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Biomedical subjects

I Hagan

Publications and source records attributed to I Hagan.

9 recordsLinked to original sources
PubMed1998

Asymmetric segregation on spindle poles of the Schizosaccharomyces pombe septum-inducing protein kinase Cdc7p.

Schizosaccharomyces pombe divides by means of a centrally placed division septum. The initiation of septation must be tightly coordinated with events in mitosis, as premature formation of the septum can lethally cut the undivided nucleus. The Spg1p GTPase and the Cdc7p kinase, with which it interacts, play a central role in signaling the initiation of septum formation. Loss-of-function mutations in either gene prevent septation, whereas inappropriate activation of Spg1p can induce septum formation from G1 or G2 interphase cells. Increased expression of either gene leads to multiple rounds of septation without cell cleavage, emphasizing the need for precise cell cycle regulation of their activity. To understand the mechanisms underlying this regulation, we have investigated whether these key initiators of septum formation are controlled by changes in their activity and/or location during mitosis and cytokinesis. We demonstrate that Spg1p localizes to the spindle pole body in interphase and to both spindle poles during mitosis. In contrast, Cdc7p shows no discrete localization during interphase, but early in mitosis it associates with both spindle pole bodies and, as the spindle extends, is seen on only one pole of the spindle during anaphase B. Spg1p activity is required for localization of Cdc7p in vivo but not for its kinase activity in vitro. Staining with an antiserum that recognizes preferentially GDP-Spg1p indicates that activated GTP-Spg1p predominates during mitosis when Cdc7p is associated with the spindle pole body. Furthermore, staining with this antibody shows that asymmetric distribution of Cdc7p may be mediated by inactivation of Spg1p on one spindle pole. Deregulated septation in mutant cells correlates with segregation of Cdc7p to both spindle poles.

Cell Cycle Proteins↗
PubMed1997

Evidence for cell cycle-specific, spindle pole body-mediated, nuclear positioning in the fission yeast Schizosaccharomyces pombe.

Specific changes in spatial order occur during cell cycle progression in fission yeast. Growth of the rod-shaped cells is highly regulated and undergoes a cell cycle and size-regulated switch from monopolar to bipolar tip extension. During both phases of growth, the interphase nucleus is maintained in a central location. Following the separation of the genome to the cell tips in mitosis, the two nuclei migrate back towards the cell equator before stopping in two new positions that will become the middle of the two new cells. Here we use simultaneous labeling of microtubules, chromatin and spindle pole bodies in wild-type and cdc mutants, to show that nuclear positioning is achieved by regulation of spindle pole body-mediated nuclear migration. We show that the number and location of nuclear positioning signals is regulated in a cell cycle-specific manner and that spindle pole body-mediated forces are likely to be responsible for maintaining correct nuclear position once the nuclei have reached the appropriate position in the cell. Accentuating the movement of the nuclei back towards the cell equator after mitosis by artificially increasing cell length shows that the spindle pole body leads the nucleus during this migration. When multiple spindle pole bodies are associated with the same or different nuclei they all go to the same point indicating that the different spindle pole bodies are responding to the same positional cue. In a septation-defective mutant cell, which contains four nuclei, the spindle pole bodies on the four different nuclei initially group as two pairs in regions that would become the middle of the new cells, were the cell able to divide. In the subsequent interphase, the nuclei aggregate as a group of four in the centre of the cell. The presence of two or three clusters of spindle pole bodies in larger cells with eight nuclei suggests that the mechanisms specifying the normally central location for multiple nuclei may be unable to operate properly as the cells get larger. Perturbation of microtubules with the microtubule poison thiabendazole prevents the spindle pole body clustering in septation mutants, demonstrating that nuclear positioning requires a functional microtubule cytoskeleton.

Anaphase↗
PubMed1995

The product of the spindle formation gene sad1+ associates with the fission yeast spindle pole body and is essential for viability.

Spindle formation in fission yeast occurs by the interdigitation of two microtubule arrays extending from duplicated spindle pole bodies which span the nuclear membrane. By screening a bank of temperature-sensitive mutants by anti-tubulin immunofluorescence microscopy, we previously identified the sad1.1 mutation (Hagan, I., and M. Yanagida. 1990. Nature (Lond.). 347:563-566). Here we describe the isolation and characterization of the sad1+ gene. We show that the sad1.1 mutation affected both spindle formation and function. The sad1+ gene is a novel essential gene that encodes a protein with a predicted molecular mass of 58 kD. Deletion of the gene was lethal resulting in identical phenotypes to the sad1.1 mutation. Sequence analysis predicted a potential membrane-spanning domain and an acidic amino terminus. Sad1 protein migrated as two bands of 82 and 84 kD on SDS-PAGE, considerably slower than its predicted mobility, and was exclusively associated with the spindle pole body (SPB) throughout the mitotic and meiotic cycles. Microtubule integrity was not required for Sad1 association with the SPB. Upon the differentiation of the SPB in metaphase of meiosis II, Sad1-staining patterns similarly changed from a dot to a crescent supporting an integral role in SPB function. Moderate overexpression of Sad1 led to association with the nuclear periphery. As Sad1 was not detected in the cytoplasmic microtubule-organizing centers activated at the end of anaphase or kinetochores, we suggest that Sad1 is not a general component of microtubule-interacting structures per se, but is an essential mitotic component that associates with the SPB but is not required for microtubule nucleation. Sad1 may play a role in SPB structure, such as maintaining a functional interface with the nuclear membrane or in providing an anchor for the attachment of microtubule motor proteins.

