DNA content of a functioning chicken kinetochore

© The Author(s) 2014. In order to understand the three-dimensional structure of the functional kinetochore in vertebrates, we require a complete list and stoichiometry for the protein components of the kinetochore, which can be provided by genetic and proteomic experiments. We also need to know how the chromatin-containing CENP-A, which makes up the structural foundation for the kinetochore, is folded, and how much of that DNA is involved in assembling the kinetochore. In this MS, we demonstrate that functioning metaphase kinetochores in chicken DT40 cells contain roughly 50 kb of DNA, an amount that corresponds extremely closely to the length of chromosomal DNA associated with CENP-A in ChIP-seq experiments. Thus, during kinetochore assembly, CENP-A chromatin is compacted into the inner kinetochore plate without including significant amounts of flanking pericentromeric heterochromatin. © 2014 The Author(s).Wellcome Trust [grant number 073915]; Wellcome Trust Centre for Cell Biology (core grant numbers 077707 and 092076); Darwin Trust of Edinburg

Ska3 Ensures Timely Mitotic Progression by Interacting Directly With Microtubules and Ska1 Microtubule Binding Domain

The establishment of physical attachment between the kinetochore and dynamic spindle microtubules, which undergo cycles of polymerization and depolymerization generating straight and curved microtubule structures, is essential for accurate chromosome segregation. The Ndc80 and Ska complexes are the major microtubule-binding factors of the kinetochore responsible for maintaining chromosome-microtubule coupling during chromosome segregation. We previously showed that the Ska1 subunit of the Ska complex binds dynamic microtubules using multiple contact sites in a mode that allows conformation-independent binding. Here, we show that the Ska3 subunit is required to modulate the microtubule binding capability of the Ska complex (i) by directly interacting with tubulin monomers and (ii) indirectly by interacting with tubulin contacting regions of Ska1 suggesting an allosteric regulation. Perturbing either the Ska3-microtubule interaction or the Ska3-Ska1 interactions negatively influences microtubule binding by the Ska complex in vitro and affects the timely onset of anaphase in cells. Thus, Ska3 employs additional modulatory elements within the Ska complex to ensure robust kinetochore-microtubule attachments and timely progression of mitosis

TRIP13 and APC15 drive mitotic exit by turnover of interphase- and unattached kinetochore-produced MCC

The mitotic checkpoint ensures accurate chromosome segregation through assembly of the mitotic checkpoint complex (MCC), a soluble inhibitor of the anaphase-promoting complex/cyclosome (APC/C) produced by unattached kinetochores. MCC is also assembled during interphase by Mad1/Mad2 bound at nuclear pores, thereby preventing premature mitotic exit prior to kinetochore maturation and checkpoint activation. Using degron tagging to rapidly deplete the AAA+ ATPase TRIP13, we show that its catalytic activity is required to maintain a pool of open-state Mad2 for MCC assembly, thereby supporting mitotic checkpoint activation, but is also required for timely mitotic exit through catalytic disassembly of MCC. Strikingly, combining TRIP13 depletion with elimination of APC15-dependent Cdc20 ubiquitination/degradation results in a complete inability to exit mitosis, even when MCC assembly at unattached kinetochores is prevented. Thus, mitotic exit requires MCC produced either in interphase or mitosis to be disassembled by TRIP13-catalyzed removal of Mad2 or APC15-driven ubiquitination/degradation of its Cdc20 subunit

Conditional targeting of MAD1 to kinetochores is sufficient to reactivate the spindle assembly checkpoint in metaphase

