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Alex McDougall

Publications and source records attributed to Alex McDougall.

12 recordsLinked to original sources
PubMed2005

Microdomains bounded by endoplasmic reticulum segregate cell cycle calcium transients in syncytial Drosophila embryos.

Cell cycle calcium signals are generated by the inositol trisphosphate (InsP3)-mediated release of calcium from internal stores (Ciapa, B., D. Pesando, M. Wilding, and M. Whitaker. 1994. Nature. 368:875-878; Groigno, L., and M. Whitaker. 1998. Cell. 92:193-204). The major internal calcium store is the endoplasmic reticulum (ER); thus, the spatial organization of the ER during mitosis may be important in shaping and defining calcium signals. In early Drosophila melanogaster embryos, ER surrounds the nucleus and mitotic spindle during mitosis, offering an opportunity to determine whether perinuclear localization of ER conditions calcium signaling during mitosis. We establish that the nuclear divisions in syncytial Drosophila embryos are accompanied by both cortical and nuclear localized calcium transients. Constructs that chelate InsP3 also prevent nuclear division. An analysis of nuclear calcium concentrations demonstrates that they are differentially regulated. These observations demonstrate that mitotic calcium signals in Drosophila embryos are confined to mitotic microdomains and offer an explanation for the apparent absence of detectable global calcium signals during mitosis in some cell types.

Animals↗
PubMed2005

RNA interference in meiosis I human oocytes: towards an understanding of human aneuploidy.

Although female meiosis I errors account for the majority of human aneuploidy, their molecular basis is largely unknown. By elucidating gene function, gene knockdown using RNA interference (RNAi) could shed light on this enigmatic process. In practice, however, the extreme paucity of immature human oocytes makes the evaluation of gene-targeting tools difficult. Here, we undertake RNAi in human oocytes and describe an approach employing mouse oocytes which could overcome the problem of limited biological material. We designed a short interfering RNA (siRNA) designated si539 to target the human mitotic arrest deficient 2 (hMad2) spindle checkpoint component. In human oocytes microinjected with si539, the hMad2 signal detected by Western blotting was 85-92% less intense than in oocytes injected with control siRNA indicating efficient silencing. Further examination of si539's targeting efficiency was undertaken using a green fluorescent protein (GFP)-tagged hMad2 mRNA construct in mouse oocytes. Consistent with Western blot analysis, si539 reduced hMad2-GFP expression in mouse oocytes by approximately 94% and relieved the meiosis I arrest otherwise induced by hMad2-GFP in mouse oocytes. By facilitating the investigation of candidate genes involved in regulating human female meiosis I, this approach can bring us closer to understanding the origins of aneuploidies such as Down's syndrome.

Aneuploidy↗
PubMed2005

Restaging the spindle assembly checkpoint in female mammalian meiosis I.

In mammalian somatic cells, the spindle assembly checkpoint (SAC) is indispensable for ensuring the fidelity of chromosome segregation by delaying cell-cycle progression in the face of even a single misaligned chromosome. In contrast, the role of the SAC in unperturbed mammalian oocytes is less well defined as progression through meiosis I is unaltered in mouse oocytes in the presence of one or a few misaligned chromosomes. Furthermore, attempts to disable the function of the SAC protein, Mad2, in mouse oocytes have produced conflicting results. To gain further insight into SAC function during female mammalian meiosis I, we recently utilised a morpholino-based antisense approach to deplete the majority of Mad2 in mouse oocytes. Our results define a clear role for Mad2 in ensuring the proper timing of meiosis I events and ultimately, in ensuring the fidelity of homologue disjunction. We discuss the implications of these results for the regulation of meiosis I in mammalian oocytes and for the genesis of human aneuploidy.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2005

Mad2 prevents aneuploidy and premature proteolysis of cyclin B and securin during meiosis I in mouse oocytes.

In mitosis, the spindle checkpoint protein Mad2 averts aneuploidy by delaying anaphase onset until chromosomes align. Here we show that depletion of Mad2 in meiosis I mouse oocytes induced an increased incidence of aneuploidy. Proteolysis of cyclin B and securin commenced earlier in Mad2-depleted oocytes, resulting in a shortened duration of meiosis I. Furthermore, overexpression of Mad2 inhibited homolog disjunction. We conclude that Mad2 delays the onset of cyclin B and securin degradation and averts aneuploidy during meiosis I in mammalian oocytes. The data suggest a link between trisomies such as Down syndrome and defective oocyte spindle checkpoint function.

Anaphase↗
PubMed2005

Mad2 is required for inhibiting securin and cyclin B degradation following spindle depolymerisation in meiosis I mouse oocytes.

