La première augmentation de la longévité en laboratoire fût observée à la suite d’une intervention nutritionnelle consistant en une réduction de l’apport alimentaire chez le rat. Plus tard, ce phénomène a été reproduit dans de très nombreuses espèces et référé en tant que restriction calorique. Le développement des techniques de biologie moléculaire moderne a permis de montrer dans des organismes modèles simples que cette flexibilité du processus de vieillissement était régulée par des facteurs génétiques. De fait, plusieurs mécanismes cellulaires ont alors pu être identifiés comme responsables de ce contrôle du vieillissement. Ces voies de régulation ont révélées être conservées entre les espèces, depuis les levures jusqu’aux organismes multicellulaires tels que le nématode, la mouche ou la souris, suggérant l’existence d’un programme universel de vieillissement dans le vivant. La levure s’est avéré à plusieurs reprises être un modèle puissant et fiable pour la découverte de gènes impliqués dans ce phénomène. Mon étude a consisté au développement d’un nouveau modèle unicellulaire d’étude du vieillissement à travers l’espèce Schizosaccharomyces pombe appelée aussi levure à fission. La première étape de mon travail a montré que les voies de détection des nutriments gouvernées par la sérine/thréonine protéine kinase A (Pka1) et la sérine/thréonine kinase Sck2 contrôlent le vieillissement chronologique de ces cellules comme il était connu dans la levure Saccharomyces cerevisiae. Ceci permit de valider l’utilisation de la levure à fission pour l’étude du vieillissement. Ensuite, nous avons analysé plus en détail l’effet pro-vieillissement du glucose en étudiant le rôle de sa détection par le récepteur membranaire Git3 couplé à la protéine G (Gpa2) en amont de la kinase Pka1. La perte du signal du glucose par la délétion de Git3 imite partiellement l’effet d’augmentation de longévité obtenu par baisse de la concentration en glucose dans le milieu. De plus, l’effet néfaste du signal du glucose est maintenu en absence de tout métabolisme du glucose suite à la mutation des hexokinases, premières enzymes de la glycolyse. L’ensemble de ces résultats suggèrent que la signalisation du glucose est prédominante sur son métabolisme pour son effet pro-vieillissement. D’autre part, à la fois la suppression de cette signalisation et la baisse de niveau de glucose disponible allongent la durée de vie en corrélation avec une augmentation de la résistance au stress, une hausse d’activité mitochondriale et une baisse de production de radicaux libres. Finalement, le criblage d’une banque de surexpression d’ADNc a permis d’identifier plusieurs gènes candidats responsables de ces effets en aval de la voie de signalisation Git3/PKA. La recherche sur les mécanismes moléculaires du vieillissement propose une nouvelle approche, un nouvel angle de vue, pour la compréhension des fonctions cellulaires et promet d’apporter de précieuses clefs pour mieux comprendre certaines maladies. En effet, le vieillissement est la première cause d’apparition de nombreuses affections comme les cancers, les maladies cardiovasculaires et métaboliques ou les maladies neurodégénératives tels que les syndromes d’Alzheimer et de Parkinson. / The first increase in life span due to man’s intervention was obtained with rats subjected to a diet reduced in calorie intake. Later, this phenomenon was repeated with many other species and referred as diet restriction or calorie restriction. The development of modern Molecular Biology approaches and the use of simple model organisms demonstrated that the rate of aging was regulated by genetic traits. Indeed, several cellular mechanisms were identified as responsible for the control of aging. These regulatory pathways appear to be conserved throughout species, from yeast to multicellular organisms like nematode, fly and mice, thus suggesting the existence of a universal program of aging. Yeast proved several times to be a powerful and reliable model for discovering genes involved in the regulation of aging. My study consisted in developing Schizosaccharomyces pombe (also called fission yeast) as a new unicellular model to study aging. The first step of my work was to show that pathways of nutrient detection through kinases involving Pka1 and Sck2 control chronological aging in S. pombe, as it was previously demonstrated in Saccharomyces cerevisiae. This first work validated the use of fission yeast for the study of aging. Subsequently, we analysed in more detail the pro-aging effect of glucose focusing on the role of its signalling through the G-protein Gpa2-coupled membrane receptor Git3, which acts upstream of Pka1. The loss of the glucose signal due to deletion of Git3 mimics partially the effect of increasing longevity by reducing glucose in the medium. Moreover, detrimental effects of glucose signal are maintained in absence of sugar metabolism following loss of hexokinases, the first enzymes of glycolysis. Together, these results suggest that the pro-aging effects of glucose signalling are predominant over those due to metabolism of this sugar. Moreover, both obliteration of this signalling pathway and decrease of glucose availability extend life span, and correlate with an increase in stress resistance, in mitochondrial activity and a lower production of free radicals. Finally, screening a cDNA-overexpression library allowed us to identify several genes candidates responsible for the effects on longevity downstream of Git3/Pka1. Research in the molecular mechanisms of aging propose holds the promise to bring precious clues as to this mysterious processes affecting all living creatures, and paves the way to unravel the underlying causes of many human diseases. Indeed, aging is the first cause of numerous late-onset pathologies including cancers, cardiovascular diseases or neurodegenerative diseases like Alzheimer and Parkinson syndromes.

