Chromosome alignment is highly conserved in all eukaryotic cell divisions. Microtubule (MT) -based forces generated by the mitotic spindle are integral for proper chromosome alignment and equal chromosome segregation. The kinetochore is a multi-subunit protein complex that assembles on centromeric regions of chromosomes. Kinetochores tether chromosomes to MTs (K fibers) that emanate from opposite poles, in a process called biorientation. This linkage translates K fiber dynamics into chromosome movements during alignment and segregation. Stable, high-affinity kinetochore attachments promote spindle assembly checkpoint (SAC) silencing, which is active when unattached kinetochores are present. During chromosome alignment, 1) K fiber plus-end dynamics decrease, confining chromosome movements near the spindle equator, and 2) electrostatic interactions between kinetochore proteins and MTs increase. Chromosome segregation occurs as soon as all chromosomes are stably attached to microtubules and the SAC has been silenced. SAC silencing and chromosome alignment are temporally coordinated during normal divisions, implying that the mechanisms regulating K fiber dynamics and kinetochore affinity must be linked. Interestingly, HeLa cells depleted of a kinesin-8 motor Kif18A, known for its role in promoting chromosome alignment, display a SAC-dependent mitotic delay due to kinetochore-MT attachment defects. This is puzzling, as Kif18A's function in chromosome alignment is to suppress MT growth by stably associating with MT plus-ends. Whether Kif18A is required for attachment in all cells and how it promotes kinetochore microtubule linkages are not understood. The work presented in this dissertation supports a model in which Kif18A functions as a molecular link that coordinates chromosome alignment and anaphase onset. We find that Kif18A is required to stabilize kinetochore-MT attachments during mammalian germline development, as germline precursor cells in Kif18A mutant mice are unable to divide during embryogenesis due to an active SAC. However, while all cell types require functional Kif18A for chromosome alignment, mouse primary somatic cells can still divide with normal timing. This finding indicates a cell-type specific dependence on Kif18A for stabilizing kinetochore-MT attachments, and provides evidence that this function might be separate from Kif18A's known role in chromosome alignment. Consistent with this idea, we find that an evolutionarily conserved binding motif for protein phosphatase 1 (PP1) is required for Kif18A's novel role in regulating kinetochore microtubule attachments. Kif18A-PP1 interaction is required for Kif18A-mediated dephosphorylation of the kinetochore protein Hec1, which enhances attachment. However, Kif18A's interaction with PP1 is dispensable for chromosome alignment. Thus, point mutations that disrupt PP1 binding separate Kif18A's role in stabilizing kinetochore attachments from its function in promoting chromosome alignment. Additionally, through structure function studies of the motor domain, we identified a long surface loop (Loop2) that is required for Kif18A's unique MT plus-end binding activity, which is essential for its function in confining chromosome movements. Taken together, we find that Kif18A is molecularly tuned to provide temporal control of chromosome alignment and anaphase entry.

Das im Cytosol liegende Dictyostelium Centrosom ist aus einer geschichteten Core-Region aufgebaut, die von einer Mikrotubuli-nukleierenden Corona umgeben ist. Zudem ist es über eine spezifische Verbindung eng an den Kern geknüpft und durch die Kernmembran hindurch mit den geclusterten Centromeren verbunden. Beim G2/M Übergang dissoziiert die Corona vom Centrosom und der Core verdoppelt sich so dass zwei Spindelpole entstehen. CP55 und CP148 wurden in einer Proteom-Analyse des Centrosoms identifiziert. CP148 ist ein neues coiled-coil Protein der centrosomalen Corona. Es zeigt eine zellzyklusabhängige An- und Abwesenheit am Centrosom, die mit der Dissoziation der Corona in der Prophase und ihrer Neubildung in der Telophase korreliert. Während der Telophase erschienen in GFP-CP148 exprimierenden Zellen viele, kleine GFP-CP148-Foci im Cytoplasma, die zum Teil miteinander fusionierten und zum Centrosom wanderten. Daraus resultierte eine hypertrophe Corona in Zellen mit starker GFP-CP148 Überexpression. Ein Knockdown von CP148 durch RNAi führte zu einem Verlust der Corona und einem ungeordneten Interphase Mikrotubuli-Cytoskelett. Die Bildung der mitotischen Spindel und der astralen Mikrotubuli blieb davon unbeeinflusst. Das bedeutet, dass