Recruitment of Mad2 to the Kinetochore Requires the Rod/Zw10 Complex

SummaryCompromising the activity of the spindle checkpoint permits mitotic exit in the presence of unattached kinetochores and, consequently, greatly increases the rate of aneuploidy in the daughter cells [1–3]. The metazoan checkpoint mechanism is more complex than in yeast in that it requires additional proteins and activities besides the classical Mads and Bubs. Among these are Rod, Zw10, and Zwilch, components of a 700 Kdal complex (Rod/Zw10) [4–6] that is required for recruitment of dynein/dynactin to kinetochores [7, 8] but whose role in the checkpoint is poorly understood. The dynamics of Rod and Mad2, examined in different organisms, show intriguing similarities as well as apparent differences [7, 9] . Here we simultaneously follow GFP-Mad2 and RFP-Rod and find they are in fact closely associated throughout early mitosis. They accumulate simultaneously on kinetochores and are shed together along microtubule fibers after attachment. Their behavior and position within attached kinetochores is distinct from that of BubR1; Mad2 and Rod colocalize to the outermost kinetochore region (the corona), whereas BubR1 is slightly more interior. Moreover, Mad2, but not BubR1, Bub1, Bub3, or Mps1, requires Rod/Zw10 for its accumulation on unattached kinetochores. Rod/Zw10 thus contributes to checkpoint activation by promoting Mad2 recruitment and to checkpoint inactivation by recruiting dynein/dynactin that subsequently removes Mad2 from attached kinetochores

Extra centrosomes and/or chromosomes prolong mitosis in human cells

Author Posting. © The Author(s), 2008. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in Nature Cell Biology 10 (2008): 748-751, doi:10.1038/ncb1738.Using laser microsurgery and cell fusion we have explored how additional centrosomes and/or chromosomes influence the duration of mitosis in human cells. We find that doubling the chromosome number adds ~10 minutes to a 20 minute division while doubling the number of centrosomes adds ~30 minutes more, and extra centrosomes and/or chromosomes prolong mitosis by delaying satisfaction of the spindle assembly checkpoint. Thus mitosis can be prolonged by non genetic means and extra chromosomes and centrosomes likely contribute to the elevated mitotic index seen in many tumors.This work was supported by National Institutes of General Medical Sciences grants 40198 (to C.L.R.) and 59363 (to A.K.)

A tumor suppressor role of the Bub3 spindle checkpoint protein after apoptosis inhibition.

Most solid tumors contain aneuploid cells, indicating that the mitotic checkpoint is permissive to the proliferation of chromosomally aberrant cells. However, mutated or altered expression of mitotic checkpoint genes accounts for a minor proportion of human tumors. We describe a Drosophila melanogaster tumorigenesis model derived from knocking down spindle assembly checkpoint (SAC) genes and preventing apoptosis in wing imaginal discs. Bub3-deficient tumors that were also deficient in apoptosis displayed neoplastic growth, chromosomal aneuploidy, and high proliferative potential after transplantation into adult flies. Inducing aneuploidy by knocking down CENP-E and preventing apoptosis does not induce tumorigenesis, indicating that aneuploidy is not sufficient for hyperplasia. In this system, the aneuploidy caused by a deficient SAC is not driving tumorigenesis because preventing Bub3 from binding to the kinetochore does not cause hyperproliferation. Our data suggest that Bub3 has a nonkinetochore-dependent function that is consistent with its role as a tumor suppressor

Near-bed and surface flow division patterns in experimental river bifurcations

Understanding channel bifurcation mechanics is of great importance for predicting and managing multichannel river processes and avulsion in distributary river deltas. To date, research on river channel bifurcations has focused on factors determining the stability and evolution of bifurcations. It has recently been shown that, theoretically, the nonlinearity of the relation between sediment transport and flow discharge causes one of the two distributaries of a (slightly) asymmetrical bifurcation to grow and the other to shrink. The positive feedback introduced by this effect results in highly asymmetrical bifurcations. However, there is a lack of detailed insight into flow dynamics within river bifurcations, the consequent effect on bed load flux through bifurcating channels, and thus the impact on bifurcation stability over time. In this paper, three key parameters (discharge ratio, width-to-depth ratio, and bed roughness) were varied in order to examine the secondary flow field and its effect on flow partitioning, particularly near-bed and surface flow, at an experimental bifurcation. Discharge ratio was controlled by varying downstream water levels. Flow fields were quantified using both particle image velocimetry and ultrasonic Doppler velocity profiling. Results show that a bifurcation induces secondary flow cells upstream of the bifurcation. In the case of unequal discharge ratio, a strong increase in the secondary flow near the bed causes a larger volume of near-bed flow to enter the dominant channel compared to surface and depth-averaged flow. However, this effect diminishes with larger width-to-depth ratio and with increased bed roughness. The flow structure and division pattern will likely have a stabilizing effect on river channel bifurcations. The magnitude of this effect in relation to previously identified destabilizing effects is addressed by proposing an adjustment to a widely used empirical bed load nodal-point partition equation. Our finding implies that river bifurcations can be stable under a wider range of conditions than previously thought. Key Points Secondary flow in symmetrical bifurcations causes strong near-bed flow curvature A disproportional amount of near-bed flow enters the dominant downstream channel Flow curvature adds a stabilizing feedback on bifurcation evolution

