The DNA damage checkpoint kinase Rad53 is important for the survival of budding yeast under genotoxic stresses. We performed a biochemical screen to identify proteins with specific affinity for the ...two Forkhead associated (FHA) domains of Rad53. The N-terminal FHA1 domain was found to coordinate a complex protein interaction network, which includes nuclear proteins involved in DNA damage checkpoints and transcriptional regulation. Unexpectedly, cytosolic proteins involved in cytokinesis, including septins, were also found as FHA1 binding proteins. Consistent with this interaction, a Rad53 mutant defective in its nuclear localization was found to localize to the bud neck. Abnormal morphology was observed in cells overexpressing the FHA1 domain and in rad53Δ cells under DNA replication stress. Further, septin Shs1 appears to have an important role in the response to DNA replication stress. Collectively, the results suggest a novel function of Rad53 in the regulation of polarized cell growth in response to DNA replication stress.
We have used local fluorescence photoactivation to mark the lattice of spindle microtubules during anaphase A in Xenopus extract spindles. We find that both poleward spindle microtubule flux and ...anaphase A chromosome movement occur at similar rates (∼2 μm/min). This result suggests that poleward microtubule flux, coupled to microtubule depolymerization near the spindle poles, is the predominant mechanism for anaphase A in Xenopus egg extracts. In contrast, in vertebrate somatic cells a "Pacman" kinetochore mechanism, coupled to microtubule depolymerization near the kinetochore, predominates during anaphase A. Consistent with the conclusion from fluorescence photoactivation analysis, both anaphase A chromosome movement and poleward spindle microtubule flux respond similarly to pharmacological perturbations in Xenopus extracts. Furthermore, the pharmacological profile of anaphase A in Xenopus extracts differs from the previously established profile for anaphase A in vertebrate somatic cells. The difference between these profiles is consistent with poleward microtubule flux playing the predominant role in anaphase chromosome movement in Xenopus extracts, but not in vertebrate somatic cells. We discuss the possible biological implications of the existence of two distinct anaphase A mechanisms and their differential contributions to poleward chromosome movement in different cell types.
Background: The PAR proteins are known to be localized asymmetrically in polarized C. elegans, Drosophila, and human cells and to participate in several cellular processes, including asymmetric cell ...division and spindle orientation. Although astral microtubules are known to play roles in these processes, their behavior during these events remains poorly understood.
Results: We have developed a method that makes it possible to examine the residence time of individual astral microtubules at the cell cortex of developing embryos. Using this method, we found that microtubules are more dynamic at the posterior cortex of the C. elegans embryo compared to the anterior cortex during spindle displacement. We further observed that this asymmetry depends on the PAR-3 protein and heterotrimeric G protein signaling, and that the PAR-2 protein affects microtubule dynamics by restricting PAR-3 activity to the anterior of the embryo.
Conclusions: These results indicate that PAR proteins function to regulate microtubule dynamics at the cortex during microtubule-dependent cellular processes.
Differential interference contrast (DIC) imaging of Caenorhabditis elegans embryogenesis led to a Nobel Prize in Physiology or Medicine (Sulston et al., 1983) as did the first use of green ...fluorescent protein (GFP) in a transgenic C. elegans (Chalfie et al., 1994). Given that C. elegans is free living, does not require exceptional environmental control, and is optically clear, live imaging is a powerful tool in for this model system. Combining genetics with high-resolution imaging has continued to make important contributions to many fields. In this chapter, we discuss how certain aspects of high-resolution microscopy are implemented. This is not an exhaustive review of microscopy; it is meant to be a helpful guide and point of reference for some basic concepts in imaging. While these concepts are largely true for all biological imaging, they are chosen as particularly important for C. elegans.
The budding yeast shmoo tip is a model system for analyzing mechanisms coupling force production to microtubule plus-end polymerization/depolymerization. Dynamic plus ends of astral microtubules ...interact with the shmoo tip in mating yeast cells, positioning nuclei for karyogamy 1. We have used live-cell imaging of GFP fusions to identify proteins that couple dynamic microtubule plus ends to the shmoo tip. We find that Kar3p, a minus end-directed kinesin motor protein, is required, whereas the other cytoplasmic motors, dynein and the kinesins Kip2p and Kip3p, are not. In the absence of Kar3p, attached microtubule plus ends released from the shmoo tip when they switched to depolymerization. Furthermore, microtubules in cells expressing kar3-1, a mutant that results in rigor binding to microtubules 2, were stabilized specifically at shmoo tips. Imaging of Kar3p-GFP during mating revealed that fluorescence at the shmoo tip increased during periods of microtubule depolymerization. These data are the first to localize the activity of a minus end-directed kinesin at the plus ends of microtubules. We propose a model in which Kar3p couples depolymerizing microtubule plus ends to the cell cortex and the Bim1p-Kar9p protein complex maintains attachment during microtubule polymerization. In support of this model, analysis of Bim1p-GFP at the shmoo tip results in a localization pattern complementary to that of Kar3p-GFP.
