. Here, one of two macromolecules are coassembled into a Fmoc-FRGDF network. The first macromolecule is fucoidan, a seaweed-derived polysaccharide with known anti-inflammatory properties, while the second is versican, a developmentally important proteoglycan which plays a variety of roles in muscle development. Versican was selected owing to its charge similarity to fucoidan, yet vastly different biological function. Fucoidan addition was found to increase fibre bundling and alter hydrogel mechanical properties, while versican addition had no substantial effect on hydrogel mechanics when compared to an Fmoc-FRGDF empty-vector control. Cell morphology was substantially altered by macromolecule addition, with fucoidan samples resulting in smaller, rounder cells with fewer multinucleated syncytia compared to an Fmoc-FRGDF control, while versican hydrogels showed an initial decrease in cell-size and multinucleation after 24h and a comparable cell-size and multinucleation following 72h. Here, it is possible that macromolecule addition perturbs cells attachment, and therefore, macromolecule selection is a key consideration. Interestingly, the regain of cell morphological characteristics in versican-containing hydrogels following 72h indicates the ability of cells to break-down versican, while the maintenance of small, round cells in the fucoidan hydrogels shows an inability for cells to break down fucoidan. The ability of Fmoc-SAPs to form components in bioinks is investigated through assembly with gelatin methacryloyl (GelMA) macromolecules. Initially, GelMA nanostructure and mechanical properties are investigated in response to increased degree of methacrylation or increased control. Here, structure-function relationships are drawn, and 18% methacryloyl Gelma (LM-GelMA) is selected for further bioink development owing to favourable thermoresponsive viscoelastic properties and improved strain tolerance. LM-GelMA assembly with coassembled Fmoc-FRGDF/Fmoc-PHSRN is investigated as a potential avenue to develop biologically and mechanically tuneable hydrogels. The incorporation of Fmoc-SAPs allows for control over sequence selection, while control over mechanical properties is offered through GelMA inclusion. LM-GelMA/Fmoc-FRGDF/Fmoc-PHSRN (FPG-Hybrid) bioinks demonstrate enhanced printability and are shown to support primary myoblast differentiation. The potential of Fmoc-SAP/GelMA bioinks to act as a modular bioink toolkit is further investigated through Fmoc-FRGDF/Fmoc-PHSRN substitution with Fmoc-DIKVAV, to develop a neural-suitable bioink (DIKVAV-Hybrid). This DIKVAV-Hybrid bioink demonstrated unique mechanical morphological properties and is shown to support rat cortical neurosphere viability. Throughout this project, the networks have been vigorously characterised through various analytical techniques, including micro/nanoimaging (Transmission electron microscopy, Atomic force microscopy, Cryo-scanning electron microscopy), Small-angle X-ray scattering, Small-angle neutron scattering, rheology, and spectroscopy; while the overall effectiveness of these systems have been analysed through in vitro muscle and neural cultures. Work detailed through this thesis aims to vigorously characterise Fmoc-SAP hydrogels and bioinks, providing the foundations for further biological studies and material optimisation

Heat-induced changes in the material properties of cytoplasm

Organisms are frequently exposed to fluctuating environmental conditions and might consequently experience stress. Environmental stress can damage cellular components, which can threaten especially single-celled organisms, such as yeast, as they cannot escape. To survive, cells mount protective stress responses, which serve to preserve cellular components and architecture. Recent findings in yeast show that the stress response upon energy depletion stress involves a gelation of the cytoplasm due to macromolecular protein assembly, characterized by drastic changes in cytoplasmic material properties. Remarkably, the stress-induced cytoplasmic gelation is protective, raising the question whether this could be a common strategy of cells to cope with severe