Mad2 is a pivotal component of the spindle assembly checkpoint (SAC) which inhibits anaphase promoting complex/cyclo-some (APC/C) activity by sequestering Cdc20 thereby regulating the destruction of securin and cyclin B. During mitosis, spindle depolymerisation induces a robust Mad2-dependent arrest due to inhibition of securin and cyclin B destruction. In contrast to mitosis, the molecular details underpinning the meiosis I arrest experienced by mouse oocytes exposed to spindle depolymerisation remain incompletely characterised. Notably, the role of Mad2 and the fate of the anaphase-marker, securin, are unexplored. As shown previously, we find that spindle depolymerisation by nocodazole inhibits first polar body extrusion (PBE) and stabilises cyclin B and cyclin-dependent kinase 1 activity in mouse oocytes. Here we show that stabilisation of cyclin B in nocodazole can be sustained for several hours and is associated with stabilisation of securin. These effects are SAC-mediated as, in oocytes depleted of the majority of Mad2 by morpholino antisense, securin and cyclin B are destabilised and 15% of oocytes undergo PBE. This reflects premature APC/C activation as a mutant form of cyclin B lacking its APC/C degradation signal is stable in Mad2-depleted oocytes. Moreover, homologues do not disjoin during the prolonged meiosis I arrest (> 18 h) induced by nocodaozole indicating that a non-cleavage mechanism is insufficient on its own for resolution of arm cohesion in mammalian oocytes. In conclusion, when all kinetochores lack attachment and tension, mouse oocytes mount a robust Mad2-dependent meiosis I arrest which inhibits the destruction of securin and cyclin B.

Animals↗
PubMed2004

Ca(2+)-promoted cyclin B1 degradation in mouse oocytes requires the establishment of a metaphase arrest.

CDK1-cyclin B1 is a universal cell cycle kinase required for mitotic/meiotic cell cycle entry and its activity needs to decline for mitotic/meiotic exit. During their maturation, mouse oocytes proceed through meiosis I and arrest at second meiotic metaphase with high CDK1-cyclin B1 activity. Meiotic arrest is achieved by the action of a cytostatic factor (CSF), which reduces cyclin B1 degradation. Meiotic arrest is broken by a Ca2+ signal from the sperm that accelerates it. Here we visualised degradation of cyclin B1::GFP in oocytes and found that its degradation rate was the same for both meiotic divisions. Ca2+ was the necessary and sufficient trigger for cyclin B1 destruction during meiosis II; but it played no role during meiosis I and furthermore could not accelerate cyclin B1 destruction during this time. The ability of Ca2+ to trigger cyclin B1 destruction developed in oocytes following a restabilisation of cyclin B1 levels at about 12 h of culture. This was independent of actual first polar body extrusion. Thus, in metaphase I arrested oocytes, Ca2+ would induce cyclin B1 destruction and the first polar body would be extruded. In contrast to some reports in lower species, we found no evidence that oocyte activation was associated with an increase in 26S proteasome activity. We therefore conclude that Ca2+ mediates cyclin B1 degradation by increasing the activity of an E3 ubiquitin ligase. However, this stimulation occurs only in the presence of the ubiquitin ligase inhibitor CSF. We propose a model in which Ca2+ directly stimulates destruction of CSF during mammalian fertilisation.

Animals↗
PubMed2004

Fertilisation calcium signals in the ascidian egg.

The egg of ascidians (urochordate), as virtually all animal and plant species, displays Ca2+ signals upon fertilisation. These Ca2+ signals are repetitive Ca2+ waves that initiate in the cortex of the egg and spread through the whole egg interior. Two series of Ca2+ waves triggered from two distinct Ca2+ wave pacemakers entrain the two meiotic divisions preceding entry into the first interphase. The second messenger inositol (1,4,5) trisphosphate (IP3) is the main mediator of these global Ca2+ waves. Other Ca2+ signalling pathways (RyR and NAADPR) are functional in the egg but they mediate localised cortical Ca2+ signals whose physiological significance remains unclear. The meiosis I Ca2+ wave pacemaker is mobile and relies on intracellular Ca2+ release from the endoplasmic reticulum (ER) induced by a large production of IP3 at the sperm aster site. The meiosis II Ca2+ wave pacemaker is stably localised in a vegetal protrusion called the contraction pole. It is probable that a local production of IP3 in the contraction pole determines the site of this second pacemaker while functional interactions between ER and mitochondria regulate its activity. Finally, a third ectopic pacemaker can be induced by a global increase in IP3, making the ascidian egg a unique system where three different Ca2+ wave pacemakers coexist in the same cell.

Animals↗
PubMed2003

Exploring the mechanism of action of the sperm-triggered calcium-wave pacemaker in ascidian zygotes.