Spatiotemporal control of mitosis by the conserved spindle matrix protein Megator

A putative spindle matrix has been hypothesized to mediate chromosome motion, but its existence and functionality remain controversial. In this report, we show that Megator (Mtor), the Drosophila melanogaster counterpart of the human nuclear pore complex protein translocated promoter region (Tpr), and the spindle assembly checkpoint (SAC) protein Mad2 form a conserved complex that localizes to a nuclear derived spindle matrix in living cells. Fluorescence recovery after photobleaching experiments supports that Mtor is retained around spindle microtubules, where it shows distinct dynamic properties. Mtor/Tpr promotes the recruitment of Mad2 and Mps1 but not Mad1 to unattached kinetochores (KTs), mediating normal mitotic duration and SAC response. At anaphase, Mtor plays a role in spindle elongation, thereby affecting normal chromosome movement. We propose that Mtor/Tpr functions as a spatial regulator of the SAC, which ensures the efficient recruitment of Mad2 to unattached KTs at the onset of mitosis and proper spindle maturation, whereas enrichment of Mad2 in a spindle matrix helps confine the action of a diffusible “wait anaphase” signal to the vicinity of the spindle

Recommendations for the Establishment and Operation of Human Milk Banks in Europe: A Consensus Statement From the European Milk Bank Association (EMBA)

Objectives: To develop recommendations from the European Milk Bank Association (EMBA) for the establishment and operation of human milk banks (HMB) in Europe.Method: A working group comprising members of the EMBA was convened in 2015 to develop Europe-wide recommendations for milk banks. Each member had experience of guideline development and/or milk banking operations. An initial survey was agreed using collated published global recommendations. A total of 108 potential recommendations were included in the survey; responders noted which were included in their national guidelines. The responses were collated, compared, and discussed and the group determined where there was consensus and where substantial or minor differences were identified. Where there was consensus or robust published evidence on which to base recommendations these were included. When there was no consensus and no clear evidence base, a statement of explanation based on collective expert opinion was agreed.Results: Published, internationally available guidelines with recommendations for human milk banks from France, Italy, and the UK, together with guidelines from Austria, Denmark, Germany, Norway, Slovakia, Spain, Sweden, and Switzerland were included as source materials. These covered: General recommendations; Donor recruitment and screening; Expression, handling, and storage of donor human milk (DHM); Pooling of DHM; Milk screening; Milk treatment (pasteurization); Delivery of DHM to recipients.Conclusions: Evidence based recommendations and consensus statements from the EMBA will now be published on the EMBA website to assist in the safe establishment and operation of HMBs throughout Europe. These have also been used to inform the chapter on human milk to be included in the 2019 edition of the Guide to the quality and safety of tissues and cells for human application, published by the European Directorate for the Quality of Medicines & HealthCare (EDQM)

Aneuploidy in intestinal stem cells promotes gut dysplasia in Drosophila

Aneuploidy is associated with different human diseases including cancer. However, different cell types appear to respond differently to aneuploidy, either by promoting tumorigenesis or causing cell death. We set out to study the behavior of adult Drosophila melanogaster intestinal stem cells (ISCs) after induction of chromosome missegregation either by abrogation of the spindle assembly checkpoint or through kinetochore disruption or centrosome amplification. These conditions induce moderate levels of aneuploidy in ISCs, and we find no evidence of apoptosis. Instead, we observe a significant accumulation of ISCs associated with increased stem cell proliferation and an excess of enteroendocrine cells. Moreover, aneuploidy causes up-regulation of the JNK pathway throughout the posterior midgut, and specific inhibition of JNK signaling in ISCs is sufficient to prevent dysplasia. Our findings highlight the importance of understanding the behavior of different stem cell populations to aneuploidy and how these can act as reservoirs for genomic alterations that can lead to tissue pathologies.This article is a result of the project Norte Portugal Regional Operational Program (NORTE 2020) Norte-01-0145-FEDER-000029 – Advancing Cancer Research: From basic knowledge to application, under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund, and it is also funded by National Funds through Fundação para a Ciência e a Tecnologia under the project PTDC/BEX-BCM/1921/2014

Simple large wood structures promote hydromorphological heterogeneity and benthic macroinvertebrate diversity in low-gradient rivers

This work has been carried out within the SMART Joint Doctorate Programme ‘Science for the MAnagement of Rivers and their Tidal systems’ funded by the Erasmus Mundus programme of the European Union