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Biomedical subjects

I M Hagan

Publications and source records attributed to I M Hagan.

27 records · Page 2Linked to original sources
PubMed1997

Mal3, the fission yeast homologue of the human APC-interacting protein EB-1 is required for microtubule integrity and the maintenance of cell form.

Through a screen designed to isolate novel fission yeast genes required for chromosome segregation, we have identified mal3+. The mal3-1 mutation decreased the transmission fidelity of a nonessential minichromosome and altered sensitivity to microtubule-destabilizing drugs. Sequence analysis revealed that the 35-kD Mal3 is a member of an evolutionary conserved protein family. Its human counterpart EB-1 was identified in an interaction screen with the tumour suppressor protein APC. EB-1 was able to substitute for the complete loss of the mal3+ gene product suggesting that the two proteins might have similar functions. Cells containing a mal3 null allele were viable but showed a variety of phenotypes, including impaired control of cell shape. A fusion protein of Mal3 with the Aequorea victoria green fluorescent protein led to in vivo visualization of both cytoplasmic and mitotic microtubule structures indicating association of Mal3 with microtubules. The absence of Mal3 protein led to abnormally short, often faint cytoplasmic microtubules as seen by indirect antitubulin immunofluorescence. While loss of the mal3+ gene product had no gross effect on mitotic spindle morphology, overexpression of mal3+ compromised spindle formation and function and led to severe growth inhibition and abnormal cell morphology. We propose that Mal3 plays a role in regulating the integrity of microtubules possibly by influencing their stability.

Adenomatous Polyposis Coli↗
PubMed1996

Forces acting on the fission yeast anaphase spindle.

The fission yeast mitotic spindle consists of three sets of microtubules: one that extends between the chromosomes and the spindle pole bodies (SPBs); one that extends between the two SPBs forming a region of overlap; and a third, the so-called astral microtubules, that associates laterally with the cytoplasmic face of the SPBs, during anaphase B. The major bundles of the latter can exist with equal probability in two configurations which we have termed parallel and convergent. Mitosis in fission yeast is characterised by an extended anaphase B during which the spindle elongates from 2 mu m (the diameter of the interphase nucleus) to about 14 mu m, spanning the entire length of the cell. Anaphase B spindles viewed by indirect immunofluorescence microscopy frequently appeared bowed but only when the astral microtubules were in the convergent orientation. To investigate the possible significance of this observation, we have examined the situation in the abnormally long spindles that are formed in cells in which cell length has been extended either by overexpression of the weel + gene or by inactivation of the cdc25 + gene. The spindles in these strains were often considerably longer (up to 30 mu m) than in wild type cells but, unlike the latter, did not extend the entire length of the cell. Bowed spindles were again observed but only when the astral microtubules were convergent. We discuss these findings in the context of the astral microtubules either exerting a pulling force on the poles of the anaphase B spindle, or counteracting a pushing force generated by sliding of anti-parallel pole to pole microtubules of the mitotic spindle, or both of the above.

Anaphase↗
PubMed1995

The chk1 pathway is required to prevent mitosis following cell-cycle arrest at 'start'.

BACKGROUND: The G2-M-phase transition is controlled by cell-cycle checkpoint pathways which inhibit mitosis if previous events are incomplete or if the DNA is damaged. Genetic analyses in yeast have defined two related, but distinct, pathways which prevent mitosis--one which acts when S phase is inhibited, and one which acts when the DNA is damaged. In the fission yeast Schizosaccharomyces pombe, many of the gene products involved have been identified. Six 'radiation checkpoint' (rad) gene products are required for both the S-M and DNA-damage checkpoints, whereas Chk1, a putative protein kinase, is required only for the DNA-damage checkpoint and not for the S-M checkpoint following the inhibition of DNA synthesis. RESULTS: We have genetically defined a third mitotic control checkpoint pathway in fission yeast which prevents mitosis when passage through 'start' (the commitment point in G1) is compromized. In cycling cells arrested at start, mitosis is prevented by a Chk1-dependent pathway. In the absence of Chk1, G1 cells attempt an abortive mitosis with a 1C DNA content without entering S phase. Similar results are seen in the absence of Rad17, a typical example of a rad gene product. CONCLUSIONS: Genetic dissection of checkpoints in logarithmically growing fission yeast has identified a pathway that couples mitosis to correct passage through start. This pathway is related to the DNA-structure check-points which ensure that mitosis is dependent on the completion of replication and the integrity of the DNA. We propose that all three mitotic control checkpoints monitor distinct DNA or protein structures at different stages in the cell cycle.

Cell Cycle↗
PubMed1995

The conserved Schizosaccharomyces pombe kinase plo1, required to form a bipolar spindle, the actin ring, and septum, can drive septum formation in G1 and G2 cells.

We have identified a Schizosaccharomyces pombe gene with homology to the budding yeast gene CDC5, the Drosophila gene polo, and the mammalian family of genes encoding polo-like kinases. Disruption of this gene, plo1+, indicates that it is essential. Loss of plo1+ function leads to a mitotic arrest in which condensed chromosomes are associated with a monopolar spindle or to the failure of septation following the completion of nuclear division. In the latter case, cells show a failure both in the formation of an F-actin ring and in the deposition of septal material, suggesting that plo1+ function is required high in the regulatory cascade that controls septation. The overexpression of plo1+ in wild-type cells also results in the formation of monopolar spindles but also induces the formation of multiple septa without nuclear division. Septation can also be induced in the absence of mitotic commitment and concomitant spindle formation by the overexpression of plo1+ in cdc25-22 or cdc2-33 cells arrested in G2; in G1 cells arrested at Start by the cdc10-V50 mutation, or in cells lacking the cyclin B homolog cdc13 that undergo repeated S phases in the absence of mitosis.

