Observation of Target Electron Momentum Effects in Single-Arm M\o ller Polarimetry

In 1992, L.G. Levchuk noted that the asymmetries measured in M\o ller scattering polarimeters could be significantly affected by the intrinsic momenta of the target electrons. This effect is largest in devices with very small acceptance or very high resolution in laboratory scattering angle. We use a high resolution polarimeter in the linac of the polarized SLAC Linear Collider to study this effect. We observe that the inclusion of the effect alters the measured beam polarization by -14% of itself and produces a result that is consistent with measurements from a Compton polarimeter. Additionally, the inclusion of the effect is necessary to correctly simulate the observed shape of the two-body elastic scattering peak.Comment: 29 pages, uuencoded gzip-compressed postscript (351 kb). Uncompressed postscript file (898 kb) available to DECNET users as SLC::USER_DISK_SLC1:[MORRIS]levpre.p

Mad3 KEN Boxes Mediate both Cdc20 and Mad3 Turnover, and Are Critical for the Spindle Checkpoint

Mitotic progression is controlled by proteolytic destruction of securin and cyclin. The mitotic E3 ubiquitin ligase, known as the anaphase promoting complex or cyclosome (APC/C), in partnership with its activators Cdc20p and Cdh1p, targets these proteins for degradation. In the presence of defective kinetochore-microtubule interactions, APC/C(Cdc20) is inhibited by the spindle checkpoint, thereby delaying anaphase onset and providing more time for spindle assembly. Cdc20p interacts directly with Mad2p, and its levels are subject to careful regulation, but the precise mode(s) of APC/C( Cdc20) inhibition remain unclear. The mitotic checkpoint complex (MCC, consisting of Mad3p, Mad2p, Bub3p and Cdc20p in budding yeast) is a potent APC/C inhibitor. Here we focus on Mad3p and how it acts, in concert with Mad2p, to efficiently inhibit Cdc20p. We identify and analyse the function of two motifs in Mad3p, KEN30 and KEN296, which are conserved from yeast Mad3p to human BubR1. These KEN amino acid sequences resemble ‘degron’ signals that confer interaction with APC/C activators and target proteins for degradation. We show that both Mad3p KEN boxes are necessary for spindle checkpoint function. Mutation of KEN30 abolished MCC formation and stabilised Cdc20p in mitosis. In addition, mutation of Mad3-KEN30, APC/C subunits, or Cdh1p, stabilised Mad3p in G1, indicating that the N-terminal KEN box could be a Mad3p degron. To determine the significance of Mad3p turnover, we analysed the consequences of MAD3 overexpression and found that four-fold overproduction of Mad3p led to chromosome bi-orientation defects and significant chromosome loss during recovery from anti-microtubule drug induced checkpoint arrest. In conclusion, Mad3p KEN30 mediates interactions that regulate the proteolytic turnover of Cdc20p and Mad3p, and the levels of both of these proteins are critical for spindle checkpoint signaling and high fidelity chromosome segregation

Multiancestry analysis of the HLA locus in Alzheimer’s and Parkinson’s diseases uncovers a shared adaptive immune response mediated by HLA-DRB1*04 subtypes

Across multiancestry groups, we analyzed Human Leukocyte Antigen (HLA) associations in over 176,000 individuals with Parkinson’s disease (PD) and Alzheimer’s disease (AD) versus controls. We demonstrate that the two diseases share the same protective association at the HLA locus. HLA-specific fine-mapping showed that hierarchical protective effects of HLA-DRB1*04 subtypes best accounted for the association, strongest with HLA-DRB1*04:04 and HLA-DRB1*04:07, and intermediary with HLA-DRB1*04:01 and HLA-DRB1*04:03. The same signal was associated with decreased neurofibrillary tangles in postmortem brains and was associated with reduced tau levels in cerebrospinal fluid and to a lower extent with increased Aβ42. Protective HLA-DRB1*04 subtypes strongly bound the aggregation-prone tau PHF6 sequence, however only when acetylated at a lysine (K311), a common posttranslational modification central to tau aggregation. An HLA-DRB1*04-mediated adaptive immune response decreases PD and AD risks, potentially by acting against tau, offering the possibility of therapeutic avenues

Ipl1p-dependent phosphorylation of Mad3p is required for the spindle checkpoint response to lack of tension at kinetochores

The spindle checkpoint delays anaphase onset until all chromosomes are correctly attached to microtubules. Ipl1 protein kinase (Aurora B) is required to correct inappropriate kinetochore–microtubule attachments and for the response to lack of tension between sister kinetochores. Here we identify residues in the checkpoint protein Mad3p that are phosphorylated by Ipl1p. When phosphorylation of Mad3p at two sites is prevented, the cell’s response to reduced kinetochore tension is dramatically curtailed. Our data provide strong evidence for a distinct checkpoint pathway responding to lack of sister kinetochore tension, in which Ipl1p-dependent phosphorylation of Mad3p is a key step

Over-expression of Mad3p induces bi-orientation and chromosome segregation defects.

