Entropy Production Associated with Aggregation into Granules in a Subdiffusive Environment

We study the entropy production that is associated with the growing or shrinking of a small granule in, for instance, a colloidal suspension or in an aggregating polymer chain. A granule will fluctuate in size when the energy of binding is comparable to kBT , which is the “quantum� of Brownian energy. Especially for polymers, the conformational energy landscape is often rough and has been commonly modeled as being self-similar in its structure. The subdiffusion that emerges in such a high-dimensional, fractal environment leads to a Fokker–Planck Equation with a fractional time derivative. We set up such a so-called fractional Fokker–Planck Equation for the aggregation into granules. From that Fokker–Planck Equation, we derive an expression for the entropy production of a growing granul

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

Bacterial cell cycle dynamics: size regulation during exponential growth and role of polyphosphate during starvation response

Bacteria are the most diverse and abundant kingdom of life and have adapted to survive and thrive in habitats around the globe. When provided with ample nutrients they grow and divide at staggering rates, increasing their population exponentially. Upon nutrient depletion on the other hand, they quickly adapt by drastically altering their metabolism, halting growth to survive for a very long time. Since bacteria are tiny -about a few micrometers-, visualizing these processes requires microscopy. To measure the dynamics of their shape and inner structures precisely, one needs to choose a technique that balances spatial resolution, temporal resolution and photo-toxicity. In this thesis, I present two projects using advanced dynamic microscopy, first to study cell size regulation during exponential growth in the abundance of nutrients and then to elucidate the role and positioning of polyphosphate granules during cell cycle exit in response to nutrient starvation. During exponential growth, bacteria balance growth and division to regulate their size, resulting in a narrow size distribution, referred to as cell size homeostasis. Recent work tried to uncover what cells sense to decide to divide in order to achieve size homeostasis: time, size, growth or a combination of those. Control of cell division is often equated to control of constriction onset; however, the constriction period still accounts for up to 40% of cell growth and could thus contribute significantly to cell size regulation. We used SIM microscopy to measure constriction kinetics and their impact on cell size regulation in Caulobacter crescentus. We found that constriction rate regulation can determine cell size. Moreover, constriction rate modulation compensates for variability in elongation before constriction, allowing a higher fidelity cell size homeostasis. We suggest a parsimonious model where excess cell wall precursors accumulate proportionally to elongation before constriction and set the rate of constriction. This is the first direct demonstration that constriction rate can contribute to cell size control and homeostasis in bacteria. Upon nutrient starvation, bacteria exit their cell cycle to preserve energy and nutrients. In many bacteria, such as Pseudomonas aeruginosa, this is associated with the accumulation of polyphosphate (polyP) in intracellular granules. PolyP is created by polyphosphate kinases (ppkâs), which are required for successful cell cycle exit and survival of and recovery from long-term starvation. Interestingly, these polyP granules are regularly spaced within the nucleoid. To date, it is not known during which stage polyP is required for cell cycle exit, and what causes the spacing of the granules. Here, we use fluorescence microscopy to probe the cell cycle stage of Îppk cells arrested during nutrient starvation as well as the localization and dynamics of ppkâs. We show that a majority of Îppk cells are arrested with open replication forks. Furthermore, we find that ppkâs localize in distinct patterns, already prior to starvation and polyP granule production, which could be responsible for the positioning of polyP granules. To this end, we developed a background subtraction algorithm to remove cytoplasmic fluorescence, improving accuracy of spot detection and localization

25 Years of Self-Organized Criticality: Solar and Astrophysics

Shortly after the seminal paper {\sl "Self-Organized Criticality: An explanation of 1/f noise"} by Bak, Tang, and Wiesenfeld (1987), the idea has been applied to solar physics, in {\sl "Avalanches and the Distribution of Solar Flares"} by Lu and Hamilton (1991). In the following years, an inspiring cross-fertilization from complexity theory to solar and astrophysics took place, where the SOC concept was initially applied to solar flares, stellar flares, and magnetospheric substorms, and later extended to the radiation belt, the heliosphere, lunar craters, the asteroid belt, the Saturn ring, pulsar glitches, soft X-ray repeaters, blazars, black-hole objects, cosmic rays, and boson clouds. The application of SOC concepts has been performed by numerical cellular automaton simulations, by analytical calculations of statistical (powerlaw-like) distributions based on physical scaling laws, and by observational tests of theoretically predicted size distributions and waiting time distributions. Attempts have been undertaken to import physical models into the numerical SOC toy models, such as the discretization of magneto-hydrodynamics (MHD) processes. The novel applications stimulated also vigorous debates about the discrimination between SOC models, SOC-like, and non-SOC processes, such as phase transitions, turbulence, random-walk diffusion, percolation, branching processes, network theory, chaos theory, fractality, multi-scale, and other complexity phenomena. We review SOC studies from the last 25 years and highlight new trends, open questions, and future challenges, as discussed during two recent ISSI workshops on this theme.Comment: 139 pages, 28 figures, Review based on ISSI workshops "Self-Organized Criticality and Turbulence" (2012, 2013, Bern, Switzerland