Actins↗
PubMed1995

The Schizosaccharomyces pombe hus5 gene encodes a ubiquitin conjugating enzyme required for normal mitosis.

Normal eukaryotic cells do not enter mitosis unless DNA is fully replicated and repaired. Controls called 'checkpoints', mediate cell cycle arrest in response to unreplicated or damaged DNA. Two independent Schizosaccharomyces pombe mutant screens, both of which aimed to isolate new elements involved in checkpoint controls, have identified alleles of the hus5+ gene that are abnormally sensitive to both inhibitors of DNA synthesis and to ionizing radiation. We have cloned and sequenced the hus5+ gene. It is a novel member of the E2 family of ubiquitin conjugating enzymes (UBCs). To understand the role of hus5+ in cell cycle control we have characterized the phenotypes of the hus5 mutants and the hus5 gene disruption. We find that, whilst the mutants are sensitive to inhibitors of DNA synthesis and to irradiation, this is not due to an inability to undergo mitotic arrest. Thus, the hus5+ gene product is not directly involved in checkpoint control. However, in common with a large class of previously characterized checkpoint genes, it is required for efficient recovery from DNA damage or S-phase arrest and manifests a rapid death phenotype in combination with a temperature sensitive S phase and late S/G2 phase cdc mutants. In addition, hus5 deletion mutants are severely impaired in growth and exhibit high levels of abortive mitoses, suggesting a role for hus5+ in chromosome segregation. We conclude that this novel UBC enzyme plays multiple roles and is virtually essential for cell proliferation.

Amino Acid Sequence↗
PubMed1990

Intramitotic controls in the fission yeast Schizosaccharomyces pombe: the effect of cell size on spindle length and the timing of mitotic events.

We have used a new cinemicroscopy technique in combination with antitubulin immunofluorescence microscopy to investigate the timing of mitotic events in cells of the fission yeast Schizosaccharomyces pombe having lengths at division between 7 and 60 microns. Wild-type fission yeast cells divide at a length of 14 microns. Separation of daughter nuclei (anaphase B) proceeds at a rate of 1.6 +/- 0.2 microns min-1, until the spindle extends the length of the cell. Coincident with spindle depolymerization, the nuclei reverse direction and take up positions that will become the center of the two daughter cells. This post-mitotic nuclear migration occurs at a rate of 1.4 +/- 0.5 microns-1. In cells in which the weel+ gene is overexpressed fivefold and that have an average length at mitosis of 28 microns, the rate of nuclear separation was only slightly reduced but, as spindles in these cells measure 20-22 microns, the duration of anaphase B was extended by approximately 40%. By contrast, in the mutant weel.50, which divides at 7 microns, both the rate and duration of anaphase B were indistinguishable from wild type. Nuclei reach the ends of these cells earlier but remain there until a point corresponding to the time of postmitotic nuclear migration in wild type. Thus, the events of mitosis can be extended but not abbreviated. These results are discussed in terms of a mitotic termination control that monitors many different events, one of which is spindle elongation.

Cell Cycle Proteins↗
PubMed1988

The use of cell division cycle mutants to investigate the control of microtubule distribution in the fission yeast Schizosaccharomyces pombe.

We have characterized the changes in microtubule organization that occur through the cell division cycle of the fission yeast Schizosaccharomyces pombe by indirect immunofluorescence microscopy. During interphase, groups of cytoplasmic microtubules, independent of the spindle pole body (SPB), form an array extending between the cell tips. These microtubules are involved in positioning the nucleus at the cell equator and in the establishment of cell polarity. At mitosis, the interphase array disappears and is replaced by an intranuclear spindle extending between the now duplicated SPBs. Elongation of the spindle sees the appearance of astral microtubules emanating from the cytoplasmic face of the SPBs. These persist until the end of anaphase whereupon the spindle microtubules depolymerize and two microtubule organizing centres (MTOCs) at the cell equator re-establish the interphase array. We have used the unique properties of various cell division cycle mutants to investigate further the function of these different microtubule arrays and their temporal and positional control.

Cell Cycle↗
PubMed1986

Growth polarity and cytokinesis in fission yeast: the role of the cytoskeleton.

The distribution of F-actin in the fission yeast Schizosaccharomyces pombe was investigated by fluorescence microscopy using rhodamine-conjugated phalloidin. Fluorescence was seen either at the ends of the cell or at the cell equator. End staining was predominantly in the form of dots whilst equatorial actin was resolved as a filamentous band. The different staining patterns showed a close correlation with the known pattern of cell wall deposition through the cell cycle. In small, newly divided cells actin was localized at the single growing cell end whilst initiation of bipolar cell growth was coincident with the appearance of actin at both ends of the cell. As cells ceased to grow and entered cell division, a ring of actin was seen to anticipate the deposition of the septum at cytokinesis. The relationship between actin and cell wall deposition was further confirmed in three temperature-sensitive cell division cycle (cdc) mutants; cdc10, cdc11 and cdc13. Immunofluorescence microscopy of S. pombe with an anti-tubulin antibody revealed a system of cytoplasmic microtubules extending between the cell ends. The function of these was investigated in the cold-sensitive, benomyl-resistant mutant ben4. In cold-grown cells actin was seen to form conspicuous filamentous rings around the nucleus. The origin of these and the possible role of microtubules in the cell-cycle-dependent rearrangements of F-actin are discussed.

Actins↗