Butylated Hydroxyanisole, BHA, and Butylated Hydroxytoluene, BHT, are commonly used as preservatives for our food as well as additives in many products such as cosmetics, petroleum, and medicine. Although their use has been approved by the Food and Drug Administration (FDA), there have been controversies and debates on whether these phenol derivatives or antioxidants are safe to use. Their accumulative toxicology and side effects need to be thoroughly investigated as we continue to consume them on a daily basis. Data obtained by genomic analysis in Tang lab suggested the involvement of DNA damage checkpoint/repair pathways in the response network to these phenol stress factors. The aims of this thesis are to examine the morphological changes and potential DNA damage induced by exposing cells to BHA and BHT using fission yeast Schizosaccharomyces pombe as a model organism. Fluorescence microscopy was used to assess DNA double-strain breaks (DSBs) by monitoring the nuclear foci formation of Rad22, a DNA repair protein, in the presence of BHA and BHT. Changes in cell morphology were also studied under microscope. Preliminary data showed that cells treated with BHA and BHT exhibited morphological changes. In addition, for the first time in S. pombe cells, Rad22 foci in the nucleus of BHA and BHT treated cells were observed. Further investigation is needed to optimal the experimental condition to continue the study. These results will not only help us to better understand the effect of these phenol derivatives in the cells, but can also establish an experimental system for future studies on the interaction of the cells with stress factors and therapeutic drugs for human-related diseases such as cancer.

The major aims of this thesis were to investigate the influence of i) antisense gene location relative to the target gene locus (?????location effect?????), ii) double-stranded RNA (dsRNA) formation, and iii) over-expression of host-encoded proteins on antisense RNA-mediated gene regulation. To test the location effect hypothesis, strains were generated which contained the target lacZ gene at a fixed location and the antisense lacZ gene at various genomic locations including all arms of the three fission yeast chomosomes and in close proximity to the target gene locus. A long inverse-PCR protocol was developed to rapidly identify the precise site of antisense gene integration in the fission yeast transformants. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus, compared with other genomic locations, indicating that target and antisense gene co-localisation is not a critical factor for efficient antisense RNA-mediated gene suppression in vivo. Instead, increased lacZ down-regulation correlated with an increase in the steady-state level of antisense RNA, which was dependent on genomic position effects and transgene copy number. In contrast, convergent transcription of an overlapping antisense lacZ gene was found to be very effective at inhibiting lacZ gene expression. DsRNA was also found to be a central component of antisense RNA-mediated gene silencing in fission yeast. It was shown that gene suppression could be enhanced by increasing the intracellular concentration of non-coding lacZ RNA, while expression of a lacZ panhandle RNA also inhibited beta-galactosidase activity. In addition, over-expression of the ATP-dependent RNA-helicase, ded1, was found to specifically enhance antisense RNA-mediated gene silencing. Through a unique overexpression screen, four novel factors were identified which specifically enhanced antisense RNA-mediated gene silencing by up to an additional 50%. The products of these antisense enhancing sequences (aes factors), all have natural associations with nucleic acids which is consistent with other proteins which have previously been identified to be involved in posttranscriptional gene silencing.