Consequences of local and global chromatin mechanics to adaption and genome stability in the budding yeast Saccharomyces cerevisiae

Le génome de la levure de boulanger Saccharomyces cerevisiae a évolué à partir d'un ancêtre chez lequel une profonde décompaction du génome s'est produite à la suite de la perte de la méthylation de la lysine 9 de l'histone H3, il y a environ 300 millions d'années. Il a été proposé que cette décompaction du génome a entraîné une capacité accrue des levures à évoluer par des mécanismes impliquant des taux de recombinaison méiotique et de mutation exceptionnellement élevés. La capacité à évoluer accrue qui en résulte pourrait avoir permis des adaptations uniques, qui en ont fait un eucaryote modèle idéal et un outil biotechnologique. Dans cette thèse, je présenterai deux exemples de la façon dont les adaptations locales et globales du génome se reflètent dans les changements des propriétés mécaniques de la chromatine qui, à leur tour, indiquent un phénomène de séparation de phase causée par les modifications post-traductionnelles des histones et des changements dans les taux d'échange des histones. Dans un premier manuscrit, je présente des preuves d'un mécanisme par lequel la relocalisation du locus INO1, gène actif répondant à la déplétion en inositol, du nucléoplasme vers l'enveloppe nucléaire, augmente la vitesse d'adaptation et la robustesse métabolique aux ressources fluctuantes, en augmentant le transport des ARNm vers le cytosol et leur traduction. La répartition d'INO1 vers l'enveloppe nucléaire est déterminée par une augmentation locale des taux d'échange d'histones, ce qui entraîne sa séparation de phase du nucléoplasme en une phase de faible densité plus proche de la périphérie nucléaire. J'ai quantifié les propriétés mécaniques de la chromatine du locus du gène dans les états réprimé et actif en analysant le déplacement de 128 sites LacO fusionnés au gène liant LacI-GFP en calculant diffèrent paramètres tel que la constante de ressort effective et le rayons de confinement du locus. De plus, j'ai mesuré l'amplitude et le taux d'expansion en fonction du temps du réseau LacO et j'ai observé une diminution significative du locus à l'état actif, ce qui est cohérent avec le comportement de ressort entropique de la chromatine décompactée. J'ai montré que les séquences d'éléments en cis dans le promoteur du locus, essentielles à la séparation de phase, sont des sites de liaison pour les complexes de remodelage de la chromatine effectuant l'acétylation des histones. Ces modifications de la chromatine entraînent une augmentation des taux d'échanges des sous-unités des complexes d'histones, et une séparation de phase locale de la chromatine. Enfin, je présente l’analyse de simulations in silico qui montrent que la séparation de phase locale de la chromatine peut être prédite à partir d'un modèle de formation/disruption des interactions multivalentes protéine-protéine et protéine-ADN qui entraîne une diminution de la dynamique de l'ADN. Ces résultats suggèrent un mécanisme général permettant de contrôler la formation rapide des domaines de la chromatine, bien que les processus spécifiques contribuant à la diminution de la dynamique de l'ADN restent à étudier. Dans un second manuscrit, je décris comment nous avons induit la « retro-évolution » de la levure en réintroduisant la méthylation de la lysine 9 de l'histone H3 par l'expression de deux gènes de la levure Schizosaccaromyces pombe Spswi6 et Spclr4. Le mutant résultant présente une augmentation de la compaction de la chromatine, ce qui entraîne une réduction remarquable des taux de mutation et de recombinaison. Ces résultats suggèrent que la perte de la méthylation de la lysine 9 de l'histone H3 pourrait avoir augmenté la capacité à l'évoluer. La stabilité inhabituelle du génome conférée par ces mutations pourrait être utile pour l'ingénierie métabolique de S. cerevisiae, dans laquelle il est difficile de maintenir des gènes exogènes intégrés pour les applications de nombreux processus biotechnologiques courants tels que la production de vin, de bière, de pain et de biocarburants. Ces résultats soulignent l'influence des propriétés physiques d'un génome sur son architecture et sa fonction globales.The genome of the budding yeast Saccharomyces cerevisiae evolved from an ancestor in which a profound genome decompaction occurred as the result of the loss of histone H3 lysine 9 methylation, approximately 300 million years ago. This decompaction may have resulted in an increased capacity of yeasts to evolve by mechanisms that include unusually high meiotic recombination and mutation rates. Resultant increased evolvability may have enabled unique adaptations, which have made it an ideal model eukaryote and biotechnological tool. In this thesis I will present two examples of how local and global genome adaptations are reflected in changes in the mechanical properties of chromatin. In a first manuscript, I present evidence for a mechanism by which partitioning of the active inositol depletion-responsive gene locus INO1 from nucleoplasm to the nuclear envelope increases the speed of adaptation and metabolic robustness to fluctuating resources, by increasing mRNA transport to the cytosol and their translation. Partitioning of INO1 to the nuclear envelope is driven by a local increase in histone exchange rates, resulting in its phase separation from the nucleoplasm into a low-density phase closer to the nuclear periphery. I quantified the mechanical properties of the gene locus chromatin in repressed and active states by monitoring mean-squared displacement of an array of 128 LacO sites fused to the gene binding LacI-GFP and calculating effective spring constants and radii of confinement of the array. Furthermore, I measured amplitude and rate of time-dependent expansion of the LacO array, and observed a significant decrease for the active-state locus which is consistent with entropic spring behavior of decompacted chromatin. I showed that cis element sequences in the promoter and upstream of the locus that are essential to phase separation are binding sites for chromatin remodeling complexes that perform histone acetylation among other modifications that result in increased histone complex exchange rates, and consequent local chromatin phase separation. Finally, I present analytical simulations that show that local phase separation of chromatin can be predicted from a model of formation/disruption of multivalent protein-protein and protein-DNA interactions that results in decreased DNA dynamics. These results suggest a general mechanism to control rapid formation of chromatin domains, although the specific processes contributing to the decreased DNA dynamics remain to be investigated. In a second manuscript, I describe how we retro-evolutionarily engineered yeast by reintroducing histone H3 lysine 9 methylation through the expression of two genes from the yeast Schizosaccaromyces pombe Spswi6 and Spclr4. This mutant shows an increase in compaction, resulting in remarkable reduced mutation and recombination rates. These results suggest that loss of histone H3 lysine 9 methylation may have increased evolvability. The unusual genome stability imparted by these mutations could be of value to metabolically engineering S. cerevisiae, in which it is difficult to maintain integrated exogenous genes for applications for many common biotechnological processes such as wine, beer, bread, and biofuels production. These results highlight the influence of the physical properties of a genome on its overall architecture and function