The interaction of kinetochores with dynamic microtubules during mitosis is essential for proper centromere motility, congression to the metaphase plate, and subsequent anaphase chromosome ...segregation. Budding yeast has been critical in the discovery of proteins necessary for this interaction. However, the molecular mechanism for microtubule-kinetochore interactions remains poorly understood. Using live cell imaging and mutations affecting microtubule binding proteins and kinetochore function, we identify a regulatory mechanism for spindle microtubule dynamics involving Stu2p and the core kinetochore component, Ndc10p. Depleting cells of the microtubule binding protein Stu2p reduces kinetochore microtubule dynamics. Centromeres remain under tension but lack motility. Thus, normal microtubule dynamics are not required to maintain tension at the centromere. Loss of the kinetochore (ndc10-1, ndc10-2, and ctf13-30) does not drastically affect spindle microtubule turnover, indicating that Stu2p, not the kinetochore, is the foremost governor of microtubule dynamics. Disruption of kinetochore function with ndc10-1 does not affect the decrease in microtubule turnover in stu2 mutants, suggesting that the kinetochore is not required for microtubule stabilization. Remarkably, a partial kinetochore defect (ndc10-2) suppresses the decreased spindle microtubule turnover in the absence of Stu2p. These results indicate that Stu2p and Ndc10p differentially function in controlling kinetochore microtubule dynamics necessary for centromere movements.
CHARISMA was a landmark randomized clinical trial that failed to demonstrate a benefit of dual antiplatelet therapy (DAPT) over aspirin alone for preventing cardiovascular events. However, subgroup ...analyses of the trial found fewer major adverse cardiovascular events (MACEs) for patients with established cardiovascular disease but more MACEs for patients with multiple risk factors without established cardiovascular disease. Our objective was to examine DAPT use in contemporary clinical practice after publication of CHARISMA results.
Retrospective analysis of a large clinical registry of outpatient cardiovascular visits to over 1000 physicians that collected data on patient clinical history, symptoms, vital signs, and medications.
Clinical characteristics and prescription rates of aspirin and clopidogrel were compared for patients with established cardiovascular disease and for patients with only multiple cardiovascular risk factors. Prescription of DAPT by calendar quarter was evaluated from 2008 to 2011 using multivariable Poisson regression models.
Of 167,839 patients with established cardiovascular disease, 20.5% were prescribed both aspirin and clopidogrel. Of 20,478 patients with multiple risk factors but no known cardiovascular disease, 3.5% were prescribed both aspirin and clopidogrel. Across 14 calendar quarters, prescription rates of DAPT did not change significantly for patients with established CVD but decreased for patients with multiple risk factors with an incidence rate ratio of 0.77.
Use of DAPT is modest in patients with established cardiovascular disease, for whom the CHARISMA trial suggested decreased MACEs, and prescription rates have remained stable over time. Use of DAPT in patients with multiple risk factors only, for whom CHARISMA suggested that DAPT may lead to increased MACE, was low and decreased over time.
Work over the last several decades has shown that kinetochores play an active part in chromosome segregation, while the chromatin and, more to the point, the DNA have gathered little attention. In ...two intriguing papers, the Bloom and Khodjakov groups show that intercentromeric chromatin plays a much more active part in chromosome segregation than previously suspected.
The National Cardiovascular Data Registry PINNACLE (Practice Innovation and Clinical Excellence) Registry is the largest outpatient cardiovascular practice registry in the world. It tracks real-world ...management and quality of 4 common cardiovascular conditions: heart failure, coronary artery disease, atrial fibrillation, and hypertension. In 2013, the PINNACLE Registry contained information on 2,898,505 patients, cared for by 4,859 providers in 431 practices. By 2017, the registry contained information on 6,040,996 patients, cared for by 8,853 providers in 724 practices. During this time period, care processes for PINNACLE patients generally improved. Among patients with heart failure, combined beta-blocker and renin-angiotensin antagonist medication rates increased from 60.7% to 72.8%. Among patients with coronary artery disease, statin medication rates increased from 66% to 80.1%. Among patients with atrial fibrillation, oral anticoagulation rates increased from 52.7% to 65.2%. In contrast, blood pressure control rates among patients with hypertension were largely stable. PINNACLE data also fueled a variety of quality measurement programs and 51 peer-reviewed publications.
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•The NCDR PINNACLE registry tracks management and quality of 4 common cardiovascular conditions: HF, CAD, AF, and HTN.•By 2017, the registry contained information on 6,040,996 patients, cared for by 8,853 providers in 724 practices. Between 2013 and 2017, care processes for PINNACLE patients generally improved.•PINNACLE registry participation should be considered by practices seeking to monitor and improve practice patterns.•The PINNACLE registry should continue to monitor current practice patterns and opportunities for improvement.
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