RNA silencing is an evolutionary conserved sequence-specific mechanism of regulation of gene expression. RNA interference (RNAi), a type of RNA silencing in animals, is based on recognition and endonucleolytic cleavage of target mRNA complimentary in sequence to 21-nucleotide (nt) small RNA guides, called small interfering RNAs (siRNAs). Another class of 21-nt small RNAs, called micro RNAs (miRNAs), is endogenously encoded in eukaryotic genomes. Both production of siRNAs from long double-stranded RNA (dsRNA) and biogenesis of miRNAs from hairpin structures are governed by the ribonuclease III enzyme Dicer. Although produced as duplex molecules, siRNAs and miRNAs are assembled into effector complex, called the RNA-induced silencing complex (RISC), as single-strands. A member of the Argonaute family of small RNA-binding proteins lies at the core of all known RNA silencing effector complexes. Plants and animals contain multiple Argonaute paralogs. In addition to endonucleolytic cleavage, Argonaute proteins can direct translational repression/destabilization of mRNA or transcriptional silencing of DNA sequences by the siRNAdirected production of silent heterochromatin. The Schizosaccharomyces pombe genome encodes only one of each of the three major classes of proteins implicated in RNA silencing: Dicer (Dcr1), RNA-dependent RNA polymerase (RdRP; Rdp1), and Argonaute (Ago1). These three proteins are required for silencing at centromeres and for the initiation of transcriptionally silent heterochromatin at the mating-type locus. That only one Dicer, RdRP and Argonaute is expressed in S. pombe might reflect the extreme specialization of RNA silencing pathways regulating targets only at the transcriptional level in this organism. We decided to test if classical RNAi can be induced in S. pombe. We introduced a dsRNA hairpin corresponding to a GFP transgene. GFP silencing triggered by dsRNA reflected a change in the steady-state concentration of GFP mRNA, but not in the rate of GFP transcription. RNAi in S. pombe required dcr1, rdp1, and ago1, but did not require chp1, tas3, or swi6, genes required for transcriptional silencing. We concluded that the RNAi machinery in S. pombecould direct both transcriptional and posttranscriptional silencing using a single Dicer, RdRP, and Argonaute protein. Our findings suggest that, in spite of specialization in distinct siRNA-directed silencing pathways, these three proteins fulfill a common biochemical function. In Drosophila, miRNA and RNAi pathways are both genetically and biochemically distinct. Dicer-2 (Dcr-2) generates siRNAs, whereas the Dicer-1 (Dcr-1)/Loquacious complex produces miRNAs. Argonaute proteins can be divided by sequence similarity into two classes: in flies, the Ago subfamily includes Argonaute1 (Ago1) and Argonaute2 (Ago2), whereas the Piwi subfamily includes Aubergine, Piwi and Argonaute 3. siRNAs and miRNAs direct posttranscriptional gene silencing through effector complexes containing Ago1 or Ago2. The third class of small RNAs, called repeat-associated small interfering RNAs (rasiRNAs), is produced endogenously in the Drosophilagerm line. rasiRNAs mediate silencing of endogenous selfish genetic elements such as retrotransposons and repetitive sequences to ensure genomic stability. We examined the genetic requirements for biogenesis of rasiRNAs in both male and female germ line of Drosophilaand silencing of 8 different selfish elements, including tree LTR retrotransposons, two non-LTR retrotransposons, and three repetitive sequences. We find that biogenesis of rasiRNAs is different from that of miRNAs and siRNAs. rasiRNA production appears not to require Dicer-1 or Dicer-2. rasiRNAs lack the 2´,3´ hydroxy termini characteristic of animal siRNA and miRNA. While siRNAs derive from both the sense and antisense strands of their dsRNA precursors, rasiRNAs accumulate in antisense polarity to their corresponding target mRNAs. Unlike siRNAs and miRNAs, rasiRNAs function through the Piwi, rather than the Ago, Argonaute protein subfamily. We find that rasiRNAs silence their target RNAs posttranscriptionally: mutations that abrogate rasiRNA function dramatically increase the steady-state mRNA level of rasiRNA targets, but do not alter their rate of transcription, measured by nuclear run-on assay. Our data suggest that rasiRNAs protect the fly germ line through a silencing mechanism distinct from both the miRNA and RNAi pathways.