Fidelity of chromosome segregation is monitored by the spindle assembly checkpoint (SAC). Key components of the SAC include MAD1, MAD2, BUB1, BUB3, BUBR1, and MPS1. These proteins accumulate on kinetochores in early prometaphase but are displaced when chromosomes attach to microtubules and/or biorient on the mitotic spindle. As a result, stable attachment of the final chromosome satisfies the SAC, permitting activation of the anaphase promoting complex/cyclosome (APC/C) and subsequent anaphase onset. SAC satisfaction is reversible, however, as addition of taxol during metaphase stops cyclin B1 degradation by the APC/C. We now show that targeting MAD1 to kinetochores during metaphase is sufficient to reestablish SAC activity after initial silencing. Using rapamycin-induced heterodimerization of FKBP-MAD1 to FRB-MIS12 and live monitoring of cyclin B1 degradation, we show that timed relocalization of MAD1 during metaphase can stop cyclin B1 degradation without affecting chromosome-spindle attachments. APC/C inhibition represented true SAC reactivation, as FKBP-MAD1 required an intact MAD2-interaction motif and MPS1 activity to accomplish this. Our data show that MAD1 kinetochore localization dictates SAC activity and imply that SAC regulatory mechanisms downstream of MAD1 remain functional in metaphase. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00412-014-0458-9) contains supplementary material, which is available to authorized users

Differential Expression of CD163 on Monocyte Subsets in Healthy and HIV-1 Infected Individuals

CD163, a haptoglobin-hemoglobin (Hp-Hb) scavenger receptor, expressed by monocytes and macrophages, is important in resolution of inflammation. Age-related non-AIDS co-morbidities in HIV-infected individuals, particularly dementia and cardiovascular disease, result in part from effects of HIV-1 infection on monocyte and macrophage biology. CD163 co-expression on CD14+CD16++ monocytes has been proposed as a useful biomarker for HIV-1 disease progression and the presence of HIV associated dementia. Here we investigated CD163 expression on monocyte subsets ex vivo, on cultured macrophages, and soluble in plasma, in the setting of HIV-1 infection. Whole blood immunophenotyping revealed CD163 expression on CD14++CD16- monocytes but not on CD14+CD16++ monocytes (P = 0.004), supported by CD163 mRNA levels. Incubation with M-CSF induced CD163 protein expression on CD14+CD16++ monocytes to the same extent as CD14++CD16− monocytes. CD163 expression on CD14++CD16+ monocytes from HIV-infected subjects was significantly higher than from uninfected individuals, with a trend towards increased expression on CD14++CD16− monocytes (P = 0.019 and 0.069 respectively), which is accounted for by HIV-1 therapy including protease inhibitors. Shedding of CD163 was shown to predominantly occur from the CD14++CD16− subset after Ficoll isolation and LPS stimulation. Soluble CD163 concentration in plasma from HIV-1 infected donors was similar to HIV-1 uninfected donors. Monocyte CD163 expression in HIV-1 infected patients showed a complicated relationship with classical measures of disease progression. Our findings clarify technical issues regarding CD163 expression on monocyte subsets and further elucidates its role in HIV-associated inflammation by demonstrating that CD163 is readily lost from CD14++CD16− monocytes and induced in pro-inflammatory CD14+CD16++ monocytes by M-CSF. Our data show that all monocyte subsets are potentially capable of differentiating into CD163-expressing anti-inflammatory macrophages given appropriate stimuli. Levels of CD163 expression on monocytes may be a potential biomarker reflecting efforts by the immune system to resolve immune activation and inflammation in HIV-infected individuals

Molecular dissection of cobra venom highlights heparinoids as an antidote for spitting cobra envenoming

Snakebites affect about 1.8 million people annually. The current standard of care involves antibody-based antivenoms, which can be difficult to access and are generally not effective against local tissue injury, the primary cause of morbidity. Here, we used a pooled whole-genome CRISPR knockout screen to define human genes that, when targeted, modify cell responses to spitting cobra venoms. A large portion of modifying genes that conferred resistance to venom cytotoxicity was found to control proteoglycan biosynthesis, including EXT1, B4GALT7, EXT2, EXTL3, XYLT2, NDST1, and SLC35B2, which we validated independently. This finding suggested heparinoids as possible inhibitors. Heparinoids prevented venom cytotoxicity through binding to three-finger cytotoxins, and the US Food and Drug Administration-approved heparinoid tinzaparin was found to reduce tissue damage in mice when given via a medically relevant route and dose. Overall, our systematic molecular dissection of cobra venom cytotoxicity provides insight into how we can better treat cobra snakebite envenoming