die Mikrotubuli-Nukleationskomplexe während der Interphase und Mitose über verschiedene Wege mit dem Core assoziiert sind. Des Weiteren bewirkte der Knockdown eine Dispersion der Centromere sowie eine veränderte Sun1 Lokalisation in der Kernhülle. Somit spielt CP148 ebenso eine Rolle in der Centrosomen-Centromer-Verbindung. Zusammengefasst ist CP148 ein essentielles Protein für die Bildung und Organisation der Corona, welche wiederum für die Centrosom/Centromer Verbindung benötigt wird. CP55 wurde als Protein der Core-Region identifiziert und verbleibt während des Zellzyklus am Centrosom. Dort besitzt es strukturelle Aufgaben, da die Mehrheit der GFP-CP55 Moleküle in der Interphase keine Mobilität zeigten. Die GFP-CP55 Überexpression führte zur Bildung von überzähligen Centrosomen mit der üblichen Ausstattung an Markerproteinen der Corona und des Cores. CP55 Knockout-Zellen waren durch eine erhöhte Ploidie, eine weniger strukturierte und leicht vergrößerte Corona sowie zusätzliche cytosolische Mikrotubuli-organisierende Zentren charakterisiert. Letztere entstanden in der Telophase und enthielten nur Corona- aber keine Core-Proteine. In CP55 k/o Zellen erfolgte die Rekrutierung des Corona-Organisators CP148 an den Spindelpol bereits in der frühen Metaphase anstatt, wie üblich, erst in der Telophase. Außerdem zeigten die Knockout-Zellen Wachstumsdefekte, deren Grund vermutlich Schwierigkeiten bei der Centrosomenverdopplung in der Prophase durch das Fehlen von CP55 waren. Darüber hinaus konnten die Knockout-Zellen phagozytiertes Material nicht verwerten, obwohl der Vorgang der Phagozytose nicht beeinträchtigt war. Dieser Defekt kann dem im CP55 k/o auftretenden dispergierten Golgi-Apparat zugeschrieben werden. / The Dictyostelium centrosome consists of a layered core structure surrounded by a microtubule-nucleating corona. A tight linkage through the nuclear envelope connects the cytosolic centrosome with the clustered centromeres within the nuclear matrix. At G2/M the corona dissociates, and the core structure duplicates yielding two spindle poles. The two proteins CP148 and CP55 were discovered in a proteomic analysis of Dictyostelium centrosomes. CP148 is a novel coiled-coil protein of the centrosomal corona. GFP-CP148 exhibited cell cycle dependent presence and absence at the centrosome, which correlates with dissociation of the corona in prophase and its reformation in late telophase. During telophase, GFP-CP148 formed cytosolic foci, which coalesced and joined the centrosome. This explains the hypertrophic appearance of the corona upon strong overexpression of GFP-CP148. Depletion of CP148 by RNAi caused virtual loss of the corona and disorganization of interphase microtubules. Surprisingly, formation of the mitotic spindle and astral microtubules was unaffected. Thus, microtubule nucleation complexes associate with centrosomal core components through different means during interphase and mitosis. Furthermore, CP148 RNAi caused dispersal of centromeres and altered Sun1 distribution at the nuclear envelope, suggesting a role of CP148 in the linkage between centrosomes and centromeres. Taken together, CP148 is an essential factor for the formation of the centrosomal corona, which in turn is required for centrosome/centromere linkage. As CP148, CP55 was also identified in a centrosomal proteome analysis. It is a component of the centrosomal core structure, and persists at the centrosome throughout the entire cell cycle. FRAP experiments revealed the majority of centrosomal GFP-CP55 is immobile indicating a structural task of CP55 at the centrosome. GFP-CP55 overexpression elicits supernumerary centrosomes containing the usual set of corona and core marker proteins. The CP55 null mutant is characterized by increased ploidy, a less structured, slightly enlarged corona, and by supernumerary, cytosolic MTOCs, containing only corona proteins and lacking a core structure. Live cell imaging showed that supernumerary MTOCs arise in telophase. Lack of CP55 also caused premature recruitment of the corona organizer CP148 to mitotic spindle poles, already in metaphase instead of telophase. Forces transmitted through astral microtubules may expel prematurely acquired or loosely attached corona fragments into the cytosol, where they act as independent MTOCs. CP55null cells were also impaired in growth, most probably due to difficulties in centrosome splitting during prophase. Furthermore, although they were still capable of phagocytosis, they appeared unable to utilize phagocytosed nutrients. This inability may be attributed to their disorganized Golgi apparatus.