Coordination of mitosis and cytokinesis is crucial to generate healthy daughter cells with equal amounts of genetic and cytoplasmic materials. In the fission yeast Schizosaccharomyces pombe, an evolutionarily conserved Cdc14-like phosphatase (Clp1) functions to couple mitosis and cytokinesis by antagonizing CDK activity. The activity of Clp1 is thought to be regulated in part by its subcellular localization. It is sequestered in the nucleolus and the spindle pole body (SPB) during interphase. Upon mitotic entry, it is released into the cytoplasm and localized to the kinetochores, the actomyosin ring, and the mitotic spindle to carry out distinct functions. It is not clear how Clp1 is released from the nucleolus, however, once released, a conserved signaling pathway termed Septation Initiation Network (SIN) functions to retain Clp1 in the cytoplasm until completion of cytokinesis. The SIN and Clp1 function together in a positive feedback loop to promote each other’s activity. That is, the SIN promotes cytoplasmic retention of Clp1, and cytoplasmic Clp1 antagonizes CDK activity and reverses CDK inhibition on the SIN pathway to promote its function and activity. However, at the start of this thesis, the mechanism by which the SIN regulated Clp1 was unknown. The SIN pathway is also required to promote constriction of the actomyosin ring, and the septum formation. However, its downstream targets were still uncharacterized. In two separate studies, we studied how Clp1 is released from the nucleolus at mitotic entry and how the SIN kinase Sid2 acts to retain Clp1 in the cytoplasm. We identified several Sid2 candidate substrates, and revealed other functions of the SIN pathway in coordinating mitotic events.

Proteins of the CSL family (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) act as effectors of the Notch signalling pathway in metazoan organisms. They function as repressors or activators of gene transcription in the framework of this pathway and influence many developmental processes. Metazoan CSL proteins can regulate gene expression Notch-independently as well. Notch-independent functions of CSL proteins might be evolutionarily ancestral and in cells and organisms may be important equally as Notch-dependent functions. Presence of CSL proteins was identified in several fungal species, organisms lacking the Notch signalling pathway components and most of known metazoan interacting partners of CSL proteins. CSL paralogs of the fission yeast Schizosaccharomyces pombe, cbf11 and cbf12, are non-essential genes encoding proteins localized in the nucleus of the cell. They exert antagonistic effects on regulation of processes like coordination of nuclear and cellular division and cell cycle progression, ploidy maintenance, cell adhesion and other. In this study, we have proved that both CSL paralogs are able to sequence-specifically bind the CSL-response element DNA in vitro and Cbf11 in vivo as well. Both proteins could activate gene expression in vivo and perform the function of transcription factors....

Faithful duplication and segregation of undamaged DNA is critical to the survival of all organisms and prevention of oncogenesis in multicellular organisms. To ensure inheritance of intact DNA, cells rely on checkpoints. Checkpoints alter cellular processes in the presence of DNA damage preventing cell cycle transitions until replication is completed or DNA damage is repaired. Several checkpoints are specific to S-phase. The S-M replication checkpoint prevents mitosis in the presence of unreplicated DNA. Rather than outright halting replication, the S-phase DNA damage checkpoint slows replication in response to DNA damage. This checkpoint utilizes two general mechanisms to slow replication. First, this checkpoint prevents origin firing thus limiting the number of replication forks traversing the genome in the presence of damaged DNA. Second, this checkpoint slows the progression of the replication forks. Inhibition of origin firing in response to DNA damage is well established, however when this thesis work began, slowing of replication fork progression was controversial. Fission yeast slow replication in response to DNA damage utilizing an evolutionarily conserved kinase cascade. Slowing requires the checkpoint kinases Rad3 (hATR) and Cds1 (hChk2) as well as additional checkpoint components, the Rad9-Rad1-Hus1 complex and the Mre11-Rad50-Nbs1 (MRN) recombinational repair complex. The exact role MRN serves to slow replication is obscure due to its many roles in DNA metabolism and checkpoint response to damage. However, fission yeast MRN mutants display defects in recombination in yeast and, upon beginning this project, were described in vertebrates to display S-phase DNA damage checkpoint defects independent of origin firing. Due to these observations, I initially hypothesized that recombination was required for replication slowing. However, two observations forced a paradigm shift in how I thought replication slowing to occur and how replication fork metabolism was altered in response to DNA damage. We found rhp51Δ mutants (mutant for the central mitotic recombinase similar to Rad51 and RecA) to slow well. We observed that the RecQ helicase Rqh1, implicated in negatively regulating recombination, was required for slowing. Therefore, deregulated recombination appeared to actually be responsible for slowing failures exhibited by the rqh1Δ recombination regulator mutant. Thereafter, I began a search for additional regulators required for slowing and developed the epistasis grouping described in Chapters II and V. We found a wide variety of mutants which either completely or partially failed to slow replication in response to DNA damage. The three members of the MRN complex, nbs1Δ, rad32Δ and rad50Δ displayed a partial defect in slowing, as did the helicase rqh1Δ and Rhp51-mediator sfr1Δ mutants. We found the mus81Δ and eme1Δ endonuclease complex and the smc6-xhypomorph to completely fail to slow. We were able to identify at least three epistasis groups due to genetic interaction between these mutants and recombinase mutants. Interestingly, not all mutants’ phenotypes were suppressed by abrogation of recombination. As introduced in Chapters II, III and IV checkpoint kinase cds1Δ, mus81Δ endonuclease, and smc6-x mutant slowing defects were not suppressed by abrogation of recombination, while the sfr1Δ, rqh1Δ, rad2Δ and nbs1Δ mutant slowing defects were. Additionally, data shows replication slowing in fission yeast is primarily due to proteins acting locally at sites of DNA damage. We show that replication slowing is lesion density-dependent, prevention of origin firing representing a global response to insult contributes little to slowing, and constitutive checkpoint activation is not sufficient to induce DNA damage-independent slowing. Collectively, our data strongly suggest that slowing of replication in response to DNA damage in fission yeast is due to the slowing of replication forks traversing damaged template. We show slowing must be primarily a local response to checkpoint activation and all mutants found to fail to slow are implicated in replication fork metabolism, and recombination is responsible for some mutant slowing defects.