Differential Expression of CD163 on Monocyte Subsets in Healthy and HIV-1 Infected Individuals

CD163, a haptoglobin-hemoglobin (Hp-Hb) scavenger receptor, expressed by monocytes and macrophages, is important in resolution of inflammation. Age-related non-AIDS co-morbidities in HIV-infected individuals, particularly dementia and cardiovascular disease, result in part from effects of HIV-1 infection on monocyte and macrophage biology. CD163 co-expression on CD14+CD16++ monocytes has been proposed as a useful biomarker for HIV-1 disease progression and the presence of HIV associated dementia. Here we investigated CD163 expression on monocyte subsets ex vivo, on cultured macrophages, and soluble in plasma, in the setting of HIV-1 infection. Whole blood immunophenotyping revealed CD163 expression on CD14++CD16- monocytes but not on CD14+CD16++ monocytes (P = 0.004), supported by CD163 mRNA levels. Incubation with M-CSF induced CD163 protein expression on CD14+CD16++ monocytes to the same extent as CD14++CD16− monocytes. CD163 expression on CD14++CD16+ monocytes from HIV-infected subjects was significantly higher than from uninfected individuals, with a trend towards increased expression on CD14++CD16− monocytes (P = 0.019 and 0.069 respectively), which is accounted for by HIV-1 therapy including protease inhibitors. Shedding of CD163 was shown to predominantly occur from the CD14++CD16− subset after Ficoll isolation and LPS stimulation. Soluble CD163 concentration in plasma from HIV-1 infected donors was similar to HIV-1 uninfected donors. Monocyte CD163 expression in HIV-1 infected patients showed a complicated relationship with classical measures of disease progression. Our findings clarify technical issues regarding CD163 expression on monocyte subsets and further elucidates its role in HIV-associated inflammation by demonstrating that CD163 is readily lost from CD14++CD16− monocytes and induced in pro-inflammatory CD14+CD16++ monocytes by M-CSF. Our data show that all monocyte subsets are potentially capable of differentiating into CD163-expressing anti-inflammatory macrophages given appropriate stimuli. Levels of CD163 expression on monocytes may be a potential biomarker reflecting efforts by the immune system to resolve immune activation and inflammation in HIV-infected individuals

Whole-proteome genetic analysis of dependencies in assembly of a vertebrate kinetochore

Kinetochores orchestrate mitotic chromosome segregation. Here, we use quantitative mass spectrometry of mitotic chromosomes isolated from a comprehensive set of chicken DT40 mutants to examine the dependencies of 93 confirmed and putative kinetochore proteins for stable association with chromosomes. Clustering and network analysis reveal both known and unexpected aspects of coordinated behavior for members of kinetochore protein complexes. Surprisingly, CENP-T depends on CENP-N for chromosome localization. The Ndc80 complex exhibits robust correlations with all other complexes in a “core” kinetochore network. Ndc80 associated with CENP-T interacts with a cohort of Rod, zw10, and zwilch (RZZ)–interacting proteins that includes Spindly, Mad1, and CENP-E. This complex may coordinate microtubule binding with checkpoint signaling. Ndc80 associated with CENP-C forms the KMN (Knl1, Mis12, Ndc80) network and may be the microtubule-binding “workhorse” of the kinetochore. Our data also suggest that CENP-O and CENP-R may regulate the size of the inner kinetochore without influencing the assembly of the outer kinetochore

Changes in symptomatology, reinfection, and transmissibility associated with the SARS-CoV-2 variant B.1.1.7: an ecological study