The Saccharomyces cerevisiae iron-responsive transcription factor, Aft1p, has a well established role in regulating iron homeostasis through the transcriptional induction of iron-regulon genes. However, recent studies have implicated Aft1p in other cellular processes independent of iron-regulation such as chromosome stability. In addition, chromosome spreads and two-hybrid data suggest that Aft1p interacts with and co-localizes with kinetochore proteins, however the cellular implications of this have not been established. Here, we demonstrate that Aft1p associates with the kinetochore complex through Iml3p. Furthermore, we show that Aft1p, like Iml3p, is required for the increased association of cohesin with the pericentromere and that aft1Δ cells display sister chromatid cohesion defects in both mitosis and meiosis. Our work defines a new role for Aft1p in the sister chromatid cohesion pathway.

The Saccharomyces cerevisiae iron-responsive transcription factor, Aft1p, has a well established role in regulating iron homeostasis through the transcriptional induction of iron-regulon genes. However, recent studies have implicated Aft1p in other cellular processes independent of iron-regulation such as chromosome stability. In addition, chromosome spreads and two-hybrid data suggest that Aft1p interacts with and co-localizes with kinetochore proteins, however the cellular implications of this have not been established. Here, we demonstrate that Aft1p associates with the kinetochore complex through Iml3p. Furthermore, we show that Aft1p, like Iml3p, is required for the increased association of cohesin with the pericentromere and that aft1Δ cells display sister chromatid cohesion defects in both mitosis and meiosis. Our work defines a new role for Aft1p in the sister chromatid cohesion pathway.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 105-117).

Mutations in the presenilin 1 gene account for most early-onset familial Alzheimer's disease (FAD). The presenilins and AD may also be related through a common involvement in the cell cycle. Here we report that one important aspect of the cell cycle---proper chromosome segregation---is dependent on presenilin function and therefore may be involved in AD pathogenesis. Specifically we find that FAD mutations in presenilin 1 (M146L and M146V) lead to chromosome missegregation and aneuploidy in vivo and in vitro: 1) Both metaphase chromosome analysis and in situ hybridization reveal significant aneuploidy in the lymphocytes and neurons of PS-1 transgenic mice. 2) Transiently transfected human cells expressing normal and, especially, mutant PS-1 develop aneuploidy within 48 hours, including trisomy 21, while cells transfected with dominant negative PS-1 genes lacking ?-secretase activity have no effect on chromosome segregation. 3) Analysis of mitotic spindles in the transfected cells reveals abnormal microtubule arrays and lagging chromosomes. The possible mechanisms by which cell cycle defects and chromosome missegregation induced by y-secretase may contribute to Alzheimer's disease will be discussed.

No description available.