In ascidians, as in mammals, sperm trigger repetitive Ca2+-waves that originate from cortical pacemakers situated in the vegetal hemisphere of the zygotes. In ascidians, a vegetal protrusion termed the contraction pole (CP) acts as the Ca2+-wave pacemaker, but the mechanism that underlies the generation of a Ca2+-wave pacemaker is not known. Here, we tested four hypotheses to determine which factors at the CP are involved in setting the pace of the ascidian Ca2+-wave pacemaker: (1) localized Ca2+ influx; (2) accumulation of phosphatidylinositol (4,5)bisphosphate [PtdIns(4,5)P2]; (3) accumulation of cortical endoplasmic reticulum (cER); and (4) enrichment of the sperm activating factor. We developed a method of dynamically monitoring the location of the CP during fertilization using a plekstrin homology (PH) domain from phospholipase Cdelta1 coupled to green fluorescent protein (GFP) that binds PtdIns(4,5)P2. We found that eggs in Ca2+-free sea water displayed Ca2+ waves that originated from the CP, showing that enhanced CP Ca2+ influx does not determine the origin of the pacemaker. Also, disruption of the PH::GFP-labelled CP once it had formed did not dislodge the Ca2+-wave pacemaker from that site. Next, when we prevented the accumulation of cER at the CP, all of the Ca2+ waves came from the site of sperm-egg fusion and the frequency of Ca2+ oscillations was unaltered. These data show that local Ca2+ influx, the accumulation of PtdIns(4,5)P2 and cER at the CP are not required for Ca2+-wave pacemaker function and instead suggest that a factor associated with the sperm determines the site of the Ca2+-wave pacemaker. Finally, when we injected ascidian sperm extract into the centre of unfertilized ascidian eggs that had been treated with microfilament- and microtubule-disrupting drugs, all the Ca2+ waves still originated from near the plasma membrane, showing that the sperm factor does not require an intact cortex if it is enriched near the plasma membrane (PM). We suggest that the Ca2+-releasing sperm factor might be tethered near or on the PM and that following the cortical contraction, it is translocated to the vegetal CP, thus making that site act as a Ca2+-wave pacemaker.

Actin Cytoskeleton↗
PubMed2003

Homologue disjunction in mouse oocytes requires proteolysis of securin and cyclin B1.

Disjunction of pairs of homologous chromosomes during the first meiotic division (MI) requires anaphase-promoting complex (APC)-mediated activation of separase in budding yeast and Caenorhabditis elegans, but not Xenopus laevis. It is not clear which model best fits the mammalian system. Here we show that homologue disjunction in mouse oocytes is dependent on proteolysis of the separase inhibitor securin and the Cdk1 regulatory sub-unit cyclin B1. Proteolysis of both proteins was entirely dependent on their conserved destruction box (D-box) motifs, through which they are targeted to the APC. These data indicate that the mechanisms regulating homologue disjunction in mammalian oocytes are similar to those of budding yeast and C.elegans.

Animals↗
PubMed2002

Ca(2+) oscillations promote APC/C-dependent cyclin B1 degradation during metaphase arrest and completion of meiosis in fertilizing mouse eggs.

Cyclin B1, the regulatory component of M phase-promoting factor (MPF), is degraded during the metaphase-anaphase transition in an anaphase-promoting complex/cyclosome (APC/C)-dependent process. MPF activity is stable in eggs, and a sperm-triggered Ca(2+) signal is needed to promote cyclin degradation. In frogs, a single Ca(2+) spike promotes cell cycle resumption, but, in mammals, the Ca(2+) signal is more complex, consisting of a series of spikes that stop several hours after sperm fusion. Using dual imaging in mouse eggs, we have examined how the Ca(2+) signal generates cyclin B1 destruction using destructible and nondestructible GFP-tagged constructs. APC/C activity was present in unfertilized eggs, giving cyclin B1 a half-life of 1.15 +/- 0.28 hr. However, APC/C-dependent cyclin degradation was elevated 6-fold when sperm raised cytosolic Ca(2+) levels above 600 nM. This activation was transitory since cyclin B1 levels recovered between Ca(2+) spikes. For continued cyclin degradation at basal Ca(2+) levels, multiple spikes were needed. APC/C-mediated degradation was observed until eggs had completed meiosis with the formation of pronuclei, and, at this time, Ca(2+) spikes stopped. Therefore, the physiological need for a repetitive Ca(2+) signal in mammals is to ensure long-term cyclin destruction during a protracted exit from meiosis.

Anaphase-Promoting Complex-Cyclosome↗
PubMedDate not supplied

Inositol 1,4,5-trisphosphate (IP3) responsiveness is regulated in a meiotic cell cycle dependent manner: implications for fertilization induced calcium signaling.

Fertilisation of mammalian eggs is known to trigger a series of transient rises in cytosolic calcium, known as calcium oscillations, the initiation and duration of which are crucial for meiotic exit and subsequent entry into embryogenesis. It is not known how these calcium oscillations are terminated when the zygote exits meiosis; it is thought that responsiveness to inositol 1,4,5-trisphosphate (IP3) is involved, since the oscillations are known to be mediated by an IP3 dependent mechanism. Here we report that IP3 responsiveness is maintained throughout meiotic maturation and falls very rapidly after meiotic exit. We also show that inhibition of the major cell cycle kinase, CDK1, has no effect on responsiveness, but that prolonging CDK1 activity prevents the decline in responsiveness normally seen at meiotic exit. We conclude that CDK1 plays a role, but is not the only factor involved in controlling IP3 responsiveness during the meiotic cell cycle.

Animals↗