Eukaryotic cells have to divide constantly in order to promote the growth of certain organs, to replace dying or damaged cells, or to set up an entire organism. These essential processes are called mitosis in the case of somatic cell division. Mitotic cell division is the process during which chromosomes, centrosomes, and microtubules (MTs) are involved to set up a bipolar structure called the “mitotic spindle”. This bipolar spindle is formed by MTs, which are presumably mainly organized from the centrosomes. However, more data are being published that suggest MTs nucleation can occur from other MTs or even a chromosome surface. These biopolymers are built from α/β-tubulin heterodimers and can dynamically grow and shrink to exert forces necessary for chromosome segregation. Previous studies of spindles during mitosis have allowed the identification of different MT classes based on their plus-ends interaction with different cellular target sites. One of the MT classes is the kinetochore microtubules (KMTs), which physically connect chromosomes and centrosomes (i.e. spindle pole) via a specialized protein structure termed the “kinetochore”. This kinetochore-to-spindle pole connection has been studied in many organisms. In budding yeast, this connection is established by only a single KMT. In contrast, multiple KMTs bind to each mammalian kinetochore and form an MT bundle also called “k-fiber”. The ultrastructural architecture of the mammalian k-fiber connection is not well documented. Currently, different models concerning the nature of the kinetochore-to-spindle pole connection via k-fibers are discussed in the literature, i.e. a direct, semi-direct or indirect connection. The widely accepted ‘direct’ model proposes that all k-fibers of the mammalian spindle are formed through tight bundles of up to 20 KMTs, with all MT minus ends associated with the centrosome. However, it is necessary to understand the k-fibers structure in order to interpret its role during chromosome segregation. Here the architecture of the k-fiber was studied in human HeLa, U2OS and RPE-1 cell lines, as these different types of cells have been widely used in studies of mitosis. This thesis aimed to systematically investigate the characteristics of mammalian k-fibers and their attachment to the kinetochore within mammalian metaphase spindles. For that, the ultrastructure of mitotic spindles and k-fibers were analyzed using serial-section electron tomography primarily in HeLa cells. Furthermore, the spindle ultrastructure was compared by electron tomography to metaphase spindles in both U2OS and RPE-1 cells. Electron tomographic analysis of the mitotic spindle in HeLa cells revealed that the kinetochore-to-spindle pole connection is formed by k-fibers consisting of ~9 KMTs. Moreover, the data revealed that not all KMTs in k-fibers are directly associated with one of the spindle poles. Instead, KMT ends were located along the length of k-fibers indicating strongly for a semi-direct connection between the kinetochores and the spindle poles. Unexpectedly, by correlating the k-fiber ultrastructure with its position in the mitotic spindle, it can be demonstrated that the k-fiber structure varied depending on the position on the metaphase plate. It can also be shown that k-fibers located in the center of the metaphase plate had a tendency to form straighter and more bundled k-fibers. In contrast, k-fibers associated with the periphery of the metaphase plate had a more loose and disorganized structure resembling a fusiform shape. Furthermore, additional analysis of U2OS and RPE-1 cells indicated ultrastructural differences between the different cell lines. Mainly, differences between HeLa and RPE-1 cells were observed. K-fibers observed in RPE-1 cells showed a lower curvature and overall a more bundled ultrastructure compared to HeLa or U2OS cells. However, due to the small sample size for U2OS and RPE-1 cells, the results have to be confirmed in future experiments to conclude that there are indeed functional and structural differences in the k-fiber organization in different mammalian cell lines. Taken together, this work presents the first detailed quantitative ultrastructural analysis of KMTs in whole spindles in three different human cell lines. The data revealed that the currently favored direct model of k-fiber ultrastructure is oversimplified and needs to be corrected in terms of the k-fibers interaction with the spindle pole and the surrounding MT network within the mitotic spindle. The data here will serve as a structural basis for further analyses of mutant situations and contribute to our understanding of the overall organization and function of MTs in mitotic spindles.:Summary 6
Zusammenfassung 8
List of figures 10
List of tables 13
List of abbreviations and symbols 14
1 Introduction 19
1.1 The morphology of the mitotic spindle 21
1.1.1 Centrosomes 22
1.1.2 Microtubules 23
1.2 Kinetochores, KMTs and k-fibers 28
1.2.1 A brief history of k-fiber formation in mammalian cells 30
1.2.2 Models of the k-fiber ultrastructure in mammalian cells 32
2 Aims of this thesis 35
3 Materials and methods 36
3.1 Materials 37
3.1.1 Mammalian cell lines 37
3.1.2 Chemicals 38
3.1.3 Instrumentation and materials 40
3.1.4 Solutions and buffers 44
3.1.5 Software 46
3.2 Methods 47
3.2.1 Handling of cell cultures 47
3.2.2 Custom-designed incubation chambers 49
3.2.3 Specimen preparation for electron microscopy 51
3.2.4 Quality assessment of samples, acquisition of the tomographic data, and the 3D reconstruction 59
3.2.5 Ultrastructural analysis of MTs in mitotic spindles 62
3.2.6 Ultrastructural analysis of the k-fiber organization 70
4 Results 76
4.1 Initial characterization of mammalian mitotic spindles 77
4.2 Ultrastructure of KMTs 84
4.3 Curvature and tortuosity of KMTs 91
4.4 Ultrastructure of k-fibers 98
4.5 Effect of metaphase position on the k-fiber ultrastructure 102
5 Discussion 110
5.1. Comparison of data sets from different cell lines 111
5.2. Establishing a data analysis pipeline for the analysis of KMTs 113
5.3 Ultrastructural characterization of KMTs and k-fibers in HeLa cells 114
5.3.1 K-fibers have an unexpectedly low number of KMTs 115
5.3.2 Semi-direct kinetochores-to-spindle pole connection 117
5.3.3 Shape of k-fibers 121
5.4 Positional effect on the k-fiber shape 124
5.5 Comparison of k-fiber ultrastructure in different mammalian cells 127
5.6 Outlook 130
References 133
Appendix 1 149
Appendix 2 150
Appendix 3 151
Appendix 4 152
Acknowledgments 153
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:76019 |
| Date | 21 September 2021 |
| Creators | Kiewisz, Robert |
| Contributors | Müller-Reichert, Thomas, Needleman, Daniel J., Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | info:eu-repo/grantAgreement/European Commission/Horizon2020/675737//A multidisciplinary approach to cell division: From human oocyte to synthetic biology/DivIDE |
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