Amino Acid Sequence↗
PubMed1993

Cell cycle-dependent specific positioning and clustering of centromeres and telomeres in fission yeast.

Fluorescence in situ hybridization (FISH) shows that fission yeast centromeres and telomeres make up specific spatial arrangements in the nucleus. Their positioning and clustering are cell cycle regulated. In G2, centromeres cluster adjacent to the spindle pole body (SPB), while in mitosis, their association with each other and with the SPB is disrupted. Similarly, telomeres cluster at the nuclear periphery in G2 and their associations are disrupted in mitosis. Mitotic centromeres interact with the spindle. They remain undivided until the spindle reaches a critical length, then separate and move towards the poles. This demonstrated, for the first time, that anaphase A occurs in fission yeast. The mode of anaphase A and B is similar to that of higher eukaryotes. In nda3 and cut7 mutants defective in tubulin of a kinesin-related motor, cells are blocked in early stages of mitosis due to the absence of the spindle, and centromeres dissociate but remain close to the SPB, whereas in a metaphase-arrested nuc2 mutant, they reside at the middle of the spindle. FISH is therefore a powerful tool for analyzing mitotic chromosome movement and disjunction using various mutants. Surprisingly, in top2 defective in DNA topoisomerase II, while most chromatid DNAs remain undivided, sister centromeres are separated. Significance of this finding is discussed. In contrast, most chromatid DNAs are separated but telomeric DNAs are not in cut1 mutant. In cut1, the dependence of SPB duplication on the completion of mitosis is abolished. In crm1 mutant cells defective in higher-order chromosome organization, the interphase arrangements of centromeres and telomeres are disrupted.

Cell Cycle↗
PubMed1992

Kinesin-related cut7 protein associates with mitotic and meiotic spindles in fission yeast.

Several mitotic and meiotic gene products are related to the microtubule motor kinesin, providing insight into the molecular basis of the complex motile events responsible for spindle formation and function. Of these genes, three have been shown to affect spindle structure when mutated. The most severe phenotype is seen in Aspergillus nidulans bimC and Schizosaccharomyces pombe cut7 mutants. In both fungi the intranuclear spindle is bipolar, with microtubules that emanate from spindle pole bodies at either pole, interdigitating in a central overlap zone. In bimC and cut7 mutants, microtubule interdigitation does not appear to take place, instead two unconnected half spindles form and chromosome separation fails. Here we report that cut7 protein concentrates on or near the spindle pole bodies throughout mitotic and meiotic nuclear division and associates with mitotic spindle microtubules in a stage-specific manner, associating with the mid-anaphase B midzone. In cut7ts mutants, spindle pole bodies stain but mitotic microtubules do not.

Anaphase↗
PubMed1990

Novel potential mitotic motor protein encoded by the fission yeast cut7+ gene.

The structure equivalent to higher eukaryotic centrosomes in fission yeast, the nuclear membrane-bound spindle pole body, is inactive during interphase. On transition from G2 to M phase of the cell cycle, the spindle pole body duplicates; the daughter pole bodies seed microtubules which interdigitate to form a short spindle that elongates to span the nucleus at metaphase. We have identified two loci which, when mutated, block spindle formation. The predicted product of one of these genes, cut7+, contains an amino-terminal domain similar to the kinesin heavy chain head domain, indicating that the cut7+ product could be a spindle motor. The cut7+ gene resembles the Aspergillus nidulans putative spindle motor gene bimC, both in terms of its organization with a homologous amino-terminal head and no obvious heptad repeats and in the morphology of the mutant phenotype. But we find no similarity between the carboxy termini of these genes, suggested that either the cut7+ gene represents a new class of kinesin genes and that fission yeast may in addition contain a bimC homologue, or that the carboxy termini of these mitotic kinesins are not evolutionarily conserved and that the cut7+ gene belongs to a subgroup of bimC-related kinesins.

Adenosine Triphosphatases↗
PubMed1988

Cloning and sequencing of the cyclin-related cdc13+ gene and a cytological study of its role in fission yeast mitosis.

We have cloned and sequenced the cdc13+ gene from fission yeast. When a major part of the cdc13+ gene is deleted from the chromosome, cells arrest in interphase, but partial loss of gene activity leads to cells containing condensed chromosomes, aberrant septa and a microtubular cytoskeleton with characteristics of both G2 and M. Expression of this phenotype is influenced by the nutritional status of the cell. Our results suggest that the cdc13+ gene function is required for the control of the G2 to M transition. It appears to play a role in regulating the separate pathways of events involved in the physical process of mitosis, for example in the reorganization of the cytoskeleton on transition from G2 to mitosis. The cdc13+ gene function interacts closely with both the yeast and human homologues of cdc2+, suggesting that mammalian cells may contain a cdc13+ homologue. The gene encodes a putative polypeptide of 482 amino acids, and a central region of 176 amino acids of this polypeptide is 50% identical with sea urchin cyclin. Therefore, the cdc13+ protein is cyclin related and could act as a regulator or substrate of the p34cdc2 protein kinase, which initiates mitosis.

Amino Acid Sequence↗