No description available.

No description available.

The centromere is the site of kinetochore assembly that ensures proper chromosome segregation. Active centromeres are formed at chromosomal locations that do not appear to share homology between different species; this and other analyses has lead to the conclusion that centromeres are epigenetically determined. In all organisms, centromere location is specified by the assembly of unusual nucleosomes containing the histone H3 variant CENP-A in place of H3. However, an apparent paradox is that CENP-A in most organisms generally occurs on certain preferred sequences. The analyses presented focuses on the influence of DNA sequence on the selection of the locus where CENP-A chromatin are formed and whether there are any particular DNA features that promote CENP-ACnp1 assembly at centromeres in Schizosaccharomyces pombe. S. pombe provides an excellent model to study the structure and function of a complex eukaryotic centromere as it possesses epigenetically regulated centromeres that are structurally related to those of metazoa. Furthermore, plasmid-based circular minichromosomes provide a useful tool for studying these centromeres and the inactivation of minichromosome centromeres does not affect cell viability. The main aim of this study was to identify those features that enable centromeric sequences to assemble CENP-A chromatin. Manipulated circular minichromosomes were utilised to investigate the minimal central core sequence requirement for establishment of CENP-ACnp1 chromatin. These analyses showed that a minimal 2kb region from the central core of cen2 could form a functional centromere. A second aim was to analyse the DNA sequence requirements for centromere function on this minimal 2 kb region. To facilitate this, the endogenous central core region of endogenous cen2 was replaced with the central core region of cen1. This modified strain allows the structural and biological properties associated with plasmid borne central core 2 sequences to be analysed. Transcription of central domain sequences has been proposed to play a role in CENP-A establishment and/or maintenance. To explore the contribution of transcription potential promoters were mapped within the minimal 2 kb sequence and their regulatory elements investigated. Mutation of the minimal DNA element impedes its ability to assemble CENP-A chromatin. Therefore the primary DNA sequence of fission yeast centromeres is important for establishing functional centromeres and thus centromere location not entirely epigenetically regulated. It remains to be determined if the characteristics associated with theses sequences, and their mode of action, are conserved at other centromeres.

In Schizosaccharomyces pombe mitosis is initiated when Cdc25 tyrosine phosphatase dephosphorylates Cdc2 (Cdk1) and in turn Cdc2 kinase phosphorylates mitotic targets. Cdc2 is thought to phosphorylate and further activate Cdc25, forming a positive feedback loop between the two for robust entry into mitosis. Pyp3 tyrosine phosphatase is essential in the absence of Cdc25. Its role is thought to be in directly dephosphorylating Cdc2 under these conditions. Pyp3 also presents a link between cell division and growth. It interacts physically and genetically with the mRNA cap-binding protein eIF4E and is thought to play the same role as mammalian 4E-binding proteins. Pyp3 has a consensus TOS motif potentially enabling nutritional input from the TOR pathway into translation regulation. Since known 4E-BPs are not phosphatases, Pyp3 may act as a 4E-binding protein independently of its phosphatase activity. Evolutionarily conserved Cdc2 phosphorylation sites in Cdc25 were substituted to non-phosphorylatable Ala, or to Glu as a phosphomimic. The T89E phosphomimic mutation creates an activated allele of Cdc25, cdc25-89w. It has a dominant semi-wee phenotype due to accelerated entry into mitosis. Pyp3 was mutagenized to remove the function of the phosphatase active site and also the putative TOS motif. The Pyp3 active site is essential for its role in cell cycle initiation. It is also essential for the genetic interaction with eIF4E, tif1. Removal of a putative Pyp3 TOS motif affects the Pyp3 localization to cytoplasmic foci following co-overexpression of eIF4E. Similar localization occurs in response to heat stress. These results make important contributions to the understanding of mitotic initiation, and link between cell growth and division. / Thesis (Master, Biology) -- Queen's University, 2013-09-12 14:40:40.384

