The Saccharomyces cerevisiae plasmid, 2 micron circle, resides in the yeast nucleus at a high copy number. It provides no apparent growth advantage to its host, nor imposes any significant growth disadvantage. The plasmid is an excellent paradigm for studying mechanisms utilized in the persistence of a eukaryotic selfish DNA element that is selectively neutral. The plasmid achieves stable propagation and copy number maintenance by combining a partitioning system and an amplification system. The partitioning proteins Rep1p and Rep2p promote the recruitment of the histone H3 variant Cse4p and the yeast cohesin complex to the partitioning locus STB during S phase, leading to the formation of a functional partitioning complex which segregates the plasmid equally during mitosis. The integrity of the mitotic spindle is a pre-requisite for the specific nuclear localization of the plasmid as well as for plasmid association with a subset of the partitioning proteins such as Cse4p and the cohesin complex. The work presented in this thesis reveals, using tools of molecular genetics and cell biology, the involvement and possible functions of a microtubule associated nuclear motor protein, Kip1p, in the 2 micron circle partitioning pathway. The plasmid missegregates in kip1[Delta] cells, but not in cells harboring deletions of genes coding for the other nuclear motors. Kip1p interacts with the plasmid partitioning system and promotes the association of Cse4p and the cohesin complex with STB. Lack of Kip1p function delocalizes the plasmid from its characteristic nuclear locale in close proximity to the spindle pole body. The distance between a reporter plasmid and the spindle pole body is nearly doubled in a kip1[Delta] host strain. We propose that, unlike the conventional roles played by nuclear motors in spindle function and chromosome segregation, the Kip1p motor assists the 2 micron circle in associating with the mitotic spindle and translocating to its ‘partitioning center’. / text

The precise positioning of the mitotic spindle to the cell center during mitosis is a fundamental process for chromosome segregation and the division plane definition. Despite its importance, the mechanism for spindle centering remains elusive. To study this mechanism, the dynamic of the microtubules was characterized at the bulk and at the cortex in the C. elegans embryo. Then, this dynamic was correlated to the centering forces of the spindle that were studied by applying calibrated magnetic forces via super-paramagnetic beads inserted into the cytoplasm of one- and two-cell C. elegans embryos. Finally, these results were confronted with the different centering models: cortical pushing model, cortical pulling model and the cytoplasmic pulling model. This thesis shows that: (i) The microtubules dynamic of the spindle aster is controlled spatially in the C. elegans embryo, with not rescues and catastrophes in the cytoplasm but in the centrosome and the cortex, respectively. (ii) The centering mechanism of the spindle behaved roughly as a damped spring with a spring constant of 18 12 pN/ m and a drag coefficient of 127 65 pN s/ m (mean SD). This viscoelastic behavior is evidence of a centering force that recovers and/or maintains the position of the spindle in the cell center. (iii) It seems to be two mechanisms that recover/maintain the spindle position. A fast one that may work for transient displacements of the spindle and a slow one that work over large and long perturbations. (iv) The centering forces scale with the cell size. The centering forces are higher in the two-cell embryo. This result argues against a centering mechanism mediated by cytoplasmic factors. It seems to be a limit for the relation of centering force to size, as the forces found in the four-cell embryo are comparable to the single-cell ones. (v) The centering forces scale with the amount of microtubules in the cell. This strengthens the belief that the microtubules are the force transmission entities of the centering mechanism. (vi) The boundary conditions are important to maintain the centering forces. A transient residency time of microtubules at the cortex, which is controlled by cortical catastrophe factors, is indispensable for a proper force transmission by the microtubules. (vii) The elimination of cortical catastrophe factors provides evidence for microtubules buckling, which is taken as a proof of polymerization forces. (viii) The cortical pulling forces mediated by the gpr-1/2 pathway do not seem to be involved in centering and it is proposed they are present in the cell for off-center positioning purposes. (ix) The forces generated by vesicle transport are enough to displace the spindle and they are suggested to be auxiliary forces to centering. (x) The forces associated with the spindle change dramatically during cell division. From metaphase to anaphase the forces associated with the spindle scale up to five times. This behavior was consistent during the development of the embryo as the same pattern was observed in the one-, two- and four-cell embryo. (xi) The higher forces found during anaphase are not cortical pulling (via pgr-1/2 pathway) depended, and it is proposed the spindle is `immobilised' by tethering or by an unknown cortical pulling pathway. To this date, this thesis presents the most complete in-vivo measurements of the centering forces in association with the microtubules dynamics. Taken together the results constrain molecular models of centering. This thesis concludes that most probably the predominant forces of the spindle centering mechanism during mitosis are generated by astral microtubules pushing against the cortex. Additionally, this thesis presents the most complete map of forces during cell division during development, which will prove to be indispensable to understand the changes the spindle undergoes when it changes its function.