Interactions of pseudorabies virus and swine influenza virus with porcine respiratory mucus

Pseudorabies virus (PrV) and swine influenza virus (SIV) both initiate their infection in the respiratory mucosa. Crossing the mucus layer which covers the respiratory mucosa is a crucial step for the mucosal invasion of PrV and SIV. In this thesis, interactions of PrV and SIV with porcine respiratory mucus were investigated, and the association between the viral behavior in mucus and the viral pathogenesis was discussed. In the first part, it is demonstrated whether and how PrV invades the host via mucus barrier. We first set up a virus tracking model using single particle tracking. The diffusion of PrV in mucus was determined and compared with that of negatively, positively and neutrally charged nanoparticles. It was shown that PrV was almost completely obstructed, with 96% of viral particles being immobilized in the porcine respiratory mucus. The negatively and positively charged particles were similarly trapped, in contrast to the neutral particles that moved freely. This suggests that immobilization of PrV was at least partly due to charge-charge interactions between its surface proteins and mucus. Since PrV had difficulties in crossing the mucus barrier, we further identified which component mediates the interactions, by using an explant model. We found that MUC5AC was a dominant mucin expressed in the apical epithelium. The content of MUC5AC on the apical epithelium was inversely related to the attachment and infection of PrV to/in porcine trachea explants, suggesting an important role of MUC5AC in blocking PrV to reach the epithelium. The MUC5AC present above the epithelium was able to block more than 50% of virus infection, which further confirmed a strong inhibition of respiratory mucus against PrV infection. However, the mucus barrier could be overcome by PrV at low temperature (4 oC), determined by a virus-mucus binding system, a virus in-capsule-mucus penetration system and the explant model. We found that, compared to 37 oC, less viral particles were bound to the respiratory mucus at 4 oC, which resulted in deeper viral penetration in mucus, leading to a higher percentage of PrV that overcame the mucus of the explants and caused infection eventually. These results may explain winter seasonality of PrV infection. In the second part, single particle tracking and the virus in-capsule-mucus penetration system were applied to track SIV H1N1 in porcine respiratory mucus. Results showed that 70% of SIV particles were entrapped, while the rest diffused freely in mucus. In addition, SIV was partially able to penetrate through the respiratory mucus over time. Moreover, both the microscopic diffusion and macroscopic penetration were enhanced by the addition of exogenous neuraminidase, while they were in contrast diminished by the use of a neuraminidase inhibitor, indicating that neuraminidase helps SIV to move through the porcine respiratory mucus. In summary, PrV was highly hindered in porcine respiratory mucus. Thus, it may depend on mucus defects or physiological changes which for example may be caused by low temperature to invade the mucus barrier. On the other hand, SIV has evolved to produce neuraminidase which is able to release SIV particles which may be bound to mucins, thereby enabling the virus to move through the respiratory mucus. The in-depth investigation of virus-mucus interaction may provide novel insights into the study of prophylactic treatment for swine influenza and Aujeszky’s diseas

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin