Cells grow in environments that can change suddenly. To cope with unpredictable perturbations, they have evolved mechanisms to adjust their metabolism according to the various types of environmental stress. Cells experiencing starvation, for example, have low energy levels and are forced to lower their metabolism and enter a protective quiescent state to survive until nutrients become available again.
Recently, it has been shown that starved yeast cells experience a marked acidification of the cytoplasm, due to a passive influx of protons. This pH drop causes multiple rearrangements in the cytoplasm: increased crowding, reduced mobility of intracellular components and formation of stress-induced non-membrane bound compartments of specific metabolic enzymes. Cytoplasm rearrangements are required for cell survival and can be reversed upon replenishment of energy. However, there is little understanding of how cytoplasmic components reorganize in stressed quiescent cells. Using high-pressure freezing, correlative light and electron microscopy (CLEM) and electron tomography, coupled to high-resolution 3D-reconstruction techniques, I investigate the structural modi cations that happen in situ in yeast cells undergoing quiescence. I observe that the cytoplasm becomes increasingly crowded, due to a massive rearrangement of membranous structures, including accumulation of intracellular vesicles and pronounced invaginations in the plasma membrane. This is proved by quantification of the difference in ribosome densities between stressed and not stressed cells.
The increased crowding, coupled to cytoplasm acidification, leads to the formation of non-membrane bound enzyme compartments, that appear as foci and elongated structures of fluorescently tagged enzymes. I prove that the fluorescent structures correspond to bundles of filaments. Among many essential enzymes, known to form mesoscale structure in stressed yeast, I demonstrate that the eukaryotic translation initiation factor 2B (eIF2B) forms bundles of filaments in situ, and the evolutionary conserved glutamine synthetase (Gln1) self-assembles into filaments in vitro.
The present study on the energy depleted cytoplasm and the structural analysis of filament-forming enzymes provides insights into an unexplored survival strategy that is used by yeast, as well as other organisms, to cope with extreme environmental conditions and stress.:1 Introduction 1
Stress, survival and quiescence 2
1.1 Cytoplasm and cellular compartments 2
1.2 Membraneless compartmentalization in the cell 3
1.3 Stress-induced non-membrane bound assemblies 4
The quiescent sleeping yeast 6
1.4 The yeast S.cerevisiae as model organism 7
1.5 Growth and metabolism of yeast 9
1.5.1 Yeast eukaryotic translation initiation factor 2B: eIF2B 11
1.5.2 Yeast glutamine synthetase: Gln1 12
3D electron microscopy 14
Aims of the Thesis 18
2 Materials and methods 21
Room temperature electron microscopy (EM) 21
2.1 Yeast strains, media and energy depletion 21
2.2 High-pressure freezing of yeast cells 22
2.2.1 EM sample preparation for untagged eIF2B yeast strains 22
2.2.2 EM sample preparation for GFP-tagged eIF2B yeast strains 23
2.3 Electron tomography 23
2.4 Subtomogram averaging 24
2.5 Fiji script for automated ribosome counting 25
2.6 Immunofluorescence of eIF2B in yeast 26
2.7 Western-blot on yeast ribosomes 27
Single particle procedures 30
2.8 Protein purification protocols 30
2.8.1 Baculovirus-insect cell expression and purification of eIF2B 30
2.8.2 Gradient of fixation for fragile complexes 31
2.8.3 Yeast expression and purification of Gln1 33
2.9 Negative staining 34
2.9.1 Image acquisition and analysis—eIF2B 35
2.9.2 Image acquisition and analysis—Gln1 36
Cryo-electronmicroscopy(cryo-EM) 37
2.10 Plunge freezing 37
2.11 Image acquisition and 3D reconstruction 37
3 Results
Visualizing yeast’s cytoplasmic reorganization 39
3.1 Quiescence is accompanied by reorganization of the cytoplasm 40
3.2 Ribosome density proves cytoplasmic crowding in starved cells 42
3.3 eIF2B organizes in bundles of filaments in energy-depleted cells 45
3.4 eIF2B filaments are polymers of the eIF2B complex 47
3.5 Filaments are found in wild-type energy-depleted cells 49
Structural analysis of filament forming enzymes 51
3.6 Purification of eIF2B complexes 51
3.7 Single particle analysis of eIF2B 53
3.8 Purification of Gln1 complexes 55
3.9 Single particle analysis of Gln1 56
3.10 Gln1 forms filaments in vitro 58
4 Discussion and Outlook 59
4.1 Yeast cytoplasm reorganizes in response of stress 59
4.2 Ribosomes density is a measure of increased macromolecular crowding 60
4.3 eIF2B forms filaments as a survival strategy 62
4.4 Molecular analysis of filament forming enzymes 64
4.5 Outlook 65
Appendix 67
Bibliography 83
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:30960 |
| Date | 17 September 2018 |
| Creators | Marini, Guendalina |
| Contributors | Pigino, Gaia, Grill, Stephan, Hyman, Anthony, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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