Errors in segregation of genetic materials are detrimental to all organisms. The budding yeast ensures accurate chromosome segregation by employing a system called the spindle checkpoint. The spindle checkpoint, which consists of proteins such as Mad1, Mad2, Mad3, Bub1, and Bub3, monitors the attachment of microtubules to the chromosomes and prevents cell cycle progression until all chromosomes are properly attached. To understand how the spindle checkpoint arrests cells in response to attachment errors at the chromosomes, we recruited different checkpoint proteins to an ectopic site on the chromosome by taking advantage of the binding of the lactose repressor (LacI) to the lactose operator (LacO). We found that cells expressing Bub1-LacI arrest in metaphase. The phenotype is in fact caused by dimerization of Bub1 when it is fused to LacI rather than the recruitment of Bub1 to chromosome. The cell cycle arrest by the Bub1 dimer depends on the presence of other checkpoint proteins, suggesting that the dimerization of Bub1 represents an upstream event in the spindle checkpoint pathway. The results with the Bub1 dimer inspired us to fuse checkpoint proteins to each other to mimic protein interactions that may contribute to checkpoint activation. We showed that fusing Mad2 and Mad3 arrests cells in mitosis and that this arrest is independent of other checkpoint proteins. We believe that combining Mad2 and Mad3 arrests cells because both proteins can bind weakly to Cdc20, the main target of the spindle checkpoint, and the sum of these two weak bindings creates a hybrid protein that binds tightly to Cdc20. We reasoned that if Mad3's role is to make Mad2 bind tightly, artificially tethering Mad2 directly to Cdc20 should also arrest cells and this arrest should not depend on any other checkpoint components. Our experiments confirmed these predictions, suggesting that Mad3 is required for the stable binding of Mad2 to Cdc20 in vivo, that this binding is sufficient to inhibit APC activity, and that this reaction is the most downstream event in spindle checkpoint activation. The interactions among spindle checkpoint proteins thus play an important role in cell cycle arrest and must be carefully regulated.

No description available.