Histone deacetylases (HDAC) 1 and 2 play crucial role in chromatin remodeling and gene expression regimes, as part of multiprotein corepressor complexes. Protein kinase CK2-driven phosphorylation of HDAC1 and 2 regulates their catalytic activities and is required to form the corepressor complexes. Phosphorylation-mediated differential distributions of HDAC1 and 2 complexes in regulatory and coding regions of transcribed genes catalyze the dynamic protein acetylation of histones and other proteins, thereby influence gene expression. During mitosis, highly phosphorylated HDAC1 and 2 heterodimers dissociate and displace from mitotic chromosomes. Our goal was to identify the kinase involved in mitotic phosphorylation of HDAC1 and 2. We postulated that CK2-mediated increased phosphorylation of HDAC1 and 2 leads to dissociation of the heterodimers, and, the mitotic chromosomal exclusions of HDAC1 and 2 are largely due to the displacement of HDAC-associated proteins and transcription factors, which recruit HDACs, from chromosomes during mitosis. We further explored the role of un- or monomodified HDAC1 and 2 complexes in immediate-early genes (IEGs), FOSL1 (FOS-like antigen-1) and MCL1 (Myeloid cell leukemia-1), regulation. Dynamic histone acetylation is an important regulator of these genes that are overexpressed in a number of diseases and cancers. We hypothesized that transcription dependent recruitment of HDAC1 and 2 complexes over the gene body regions plays a regulatory role in transcription and splicing regulation of these genes. We present evidence that CK2-catalyzed increased phosphorylation of HDAC1 and 2 regulates the formation of distinct corepressor complexes containing either HDAC1 or HDAC2 homodimers during mitosis, which might target cellular factors. Furthermore, the exclusion of HDAC-recruiting proteins is the major factor for their displacement from mitotic chromosomes. We further demonstrated that un- or monophosphorylated HDAC1 and 2 are associated with gene body of FOSL1 in a transcription dependent manner. However, HDAC inhibitors prevented FOSL1 activation independently of the nucleosome response pathway, which is required for IEG induction. Interestingly, our mass spectrometry results revealed that HDAC1 and 2 interact with a number of splicing proteins, in particular, with serine/arginine-rich splicing factor 1 (SRSF1). HDAC1 and 2 are co-occupied with SRSF1 over gene body regions of FOSL1 and MCL1, regardless of underlying splicing mechanisms. Using siRNA-mediated knockdown approaches and HDAC inhibitors, we demonstrated that alternative splicing of MCL1 is regulated by RNA-directed localized changes in the histone acetylation levels at the alternative exon. The change in histone acetylation levels correlates with the increased transcription elongation and results in change in MCL1 splicing by exon skipping mechanism. Taken together, our results contribute to further understanding of how the multi-faceted HDAC1 and 2 complexes can be regulated and function in various processes, including, but not limited to, transcription regulation and alternative splicing. This can be an exciting area of future research for therapeutic interventions.

Microtubules are tubular polymers that are involved in a variety of cellular processes such as cell movement, mitosis and intracellular transport. The dynamic behavior of microtubules makes this possible because all of these processes require quick responses. Embryonic stem (ES) cells were first isolated from mouse embryos and they have two unique characteristics; they can be kept undifferentiated for many passages with a stable karyotype and they can be differentiated into any type of cells under appropriate conditions. The pluripotency of ES cells, their ease of manipulation in culture, and their ability to contribute to the mouse germ-line provides us a model of differentiation both in vitro and in vivo. In my thesis I focused on the cell division and neuronal differentiation of ES cells and developed two methods to understand the effects of microtubule dynamics in spindle assembly and chromosome segregation and to reveal the roles of different Microtubule Associated Proteins (MAPs) in the neuronal morphology formation. In the first part, we developed a live-cell imaging method for ES cells to visualize, track and analyze the single cell behavior in a cell population over a time period. So far many techniques have been adapted and combined for imaging of cell lines, mainly for the cancer or immortalized ones. However, because ES cells are very prone to apoptosis, tend to form spheres and hard to stably label, it is quite tricky to image them in culture conditions. In our system, we combined the BAC-based gene expression with wide-field deconvolution microscopy for ES cells that are plated onto the laminin-511 coated surface and kept in CO2 independent culture conditions. This combined technique does not interfere with the growth of cells and keeps them healthy up to 24 hours on the microscope stage. In the second part, we analyzed the effects of MAPs chTOG, EB1, Kif18A and MCAK in the overall spindle morphology and mitotic progression in mES cells. For this purpose, we utilized our stable TUBB-GFP and H2A-GFP cell lines along with our live-cell imaging set-up to reveal the effects of the above-mentioned proteins and the interplay among each other. By