Durante o desenvolvimento de holometálobos alguns tecidos são eliminados/remodelados durante a metamorfose. A autofagia age nesse processo degradando componentes citoplasmáticos, inicialmente isolando-os em dupla membrana, estrutura chamada autofagossomo e esses conteúdos são degradados por hidrolases lisossomais. Porém, aspectos apoptóticos podem estar presentes nesse processo, como o envolvimento de caspases e a fragmentação nuclear. Alterações morfológicas na glândula salivar e no corpo gorduroso, que são bons exemplos de órgãos que sofrem morte celular programada (MCP) no desenvolvimento de R. americana, foram analisados por microscopia de luz e eletrônica. Durante a remoção desses órgãos, núcleos apresentam morfologia condensada e com fragmentação confirmada por TUNEL. Ambos tecidos mostraram formação de autofagossomos, mas a glândula salivar completa o processo de MCP durante a metamorfose. Genes antiapoptóticos e autofágicos que têm importante papel na MCP foram caracterizados. MCP em R. americana apresenta cooperação de aspetos da autofagia e da apoptose. / In the development of holometabolous insects, some tissues are eliminated/remodelated during metamorphosis. Autophagy acts in this process by degrading cytoplasm contents, initially by surrounding them within a double membrane, a structure called autophagosome and its contents are degraded by lysosomal hydrolases. However, some features of apoptotic cell death may be present in this process, such as the involvement of caspases and nuclear fragmentation. Morphological changes of salivary gland and fat body, good examples of organs that suffer programmed cell death (PCD) during R. americana development, were analyzed by light and electron microscopy. During the removal of these organs, nuclei present fragmented and condensed morphology, confirmed by TUNEL assay. Both tissues show the formation of autophagosomes, but the salivary gland completes the process of PCD during metamorphosis. Antiapoptotic and autophagic genes that play important function in the PCD, were characterized. R. americana PCD occurs with the cooperation of autophagy and apoptosis features.

No description available.

Die mitotische Spindel ist ideal für die Erforschung der Selbstorganisation und Plastizität molekularer Kollektive im Zytoplasma. Die Geometrie der Spindel ist entscheidend für die korrekte Chromosomentrennung, muss sich aber an die Zellgröße anpassen. Es ist unbekannt, ob und wie Zellen während ihrer Differenzierung die Spindelarchitektur anpassen, was insbesondere während der Gehirnentwicklung relevant ist. Wir untersuchten dies mit Maus-Embryonalstammzellen, die in frühe neuronale Vorläuferzellen differenziert wurden. Wir entwickelten ein automatisiertes Mikroskopieprotokoll um einen umfassenden Datensatz von mitotischen Zellen zu generieren. Außerdem entwickelten wir Spindle3D, ein Werkzeug zur dreidimensionalen Analyse von Spindeln. Überraschenderweise waren die Spindelvolumina in differenzierenden Zellen bis zu 24% kleiner als in pluripotenten Zellen. Während die Wachstumsgeschwindigkeit der Mikrotubuli unverändert blieb, verschob sich in sich differenzierenden Zellen die Nukleation von Mikrotubuli zugunsten der astralen Population. Diese Veränderung der Spindelarchitektur basierte auf der differenzierungsbedingten Verdünnung des Zytoplasmas. Dies aktivierte CPAP, ein Protein, das die Zentrosomenreifung reguliert, was zur Superskalierung des perizentriolären Materials und verstärkte Rekrutierung von gamma-Tubulin an den Zentrosomen und somit zur Umlagerung von Mikrotubuli innerhalb der Spindel führte. Diese Veränderungen der mitotischen Architektur konnten durch externe osmotische Einwirkung in undifferenzierten Zellen nachgestellt werden. Insgesamt verbinden unsere Ergebnisse zelltypspezifische zytoplasmatische Materialeigenschaften mit der Spindelarchitektur. / The mitotic spindle provides an excellent system in which to study the plasticity of molecular collectives. To segregate chromosomes accurately, the spindle’s geometry must be adaptive to changes in cell size. It is unknown whether and how differentiating cells adjust spindle architecture, specifically during brain development when spindle defects have severe pathological consequences. Using murine embryonic stem cells, we recapitulated the transition from pluripotency to early neural cell identities in vitro. Aiming at a systematic exploration of spindle and cell morphology throughout this process, we developed an automated microscopy protocol and Spindle3D, a morphometric tool for the analysis of spindles in confocal images. Intriguingly, in cells with comparable cell volume, spindle volumes were up to 24% smaller in cells undergoing differentiation. While microtubule growth speed remained equal, we measured increased nucleation of astral microtubules at the expense of the spindle bulk in differentiating cells. The shift in spindle architecture was explained by the differentiation-driven cytoplasmic dilution. This activated the centrosomal regulator CPAP, causing the superscaling of the pericentriolar material and the concomitant increased recruitment of gamma-tubulin to the centrosomes, redistributing microtubule numbers within the spindle. Mimicking the dilution effect by osmotic challenge reproduced the same mitotic architecture in undifferentiated cells. Collectively, our results link cell state-specific cytoplasmic material properties to spindle architecture.