Robust bipolar spindle formation and faithful transmission of genetic material are vital to the maintenance of genome integrity and cellular homeostasis. Chromosome segregation errors can result in aneuploidy, a hallmark of human solid tumors. The assembly of a microtubule-based mitotic spindle relies on the concerted action of centrosomes, spindle microtubules, molecular motors and nonmotor spindle proteins. Before mitosis, centrosomes need to duplicate and increase in size in order to gain sufficient microtubule nucleation activity during bipolar spindle formation. This process is called centrosome maturation and coincides with a dramatic change of centrosome structure. However, the architecture of centrosomes and the organization of centrosome components in both interphase and mitosis have long remained elusive. In this thesis, I describe the identification and characterization of novel regulators that are essential for centrosome and mitotic spindle organization in human cells. One such regulator is human Augmin, an evolutionarily conserved eight-subunit protein complex that has essential functions for centrosome and spindle integrity. I present evidence that human Augmin promotes microtubule-dependent nucleation of microtubules by targeting microtubule-nucleating complexes to the mitotic spindle. This function of Augmin is important for generation and/or stabilization of kinetochore microtubules within the mitotic spindle, and its loss results in destabilization of kinetochore microtubules and spindle assembly errors. These errors culminate in cells displaying multipolar spindles with fragmented centrosomes and mitotic arrest. A second regulator of centrosome and spindle organization described in this thesis is CEP192. I show that CEP192 is critical for recruitment of microtubule-nucleating complexes to centrosomes and, consequently, for centrosome maturation, mitotic spindle formation, and centriole duplication. Finally, I describe novel organizational features of the centrosome using a subdiffraction microscopy approach. Because of a lack of higher-order structural information, centrosomes have traditionally been described as amorphous clouds. My results now reveal that centrosome components instead occupy separable spatial domains throughout the cell cycle and highlight the role of higher-order protein organization in the regulation of centrosome assembly and function. Collectively, this work has significantly expanded our current knowledge of centrosome architecture and biogenesis and of the mechanisms that underlie robust bipolar spindle assembly.

In this thesis I investigate social processes that motivate the contemporary reproduction and public dissemination of older Central Coast Salish spindle whorls. In a case study, I develop a cultural biography of spindle whorls to examine how material culture produced by past generations informs contemporary activity. Visual materials, first- and third-person accounts and writings in three areas—material culture, the social nature of art and colonialism—are drawn together to demonstrate that spindle whorl production and circulation is grounded in social and historical contingencies specific to Central Coast Salish First Nations. I propose that in using spindle whorls, Central Coast Salish people are drawing on the past to strengthen their position within current circumstances.

Robust bipolar spindle formation and faithful transmission of genetic material are vital to the maintenance of genome integrity and cellular homeostasis. Chromosome segregation errors can result in aneuploidy, a hallmark of human solid tumors. The assembly of a microtubule-based mitotic spindle relies on the concerted action of centrosomes, spindle microtubules, molecular motors and nonmotor spindle proteins. Before mitosis, centrosomes need to duplicate and increase in size in order to gain sufficient microtubule nucleation activity during bipolar spindle formation. This process is called centrosome maturation and coincides with a dramatic change of centrosome structure. However, the architecture of centrosomes and the organization of centrosome components in both interphase and mitosis have long remained elusive. In this thesis, I describe the identification and characterization of novel regulators that are essential for centrosome and mitotic spindle organization in human cells. One such regulator is human Augmin, an evolutionarily conserved eight-subunit protein complex that has essential functions for centrosome and spindle integrity. I present evidence that human Augmin promotes microtubule-dependent nucleation of microtubules by targeting microtubule-nucleating complexes to the mitotic spindle. This function of Augmin is important for generation and/or stabilization of kinetochore microtubules within the mitotic spindle, and its loss results in destabilization of kinetochore microtubules and spindle assembly errors. These errors culminate in cells displaying multipolar spindles with fragmented centrosomes and mitotic arrest. A second regulator of centrosome and spindle organization described in this thesis is CEP192. I show that CEP192 is critical for recruitment of microtubule-nucleating complexes to centrosomes and, consequently, for centrosome maturation, mitotic spindle formation, and centriole duplication. Finally, I describe novel organizational features of the centrosome using a subdiffraction microscopy approach. Because of a lack of higher-order structural information, centrosomes have traditionally been described as amorphous clouds. My results now reveal that centrosome components instead occupy separable spatial domains throughout the cell cycle and highlight the role of higher-order protein organization in the regulation of centrosome assembly and function. Collectively, this work has significantly expanded our current knowledge of centrosome architecture and biogenesis and of the mechanisms that underlie robust bipolar spindle assembly.

Thesis (Ph.D.). / Written for the Dept. of Biology. Title from title page of PDF (viewed 2008/01/12). Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Includes bibliographical references. Also available online.