Cse4 is the budding yeast homologue of CENP-A, a modified histone H3 that specifies the base of kinetochores in all eukaryotes. Budding yeast is unique in having only one kinetochore microtubule ...attachment site per centromere. The centromere is specified by CEN DNA, a sequence-specific binding complex (CBF3), and a Cse4-containing nucleosome. Here we compare the ratio of kinetochore proximal Cse4-GFP fluorescence at anaphase to several standards including purified EGFP molecules in vitro to generate a calibration curve for the copy number of GFP-fusion proteins. Our results yield a mean of ~5 Cse4s, ~3 inner kinetochore CBF3 complexes, and ~20 outer kinetochore Ndc80 complexes. Our calibrated measurements increase 2.5-3-fold protein copy numbers at eukaryotic kinetochores based on previous ratio measurements assuming two Cse4s per budding yeast kinetochore. All approximately five Cse4s may be associated with the CEN nucleosome, but we show that a mean of three Cse4s could be located within flanking nucleosomes at random sites that differ between chromosomes.
Different aspects of attention can be assessed through psychological tests to identify stable individual or group differences as well as alterations after interventions. Aiming for a wide ...applicability of attentional assessments, Psychology Experiment Building Language (PEBL) is an open-source software system for designing and running computerized tasks that tax various attentional functions. Here, we evaluated the reliability and validity of computerized attention tasks as provided with the PEBL package: Continuous Performance Task (CPT), Switcher task, Psychomotor Vigilance Task (PVT), Mental Rotation task, and Attentional Network Test. For all tasks, we evaluated test-retest reliability using the intraclass correlation coefficient (ICC), as well as internal consistency through within-test correlations and split-half ICC. Across tasks, response time scores showed adequate reliability, whereas scores of performance accuracy, variability, and deterioration over time did not. Stability across application sites was observed for the CPT and Switcher task, but practice effects were observed for all tasks except the PVT. We substantiate convergent and discriminant validity for several task scores using between-task correlations and provide further evidence for construct validity via associations of task scores with attentional and motivational assessments. Taken together, our results provide necessary information to help design and interpret studies involving attention assessments.
Objective
Immune complex (IC) deposition activates polymorphonuclear neutrophils (PMNs), increases vascular permeability, and leads to organ damage in systemic lupus erythematosus and rheumatoid ...arthritis. The bioactive lipid sphingosine 1‐phosphate (S1P), acting via S1P receptor 1 (S1P1), is a key regulator of endothelial cell (EC) barrier function. This study was undertaken to investigate whether augmenting EC integrity via S1P1 signaling attenuates inflammatory injury mediated by ICs.
Methods
In vitro barrier function was assessed in human umbilical vein endothelial cells (HUVECs) by electrical cell‐substrate impedance sensing. Phosphorylation of myosin light chain 2 (p–MLC‐2) and VE‐cadherin staining in HUVECs were assessed by immunofluorescence. A reverse Arthus reaction (RAR) was induced in the skin and lungs of mice with S1P1 deleted from ECs (S1P1 EC‐knockout ECKO mice) and mice treated with S1P1 agonists and antagonists.
Results
S1P1 agonists prevented loss of barrier function in HUVECs treated with IC‐activated PMNs. S1P1 ECKO and wild‐type (WT) mice treated with S1P1 antagonists had amplified RAR, whereas specific S1P1 agonists attenuated skin and lung RAR in WT mice. ApoM‐Fc, a novel S1P chaperone, mitigated EC cell barrier dysfunction induced by activated PMNs in vitro and attenuated lung RAR. Expression levels of p–MLC‐2 and disruption of VE‐cadherin, each representing manifestations of cell contraction and destabilization of adherens junctions, respectively, that were induced by activated PMNs, were markedly reduced by treatment with S1P1 agonists and ApoM‐Fc.
Conclusion
Our findings indicate that S1P1 signaling in ECs modulates vascular responses to IC deposition. S1P1 agonists and ApoM‐Fc enhance the EC barrier, limit leukocyte escape from capillaries, and provide protection against inflammatory injury. The S1P/S1P1 axis is a newly identified target to attenuate tissue responses to IC deposition and mitigate end‐organ damage.
Successful completion of mitosis requires that sister kinetochores become attached end-on to the plus ends of spindle microtubules (MTs) in prometaphase, thereby forming kinetochore microtubules ...(kMTs) that tether one sister to one spindle pole and the other sister to the opposite pole. Sites for kMT attachment provide at least four key functions: robust and dynamic kMT anchorage; force generation that can be coupled to kMT plus-end dynamics; correction of errors in kMT attachment; and control of the spindle assembly checkpoint (SAC). The SAC typically delays anaphase until chromosomes achieve metaphase alignment with each sister kinetochore acquiring a full complement of kMTs. Although it has been known for over 30 years that MT motor proteins reside at kinetochores, a highly conserved network of protein complexes, called the KMN network, has emerged in recent years as the primary interface between the kinetochore and kMTs. This Commentary will summarize recent advances in our understanding of the role of the KMN network for the key kinetochore functions, with a focus on human cells.