Kaposi's sarcoma-associated herpesvirus (KSHV) expresses numerous intronless mRNAs that are unable to access splicing-dependent cellular mRNA nuclear export pathways. To circumvent this problem, KSHV encodes the open reading frame 57 (ORF57) protein, which orchestrates the formation of an export-competent virus ribonucleoprotein particle comprising the nuclear export complex hTREX, but not the exon-junction complex (EJC). Interestingly, EJCs stimulate mRNA translation, which raises the intriguing question of how intronless KSHV transcripts are efficiently translated. Herein, we show that ORF57 associates with components of the 48S pre-initiation complex and co-sediments with the 40S ribosomal subunits. Strikingly, we observed a direct interaction between ORF57 and PYM, a cellular protein that enhances translation by recruiting the 48S pre-initiation complex to newly exported mRNAs, through an interaction with the EJC. Moreover, detailed biochemical analysis suggests that ORF57 recruits PYM to intronless KSHV mRNA and PYM then facilitates the association of ORF57 and the cellular translation machinery. We, therefore, propose a model whereby ORF57 interacts directly with PYM to enhance translation of intronless KSHV transcripts.

No description available.

SMC1 and SMC3 form a high-affinity heterodimer, which provides an open backbone of the cohesin ring, to be closed by a kleisin protein. RNAi mediated knock-down of either one heterodimer partner, SMC1 or SMC3, is expected to cause very similar if not identical phenotypes. However, we observed highly distinct, protein-specific phenotypes. Upon knock-down of human SMC1, much of SMC3 remains stable, accumulates in the cytoplasm and does not associate with other cohesin proteins. Most of the excess nuclear SMC3 is highly mobile and not or only weakly chromosome-associated. In contrast, human SMC3 knock-down rendered SMC1 instable without cytoplasmic accumulation. As observed by differential protein extraction and in FRAP experiments the remaining SMC1 or SMC3 proteins in the respective SMC1 or SMC3 knock-down experiments constituted a cohesin pool, which is associated with chromatin with highest affinity, likely the least expendable. Expression of bovine EGFP-SMC1 or mouse EGFP-SMC3 in human cells under conditions of human SMC1 or SMC3 knock-down rescued the respective phenotypes, but in untreated cells over-expressed exogenous SMC proteins mis-localized. Paucity of either one of the SMC proteins causes RAD21 degradation. These results argue for great caution in interpreting SMC1 and SMC3 RNAi or over-expression experiments. Under challenged conditions these two proteins unexpectedly behave differently, which may have biological consequences for regulation of cohesin-associated functions and for human cohesin pathologies.

SMC1 and SMC3 form a high-affinity heterodimer, which provides an open backbone of the cohesin ring, to be closed by a kleisin protein. RNAi mediated knock-down of either one heterodimer partner, SMC1 or SMC3, is expected to cause very similar if not identical phenotypes. However, we observed highly distinct, protein-specific phenotypes. Upon knock-down of human SMC1, much of SMC3 remains stable, accumulates in the cytoplasm and does not associate with other cohesin proteins. Most of the excess nuclear SMC3 is highly mobile and not or only weakly chromosome-associated. In contrast, human SMC3 knock-down rendered SMC1 instable without cytoplasmic accumulation. As observed by differential protein extraction and in FRAP experiments the remaining SMC1 or SMC3 proteins in the respective SMC1 or SMC3 knock-down experiments constituted a cohesin pool, which is associated with chromatin with highest affinity, likely the least expendable. Expression of bovine EGFP-SMC1 or mouse EGFP-SMC3 in human cells under conditions of human SMC1 or SMC3 knock-down rescued the respective phenotypes, but in untreated cells over-expressed exogenous SMC proteins mis-localized. Paucity of either one of the SMC proteins causes RAD21 degradation. These results argue for great caution in interpreting SMC1 and SMC3 RNAi or over-expression experiments. Under challenged conditions these two proteins unexpectedly behave differently, which may have biological consequences for regulation of cohesin-associated functions and for human cohesin pathologies.

No description available.

No description available.

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