Microtubules (MTs) are important components of the eukaryotic cytoskeleton, with critical functions in intracellular trafficking, establishing and maintaining cell morphology, and segregating chromosomes during mitosis. MTs are hollow, cylindrical polymers composed of αβ-tubulin heterodimers. The longitudinal assembly of αβ-tubulin subunits generates protofilaments, and multiple protofilaments (typically 13 in vivo) interact laterally to form the wall of the MT. In vitro, the polymerization of MTs proceeds in two steps: nucleation and elongation. During the nucleation phase, several αβ-tubulin subunits associate to form a seed, from which further MT elongation then occurs. However, at the relatively low αβ-tubulin concentrations found in vivo, the spontaneous assembly of MTs is not favoured, due largely to the slow kinetics of MT nucleation. The nucleation of MTs in vivo requires the γ-tubulin complex (γ-TuC), a ring-like complex composed of γ-tubulin and γ-tubulin complex proteins (GCPs). Two copies of γ- tubulin associate with one copy each of GCP2 and GCP3 to produce the γ-tubulin small complex (γ-TuSC). Multiple γ-TuSCs, along with the additional GCPs 4,5, and 6, assemble to form the larger γ-tubulin ring complex (γ-TuRC). The γ-TuRC contains a ring of 13 γ-tubulins, which acts as a template for the nucleation of MTs. Typically, the γ-TuC nucleates MTs only when localized to specific subcellular sites, referred to as microtubule organizing centres (MTOCs). However, the precise mechanism by which the γ-TuC is activated at MTOCs remains unknown. In fission yeast, the proteins Mto1 and Mto2 form a complex (Mto1/2) required for the nucleation and organization of cytoplasmic MTs. Mto1/2 determines sites of MT nucleation by recruiting the γ-TuC to several different MTOCs. Different sequences in the Mto1 C-terminus independently confer γ-TuC localization to spindle pole bodies, MTs, and the cell equator. Here, I show that the Mto1 N-terminus is necessary for localization to the nuclear envelope (NE). By simultaneously removing the N- and C-terminal localization domains, I generated the "Mto1-bonsai" mutant, which fails to localize to any conventional MTOCs. In mto1-bonsai cells, MTs are still nucleated in the cytoplasm in an Mto1- dependent manner, but nucleation is spatially random. This reveals that targeting of the γ- TuC to conventional MTOCs is not necessary for MT nucleation, and suggests that Mto1/2 has a direct role in activating MT nucleation by the γ-TuC. Live-cell confocal microscopy allows us to detect individual MT nucleation events, in which newly nucleated MTs are associated with single γ-TuCs as well as Mto1/2-bonsai complexes. Fluorescence quantification reveals that these nucleating complexes contain approximately 13 molecules of both Mto1-bonsai and Mto2, matching the 13 copies of γ-tubulin anticipated for a single γ-TuC. We propose that Mto1/2 may contribute to γ-TuC activation by promoting γ-TuSC assembly and/or inducing conformational changes in the γ-TuC upon binding. I also expressed and purified recombinant Mto1/2-bonsai complex, using a baculovirus/insect cell system. This recombinant Mto1/2-bonsai self-assembles into higher-order complexes, comparable in size to the complexes analyzed in vivo by fluorescence microscopy.