Kinetochores mediate chromosome segregation at mitosis. They are thought to contain both active, force-producing and passive, frictional interfaces with microtubules whose relative locations have ...been unclear. We inferred mechanical deformation within single kinetochores during metaphase oscillations by measuring average separations between fluorescently labeled kinetochore subunits in living cells undergoing mitosis. Inter-subunit distances were shorter in kinetochores moving toward poles than in those moving away. Inter-subunit separation decreased abruptly when kinetochores switched to poleward movement and decreased further when pulling force increased, suggesting that active force generation during poleward movement compresses kinetochores. The data revealed an active force-generating interface within kinetochores and a separate passive frictional interface located at least 20 nanometers away poleward. Together, these interfaces allow persistent attachment with intermittent active force generation.
The kinetochore is a control module that both powers and regulates chromosome segregation in mitosis and meiosis. The kinetochore-microtubule interface is remarkably fluid, with the microtubules ...growing and shrinking at their point of attachment to the kinetochore. Furthermore, the kinetochore itself is highly dynamic, its makeup changing as cells enter mitosis and as it encounters microtubules. Active kinetochores have yet to be isolated or reconstituted, and so the structure remains enigmatic. Nonetheless, recent advances in genetic, bioinformatic and imaging technology mean we are now beginning to understand how kinetochores assemble, bind to microtubules and release them when the connections made are inappropriate, and also how they influence microtubule behaviour. Recent work has begun to elucidate a pathway of kinetochore assembly in animal cells; the work has revealed that many kinetochore components are highly dynamic and that some cycle between kinetochores and spindle poles along microtubules. Further studies of the kinetochore-microtubule interface are illuminating: (1) the role of the Ndc80 complex and components of the Ran-GTPase system in microtubule attachment, force generation and microtubule-dependent inactivation of kinetochore spindle checkpoint activity; (2) the role of chromosomal passenger proteins in the correction of kinetochore attachment errors; and (3) the function of microtubule plus-end tracking proteins, motor depolymerases and other proteins in kinetochore movement on microtubules and movement coupled to microtubule poleward flux.
The antiphospholipid syndrome (APS) is defined by thrombosis and recurrent pregnancy loss in the presence of antiphospholipid (aPL) antibodies and is generally treated with anticoagulation therapy. ...Because complement activation is essential and causative in aPL antibody-induced fetal injury, we hypothesized that heparin protects pregnant APS patients from complications through inhibition of complement. Treatment with heparin (unfractionated or low molecular weight) prevented complement activation in vivo and in vitro and protected mice from pregnancy complications induced by aPL antibodies. Neither fondaparinux nor hirudin, other anticoagulants, inhibited the generation of complement split products or prevented pregnancy loss, demonstrating that anticoagulation therapy is insufficient protection against APS-associated miscarriage. Our data indicate that heparins prevent obstetrical complications in women with APS because they block activation of complement induced by aPL antibodies targeted to decidual tissues, rather than by their anticoagulant effects.
Pregnancy in women with systemic lupus erythematosus (SLE) or antiphospholipid antibodies (APL Ab)--autoimmune conditions characterized by complement-mediated injury--is associated with increased ...risk of preeclampsia and miscarriage. Our previous studies in mice indicate that complement activation targeted to the placenta drives angiogenic imbalance and placental insufficiency.
We use PROMISSE, a prospective study of 250 pregnant patients with SLE and/or APL Ab, to test the hypothesis in humans that impaired capacity to limit complement activation predisposes to preeclampsia. We sequenced genes encoding three complement regulatory proteins--membrane cofactor protein (MCP), complement factor I (CFI), and complement factor H (CFH)--in 40 patients who had preeclampsia and found heterozygous mutations in seven (18%). Five of these patients had risk variants in MCP or CFI that were previously identified in atypical hemolytic uremic syndrome, a disease characterized by endothelial damage. One had a novel mutation in MCP that impairs regulation of C4b. These findings constitute, to our knowledge, the first genetic defects associated with preeclampsia in SLE and/or APL Ab. We confirmed the association of hypomorphic variants of MCP and CFI in a cohort of non-autoimmune preeclampsia patients in which five of 59 were heterozygous for mutations.
The presence of risk variants in complement regulatory proteins in patients with SLE and/or APL Ab who develop preeclampsia, as well as in preeclampsia patients lacking autoimmune disease, links complement activation to disease pathogenesis and suggests new targets for treatment of this important public health problem.
ClinicalTrials.gov NCT00198068.
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