stress. I hypothesized that protein aggregation induced by another common stress, severe heat shock, might cause a similar cytoplasmic gelation in yeast. Furthermore, I hypothesized that the reversibility of cytoplasmic gelation is provided by molecular chaperones, which are known regulators of protein aggregation. In this thesis, I therefore aimed to characterize the changes in the material properties of the cytoplasm upon severe heat shock as well as their underlying causes and how molecular chaperones affect these changes. To characterize heat-induced changes in the material properties of the cytoplasm, I monitored Schizosaccharomyces pombe cells during recovery from severe heat shock using a combination of cell mechanical assays, time-lapse microscopy and single-particle tracking. I found that the cells entered a prolonged growth arrested state upon stress, which coincided with significant cell stiffening and a long-range motion arrest of lipid droplets in the cytoplasm, while smaller cytoplasmic nanoparticles remained mostly mobile. At the same time, a significant fraction of proteins aggregated in the cytoplasm, forming insoluble inclusions such as heat shock granules. After stress cessation, the observed changes were reversed as stiffened cells softened and lipid droplets resumed long-range motion. Cell softening and lipid droplet motion recovery coincided with protein disaggregation. These processes could be delayed by impairing protein disaggregation through genetic perturbation of the molecular chaperone Hsp104, which functions as a protein disaggregase. In contrast, no influence on protein disaggregation or heat-induced cytoplasmic material property changes was detected for the small heat shock protein Hsp16. These results suggest that the cytoplasm gels upon severe heat shock due to protein aggregation and is refluidized during recovery with the help of Hsp104. Remarkably, cells resumed growth only after refluidization of the cytoplasm, suggesting that reversible cytoplasmic gelation may contribute to regulation of the heat-induced growth arrest. In addition, cytoplasmic gelation could potentially preserve cellular architecture during heat shock. Overall, the results from my thesis work indicate that reversible cytoplasmic gelation due to macromolecular protein assembly may be a universal cellular response to severe stress which is associated with a stress-protective growth arrest. A likely stress-specific part of this response is the chaperone-dependent refluidization of the cytoplasm, which might explain the prolonged growth arrest seen upon severe heat shock as compared to other stresses and might allow more time for the repair of heat-induced damage.:Abstract Zusammenfassung Table of contents Figure index List of abbreviations 1 Introduction 1.1 Heat shock affects cellular function and fitness 1.1.1 Cells respond to stress in phases 1.1.2 Heat shock threatens cellular homeostasis and structural integrity 1.1.3 Stress severity determines detrimental effects of heat shock 1.1.4 Heat stress causes protein aggregation 1.1.5 Heat shock granules are functional aggregates in yeast 1.2 The heat shock response protects cellular fitness 1.2.1 Cells change transcription to adapt to stress 1.2.2 Molecular chaperones are important in stress protection 1.2.3 Hsp104 is a protein disaggregase chaperone 1.2.4 Small heat shock proteins modulate protein aggregation 1.2.5 Stress severity determines modules of the heat shock response 1.3 Cytoplasmic material properties change during stress 1.3.1 Cells homeostatically adapt cytoplasmic material properties during stress 1.3.2 The cytoplasm is viscoelastic 1.3.3 Is the cytoplasm a gel? 1.3.4 Stress can induce cytoplasmic gelation 1.4 Research aims 2 Materials and Methods 2.1 S. pombe strains and growth conditions 2.1.1 Growth conditions 2.1.2 Construction of S. pombe strains 2.1.3 S. pombe transformation 2.1.4 S. pombe colony PCR 2.1.5 S. pombe strains used in this thesis 2.2 Plasmids and cloning 2.2.1 Plasmids used in this thesis 2.2.2 Construction of plasmid for fluorescent GEM nanoparticle expression 2.2.3 E. coli transformation 2.2.4 Plasmid purification from