using RNAi method we either single or co-depleted the genes by siRNAs and measured the spindle length and width in RNAi conditions. We further analyzed the mitotic progression in H2A-GFP cell line in terms of the metaphase timing and the percentage of chromosome segregation errors. Our results showed that, EB1 depletion did not cause any significant changes in the overall spindle morphology or in the metaphase timing. However, the co-depletion of EB1 with chTOG partially rescued the sichTOG specific mini-spindle phenotype. siKif18A produced longer spindles without any change in the spindle width. Surprisingly, the co-depletion of antagonistic chTOG and Kif18A proteins had additive effects on the spindle dynamics and on mitotic progression in a way that spindle assembly was severely disrupted by the absence of these two proteins and as a result of this, both metaphase timing and chromosome missegregation levels increased significantly. These results overall indicate that MAPs have important roles in the regulation of dynamic instability and these proteins have an interplay among each other to be able to control the morphology of the spindle as well as the correct segregation of chromosomes into daughter cells. In the last part, I will introduce you a new ES cell based differentiation and morphology model, which brings the advantages of high resolution imaging capacity, control over development and easy genetic manipulation and culturing. We have generated Tet-induced shRNA cell lines against chTOG, Kif18A and MCAK, which are also stably expressing TUBB-GFP. These labeled cells were mixed with unlabeled wild-type mES cells before differentiation at 1:1000 ratio and then they were differentiated into mouse cortical cells and spinal motor neurons. Our results showed that, all of the three genes could be successfully knocked-down by shRNA after 48 hours of Tet induction. After mixing the labeled and unlabeled cells, single neurons could be imaged at high resolution and their skeletons could be generated afterwards. The RNAi studies in shchTOG cell line showed that, the knock-down of this gene in early differentiation interferes with the neuronal differentiation.

Histone deacetylases (HDAC) 1 and 2 play crucial role in chromatin remodeling and gene expression regimes, as part of multiprotein corepressor complexes. Protein kinase CK2-driven phosphorylation of HDAC1 and 2 regulates their catalytic activities and is required to form the corepressor complexes. Phosphorylation-mediated differential distributions of HDAC1 and 2 complexes in regulatory and coding regions of transcribed genes catalyze the dynamic protein acetylation of histones and other proteins, thereby influence gene expression. During mitosis, highly phosphorylated HDAC1 and 2 heterodimers dissociate and displace from mitotic chromosomes. Our goal was to identify the kinase involved in mitotic phosphorylation of HDAC1 and 2. We postulated that CK2-mediated increased phosphorylation of HDAC1 and 2 leads to dissociation of the heterodimers, and, the mitotic chromosomal exclusions of HDAC1 and 2 are largely due to the displacement of HDAC-associated proteins and transcription factors, which recruit HDACs, from chromosomes during mitosis. We further explored the role of un- or monomodified HDAC1 and 2 complexes in immediate-early genes (IEGs), FOSL1 (FOS-like antigen-1) and MCL1 (Myeloid cell leukemia-1), regulation. Dynamic histone acetylation is an important regulator of these genes that are overexpressed in a number of diseases and cancers. We hypothesized that transcription dependent recruitment of HDAC1 and 2 complexes over the gene body regions plays a regulatory role in transcription and splicing regulation of these genes. We present evidence that CK2-catalyzed increased phosphorylation of HDAC1 and 2 regulates the formation of distinct corepressor complexes containing either HDAC1 or HDAC2 homodimers during mitosis, which might target cellular factors. Furthermore, the exclusion of HDAC-recruiting proteins is the major factor for their displacement from mitotic chromosomes. We further demonstrated that un- or monophosphorylated HDAC1 and 2 are associated with gene body of FOSL1 in a transcription dependent manner. However, HDAC inhibitors prevented FOSL1 activation independently of the nucleosome response pathway, which is required for IEG induction. Interestingly, our mass spectrometry results revealed that HDAC1 and 2 interact with a number of splicing proteins, in particular, with serine/arginine-rich splicing factor 1 (SRSF1). HDAC1 and 2 are co-occupied with SRSF1 over gene body regions of FOSL1 and MCL1, regardless of underlying splicing mechanisms. Using siRNA-mediated knockdown approaches and HDAC inhibitors, we demonstrated that alternative splicing of MCL1 is regulated by RNA-directed localized changes in the histone acetylation levels at the alternative exon. The change in histone acetylation levels correlates with the increased transcription elongation and results in change in MCL1 splicing by exon skipping mechanism. Taken together, our results contribute to further understanding of how the multi-faceted HDAC1 and 2 complexes can be regulated and function in various processes, including, but not limited to, transcription regulation and alternative splicing. This can be an exciting area of future research for therapeutic interventions.