Cytokinesis is a fundamental step of cell proliferation by which daughter cells acquire equal amounts of genetic materials and cellular components. Cytokinesis is precisely regulated in a temporal and spatial manner to ensure that cytokinesis does not occur until chromosome segregation is complete. Failed or precocious cell division causes aneuploidy and/or polyploidy, which is often associated with cancer. In order to coordinate cytokinesis with mitosis, signaling networks have evolved in eukaryotic organisms to faithfully control late cell cycle progression by triggering cytokinesis once mitotic events have been successfully accomplished. In the fission yeast Schizosaccharomyces pombe, this conserved signaling network is known as the septation initiation network (SIN), which triggers actomyosin ring constriction and septum formation after chromosome segregation. The key output of the SIN is thought to be Sid2p kinase activity because Sid2p kinase is the most downstream component of the SIN identified so far, and in addition to the spindle pole bodies Sid2p also localizes to the division site at the end of anaphase, suggesting that Sid2p kinase transmits the division signal from the SPB to the division site, thereby triggering actomyosin ring constriction and septum formation. However, how Sid2p kinase activity is regulated during the cell cycle is still unclear. The goal of this thesis is to understand how Sid2p kinase is regulated. We identified and characterized Mob1p as a novel component of the SIN and a binding partner of the Sid2p kinase. We found that Mob1p is an essential regulatory component important for Sid2p kinase function. Furthermore, we found that phosphorylation is essential for activation of Sid2p kinase and that self-association is able to antagonize Sid2p kinase activity. Thus, we conclude that Sid2p kinase may utilize multiple modes of regulation, including Mob1p binding, stimulatory phosphorylation, and self-association, to control initiation of cytokinesis. Considering the conservation of Mob1p and Sid2p families in the eukaryotes, it is likely that other eukaryotic organisms utilize similar mechanism(s) to control cytokinesis.

The proper transmission of genetic materials into progeny cells is crucial for maintenance of genetic integrity in eukaryotes and fundamental for reproduction of organisms. To achieve this goal, chromosomes must be attached to microtubules emanating from opposite poles in a bi-oriented manner at metaphase, and then should be separated equally through proper spindle elongation in anaphase. Failure to do so leads to aneuploidy, which is often associated with cancer. Despite the presence of a safety device called the spindle assembly checkpoint (SAC) to monitor chromosome bi-orientation, mammalian cells frequently possess merotelic kinetochore orientation, in which a single kinetochore binds microtubules emanating from both poles. Merotelically attached kinetochores escape from the surveillance mechanism of the SAC and when cells proceed to anaphase cause lagging chromosomes, which are a leading cause of aneuploidy in mammalian tissue cultured cells. The fission yeast monopolin complex functions in prevention of mal-orientation of kinetochores including merotelic attachments during mitosis. Despite the known importance of Cdk1 activity during mitosis, it has been unclear how oscillations in Cdk1 activity drive the dramatic changes in chromosome behavior and spindle dynamics that occur at the metaphase/anaphase transition. In two separate studies, we show how dynamic Cdk1 phosphorylation regulates chromosome segregation. First, we demonstrate that sequential phosphorylation and dephosphorylation of monopolin by Cdk1 and Cdc14 phosphatase respectively helps ensure the orderly execution of two discrete steps in mitosis, namely sister kinetochore bi-orientation at metaphase and spindle elongation in anaphase. Second, we show that elevated Cdk1 activity is crucial for correction of merotelic kinetochores produced in monopolin and heterochromatin mutants.

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