Background The SARS-CoV-2 variant B.1.1.7 was first identified in December, 2020, in England. We aimed to investigate whether increases in the proportion of infections with this variant are associated with differences in symptoms or disease course, reinfection rates, or transmissibility. Methods We did an ecological study to examine the association between the regional proportion of infections with the SARS-CoV-2 B.1.1.7 variant and reported symptoms, disease course, rates of reinfection, and transmissibility. Data on types and duration of symptoms were obtained from longitudinal reports from users of the COVID Symptom Study app who reported a positive test for COVID-19 between Sept 28 and Dec 27, 2020 (during which the prevalence of B.1.1.7 increased most notably in parts of the UK). From this dataset, we also estimated the frequency of possible reinfection, defined as the presence of two reported positive tests separated by more than 90 days with a period of reporting no symptoms for more than 7 days before the second positive test. The proportion of SARS-CoV-2 infections with the B.1.1.7 variant across the UK was estimated with use of genomic data from the COVID-19 Genomics UK Consortium and data from Public Health England on spike-gene target failure (a non-specific indicator of the B.1.1.7 variant) in community cases in England. We used linear regression to examine the association between reported symptoms and proportion of B.1.1.7. We assessed the Spearman correlation between the proportion of B.1.1.7 cases and number of reinfections over time, and between the number of positive tests and reinfections. We estimated incidence for B.1.1.7 and previous variants, and compared the effective reproduction number, Rt, for the two incidence estimates. Findings From Sept 28 to Dec 27, 2020, positive COVID-19 tests were reported by 36 920 COVID Symptom Study app users whose region was known and who reported as healthy on app sign-up. We found no changes in reported symptoms or disease duration associated with B.1.1.7. For the same period, possible reinfections were identified in 249 (0·7% [95% CI 0·6–0·8]) of 36 509 app users who reported a positive swab test before Oct 1, 2020, but there was no evidence that the frequency of reinfections was higher for the B.1.1.7 variant than for pre-existing variants. Reinfection occurrences were more positively correlated with the overall regional rise in cases (Spearman correlation 0·56–0·69 for South East, London, and East of England) than with the regional increase in the proportion of infections with the B.1.1.7 variant (Spearman correlation 0·38–0·56 in the same regions), suggesting B.1.1.7 does not substantially alter the risk of reinfection. We found a multiplicative increase in the Rt of B.1.1.7 by a factor of 1·35 (95% CI 1·02–1·69) relative to pre-existing variants. However, Rt fell below 1 during regional and national lockdowns, even in regions with high proportions of infections with the B.1.1.7 variant. Interpretation The lack of change in symptoms identified in this study indicates that existing testing and surveillance infrastructure do not need to change specifically for the B.1.1.7 variant. In addition, given that there was no apparent increase in the reinfection rate, vaccines are likely to remain effective against the B.1.1.7 variant. Funding Zoe Global, Department of Health (UK), Wellcome Trust, Engineering and Physical Sciences Research Council (UK), National Institute for Health Research (UK), Medical Research Council (UK), Alzheimer's Society

Genomic assessment of quarantine measures to prevent SARS-CoV-2 importation and transmission

Mitigation of SARS-CoV-2 transmission from international travel is a priority. We evaluated the effectiveness of travellers being required to quarantine for 14-days on return to England in Summer 2020. We identified 4,207 travel-related SARS-CoV-2 cases and their contacts, and identified 827 associated SARS-CoV-2 genomes. Overall, quarantine was associated with a lower rate of contacts, and the impact of quarantine was greatest in the 16–20 age-group. 186 SARS-CoV-2 genomes were sufficiently unique to identify travel-related clusters. Fewer genomically-linked cases were observed for index cases who returned from countries with quarantine requirement compared to countries with no quarantine requirement. This difference was explained by fewer importation events per identified genome for these cases, as opposed to fewer onward contacts per case. Overall, our study demonstrates that a 14-day quarantine period reduces, but does not completely eliminate, the onward transmission of imported cases, mainly by dissuading travel to countries with a quarantine requirement