Thesis advisor: Charles Hoffman / Cyclic adenosine monophosphate (cAMP) and the second messengers that modulate several biological processes are regulated by adenylyl cyclase (AC) and cyclic nucleotide phosphodiesterases (PDEs). ACs and PDEs are comprised of superfamilies of enzymes that are viewed as druggable targets due to their many distinct biological roles and tissue-specific distribution. As such, small molecule regulators of ACs and PDEs are important as chemical probes to study the roles of individual ACs or PDEs and as potential therapeutics. In the past, our lab has expressed 15 mammalian PDE genes in S. pombe, replacing the endogenous Cgs2 PDE. High throughput screens for PDE inhibitors identified novel compounds that show relevant biological activity in mammalian cell culture assays. The aim of this thesis is to develop tools to understand the mechanism of interaction between key regulators of the cAMP pathway and small molecules. The current study is comprised of two parts. In the first part of this thesis, I developed a genetic screen that detected alleles whose proteins remain active in the presence of BC54 and was to confirm the effect of the PDE4BT407A mutation using cell-based assays and in vitro enzyme assays. In the second part of this thesis, I developed and carried out HTSs using a PKA-repressed GFP reporter that can identify compounds that reduce PKA activity, which would include PDE activators and AC or GNAS1 inhibitors. To date, I have identified three AC inhibitors that appear to act on several of the ten different mammalian ACs. To our knowledge, this is the first time a large HTS has identified AC inhibitors, where inhibition was assessed inside the cells. The findings in this thesis will be useful in the design of more effective PDE inhibitors and in the development of novel chemical probes for studying cAMP signaling in mammalian cells. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Charles Hoffman / Cyclic adenosine monophosphate (cAMP) and the second messengers that modulate several biological processes are regulated by adenylyl cyclase (AC) and cyclic nucleotide phosphodiesterases (PDEs). ACs and PDEs are comprised of superfamilies of enzymes that are viewed as druggable targets due to their many distinct biological roles and tissue-specific distribution. As such, small molecule regulators of ACs and PDEs are important as chemical probes to study the roles of individual ACs or PDEs and as potential therapeutics. In the past, our lab has expressed 15 mammalian PDE genes in S. pombe, replacing the endogenous Cgs2 PDE. High throughput screens for PDE inhibitors identified novel compounds that show relevant biological activity in mammalian cell culture assays. The aim of this thesis is to develop tools to understand the mechanism of interaction between key regulators of the cAMP pathway and small molecules. The current study is comprised of two parts. In the first part of this thesis, I developed a genetic screen that detected alleles whose proteins remain active in the presence of BC54 and was to confirm the effect of the PDE4BT407A mutation using cell-based assays and in vitro enzyme assays. In the second part of this thesis, I developed and carried out HTSs using a PKA-repressed GFP reporter that can identify compounds that reduce PKA activity, which would include PDE activators and AC or GNAS1 inhibitors. To date, I have identified three AC inhibitors that appear to act on several of the ten different mammalian ACs. To our knowledge, this is the first time a large HTS has identified AC inhibitors, where inhibition was assessed inside the cells. The findings in this thesis will be useful in the design of more effective PDE inhibitors and in the development of novel chemical probes for studying cAMP signaling in mammalian cells. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Accurate chromosome segregation is critical as unequal distribution of the genomic DNA results in impaired cell function or cell death. Kinetochores, the multi-protein structures assembled on centromeric DNA, drive chromosome segregation. Chromosome segregation is under supervision of mitotic spindle checkpoint. The mitotic spindle checkpoint is a surveillance mechanism ensuring that cells enter anaphase with all kinetochores properly attached to spindle microtubules and thereby preventing missegregation. Some checkpoint proteins are localised at kinetochore where they generate and enhance the checkpoint signal. Mps1 (Mph1 in S. pombe) and Aurora B (Ark1 in S. pombe) kinases are required for precise chromosome segregation and mitotic spindle checkpoint in fission yeast. In this study we investigate the roles of Mph1 and Ark1 in regulating the S. pombe kinetochore protein Spc7, which is the homologue of human Blinkin/KNL1. We demonstrated that both kinases target the N-terminus of Spc7. Loss of phosphorylation on the candidate phosphosites results in sensitivity to microtubule depolymerizing drugs indicating mitotic defects. As Blinkin has been proposed to be a docking platform for checkpoint proteins, we tested the possibility that Mph1 kinase is involved in kinetochore targeting of checkpoint proteins, Bub1 and Bub3. Our results demonstrate that Mph1-dependent phosphorylation of Spc7 at conserved MELT motifs is required for Bub1 and Bub3 kinetochore localisation. We were able to reconstitute the interaction between Spc7 and the Bub proteins in vitro demonstrating that the Spc7 phosphorylation is sufficient for Bub1 and Bub3 association with Spc7, most likely with Bub3 making the Spc7 contact. Mimicking phosphorylation at the MELT motifs leads to constitutive Bub1 localisation at kinetochores. We also showed that the N-terminus of Spc7 has microtubule binding activity regulated by Ark1 kinase. Mimicking phosphorylation at Ark1 sites results in reduced amount of recombinant Spc7 co-precipitating with microtubules in microtubule binding assays. Moreover, two stretches of basic residues, that contribute to Spc7 microtubule binding activity, have been mapped in the extreme Nterminus of Spc7. Spc7 also interacts with PP1 phosphatase, Dis2 in S. pombe, which is required for checkpoint silencing, but the mechanism of this interactions remains to be determined. These findings allow us to speculate on Spc7 role(s) in coupling microtubule binding with spindle checkpoint activation and silencing.

No description available.

At the onset of cell division microtubules growing from spindle pole bodies (SPB) interact with each other to form the mitotic spindle enabling proper chromosome positioning and segregation. However, the exact mechanism of microtubule dynamics and microtubule associated proteins (MAPs) underlying spindle assembly is still not well understood. We developed an in vivo method to observe spindle assembly in the fission yeast Schizosaccharomyces pombe by inducing depolymerization of already formed and grown spindles by subjecting the cells to low temperatures, followed by subsequent repolymerization at a permissive temperature. We observed that microtubules pivot, i.e., perform angular movement around the SPB in a random manner, exploring the intranuclear space. Eventually microtubules extending from opposite SPBs come into contact and establish an antiparallel connection thus reassembling the spindle. Mutant approaches revealed that deletion of ase1 and klp5 did not prevent spindle reassembly, however introduced aberrations during the spindle formation. Amazingly, cut7p showed direct colocalization with microtubule overlap during spindle reassembly. Abrogation of cut7p led to inability to form a functional spindle. Thus, cut7p is the main regulator of spindle formation in fission yeast. None of the mutant strains affected microtubule pivoting, confirming that microtubule pivoting is a random movement unrelated to MAPs.