E. coli 2.3 S. pombe stress treatments 2.3.1 Heat shock treatment 2.3.2 Osmoadaptation 2.4 Cell biological methods 2.4.1 Viability assay 2.4.2 Growth assay 2.5 Cell bulk mechanical assays 2.5.1 Spheroplasting assay 2.5.2 Atomic force microscopy 2.5.3 Real-time deformability cytometry 2.5.4 RT-DC sample preparation 2.5.5 RT-DC setup and measurements 2.5.6 RT-DC data evaluation 2.6 Microscopy 2.6.1 Microscopy of GEM particles 2.6.2 Fluorescence microscopy of endogenously labeled Pabp-mCherry 2.6.3 Microscopy of µNS particles 2.7 Image analysis 2.7.1 Image analysis of Pabp-mCherry in vivo fluorescence microscopy 2.7.2 Differenced brightfield image analysis 2.7.3 Kymographs 2.8 Single-particle tracking analysis 2.8.1 Particle tracking 2.8.2 Mean squared displacement analysis 2.9 Optical diffraction tomography (ODT) 2.9.1 ODT sample preparation 2.9.2 ODT optical setup and measurements 2.9.3 ODT tomogram reconstruction and quantitative analysis 2.10 Lysis and sedimentation assay 2.10.1 Lysis buffer 2.10.2 S. pombe heat shock treatment and lysis 2.10.3 Sedimentation assay 2.10.4 Protein concentration measurement 2.10.5 SDS-PAGE 2.10.6 Coomassie staining 2.10.7 Western Blot 3 Results 3.1 Physical and chemical conditions affect heat shock survival and heat-induced growth arrest of S. pombe 3.1.1 S. pombe arrests growth during severe heat shock 3.1.2 Heat-induced growth arrest is dose-responsive 3.1.3 Heat-induced growth arrest depends on experimental conditions 3.1.4 Buffer pH and energy source have a strong impact on heat shock survival 3.1.5 Osmoadaptation protects cells during heat shock 3.2 Severe heat shock induces reversible cellular stiffening 3.2.1 Cellular rounding upon cell wall removal is delayed after heat shock 3.2.2 Elastic modulus of S. pombe cells is increased after heat shock 3.2.3 Recovery from heat-induced growth arrest is preceded by cell softening 3.3 Long-range particle dynamics in cytoplasm are abolished after heat shock 3.3.1 Small particle dynamics are largely independent of heat shock treatment 3.3.2 Lipid droplets are confined in space after heat shock 3.4 Cytoplasmic crowding increases during heat shock 3.5 Heat shock induces reversible protein aggregation 3.5.1 Insoluble protein fraction is increased after heat shock 3.5.2 Heat shock granules form reversibly during heat shock 3.5.3 HSG formation and dissolution are correlated with changes in cytoplasmic long-range dynamics 3.6 Molecular chaperones modulate cytoplasmic material property changes during heat stress recovery 3.6.1 Hsp104 but not Hsp16 is required for disaggregation of heat shock granules 3.6.2 Hsp104 but not Hsp16 is required for recovery from heat-induced growth arrest 3.6.3 Hsp104 but not Hsp16 is required for recovery of cytoplasmic long-range dynamics 3.6.4 Hsp104 but not Hsp16 is required for rapid reversal of cellular stiffening which coincides with growth recovery 4 Discussion 4.1 Summary and model 4.2 Which mechanism underlies cell stiffening upon heat shock? 4.2.1 Heat-induced protein aggregation might cause cell stiffening 4.2.2 Heat-induced protein aggregation might lead to cytoplasmic gelation 4.2.3 Many factors could contribute to protein aggregation and cytoplasmic gelation 4.3 The heat-induced growth arrest state is associated with reversible cytoplasmic gelation 4.3.1 Cytoplasmic material property changes mark the severe heat-induced growth arrest state 4.3.2 Is cytoplasmic gelation a common response to severe stress? 4.4 What are the biological consequences of cytoplasmic gelation? 4.4.1 Cytoplasmic gelation might obstruct processes that require motion of large structures 4.4.2 Is cytoplasmic gelation upon heat shock protective? 4.5 Heat shock recovery involves the chaperone-mediated refluidization of the cytoplasm 4.5.1 Cytoplasmic refluidization is required for growth recovery 4.5.2 Stress tolerance is marked by enhanced reversibility of cytoplasmic gelation 4.5.3 The protein disaggregase chaperone Hsp104 regulates the reversal of heat-induced cytoplasmic material property changes 4.6 Conclusion References Acknowledgements Publications and Contributions 5 Erklärung entsprechend §5.5 der Promotionsordnung