<p>A) Mad3p overexpression induces chromosome mis-segregation following checkpoint “challenge”. Strains were pre-synchronised with α-factor, released and then arrested with nocodazole. The nocodazole was then washed out and cells arrested in the next G1. Cells were fixed and analysed for the presence of GFP-marked chromosomes. Chromosome V was labelled with GFP and spindle poles with SPC42-mCherry. Arrowheads mark G1 cells containing two copies of chromosome V. B) Mad3p overexpression induces chromosome bi-orientation defects. Cells were pre-synchronised with α-factor, then depleted for Cdc20p through the addition of Methionine. Cells were then released from G1 into media containing nocodazole and benomyl. The microtubule drugs were then washed out to allow soindle assembly, but Cdc20p remained represed to arrest cells in metaphase. Cells were briefly fixed and scored for bi-orientation (breathing centromeres). Centromere IV was labelled with GFP and spindle poles with <i>SPC42</i>-tomato. Scale bars are 5 microns.</p

<i>mad3-KEN30AAA</i> stabilises Cdc20p in mitosis.

<p>A) Strains were arrested in mitosis (with nocodazole and hydroxyurea), and cycloheximide was added (time 0) to prevent new protein synthesis. Time points were taken and immunoblotted for Cdc20p levels (anti-myc) and Mad3p levels. B) Quantitation of the Cdc20p and Mad3p levels.</p

Over-expression of Mad3p induces micro-tubule drug sensitivity, but only subtly perturbs the spindle checkpoint.

<p>A) Strains expressing extra integrated copies of wild-type <i>MAD3</i> or ken mutant <i>mad3</i> were immunoblotted to quantitate their expression levels. B) Overexpression of Mad3p induces benomyl sensitivity. The over-expression strains were diluted and plated onto rich YPD media with or without the addition of 15 µg/ml of benomyl. Plates were photographed after 3 days growth at 24°C. C) Strains overexpressing Mad3p die quickly in the presence of nocodazole. The indicated strains were pre-synchronised in G1 with alpha factor, released into media containing 20 µg/ml nocodazole, and cells were plated out at indicated times. Viability was scored as the percentage of colonies formed relative to the zero time point. D) Mad3p overexpression has a mild effect on sister-chromatid cohesion. The overexpression strains, which containing GFP marked chromosome V, were synchronised in G1 with alpha factor, then released into media containing nocodazole. Sister-chromatid separation was scored as the percentage of cells containing two clearly separated GFP spots.</p

Mad3p is an APC/C substrate.

<p>A) Mad3p is unstable in G1. Cells expressing <i>GAL-MAD3</i> were first synchronised in G1 (with alpha factor) or mitosis (with nocodazole) in raffinose media, and then a pulse of Mad3p expression was induced by 30 minutes of growth in media containing 2% galactose. Cells were then washed with glucose media (YPD) and cycloheximide was added to inhibit new protein synthesis. The G1 and mitotic arrests were maintained, samples taken at the times indicated, and whole cell extracts immunoblotted with anti-Mad3p antibodies to determine the remaining level of Mad3p. B) Mad3p turnover in G1 is APC/C dependent. Cells expressing <i>GAL-MAD3</i> were synchronised in G1 (with α-factor) in raffinose media, and then a pulse of Mad3p expression was induced by 30 minutes of growth in media containing 2% galactose. Cells were then shifted to 36°C (restrictive temperature for <i>cdc16</i>-ts) for 30 minutes, washed with glucose media (YPD) and cycloheximide was added to inhibit new protein synthesis. The G1 arrest was maintained, samples taken at the times indicated, and whole cell extracts immunoblotted with α-Mad3p antibodies to determine the remaining level of Mad3p. C) SCF mutants do not stabilise Mad3p in G1. <i>cdc4</i> and <i>cdc34</i> strains expressing <i>GAL-MAD3</i> were analysed for Mad3p turnover as in B. D) <i>mad3KEN30AAA</i> stabilises Mad3p in G1. <i>mad3Δ</i> cells containing GAL driven wild-type or <i>mad3-ken</i> mutants were arrested in alpha factor, and then Mad3p was induced for 30 minutes with galactose media. Cells were then washed in glucose media and cycloheximide was added to prevent new protein synthesis. Samples were taken at the times indicated, and whole cell extracts immunoblotted with anti-Mad3p antibodies for the level of Mad3p.</p

Mad3p contains two conserved KEN boxes.

<p>A) Mad3/BubR1 domain structure–schematic diagrams of the Mad3 and Bub proteins indicate the organisation of their functional domains. B) Clustal×alignment of the two conserved KEN boxes found in Mad3/BubR1. Species indicated are <i>Saccharomyces cerevisiae</i> (Scer), <i>Saccharomyces paradoxus</i> (Spar), <i>Saccharomyces mikatae</i> (Smik), <i>Saccharomyces kudriavzevii</i> (Skud), <i>Saccharomyces bayanus</i> (Sbay), <i>Saccharomyces castellii</i> (Scas), <i>Schizosaccharomyces pombe</i> (Spom), <i>Candida albicans</i> (Calb), <i>Arabidopsis thaliana</i> (Atha), <i>Drosophila melanogaster</i> (Dmel) <i>and Homo sapiens</i> (Hsap). Numbers indicate residue position within protein sequence.</p

Models of Mad3p KEN box interactions.

<p>a) Mad3 KEN box interactions. Mad2p and Mad3-KEN30 are both required for stable Mad3p-Cdc20p binding. b) Cdc20p turnover in mitosis: this is dependent on Mad3-KEN30, and Mad2p, suggesting that this Mad3 KEN box acts “in trans” as a Cdc20p degron. c) Mad3p turnover in G1. Mad3p is degraded in a Mad3-KEN30, Cdh1, and APC/C dependent manner.</p