Tension Management in the Kinetochore

The kinetochore is the protein machine built at the centromere that integrates mechanical force and chemical energy from dynamic microtubules into directed chromosome motion. The kinetochore also provides a powerful signaling function that is able to alter the properties of the spindle checkpoint and initiate a signal transduction cascade that leads to inhibition of the anaphase promoting complex and cell cycle arrest. Together, the kinetochore accomplishes the feat of chromosome segregation with unparalleled accuracy. Errors in segregation lead to Down’s syndrome, the most frequent inherited birth defect, pregnancy loss, and cancer. Over a century after the discovery of the kinetochore, an architectural map comprising greater than 100 proteins is emerging. Understanding the architecture and physical biology of the key components provides new insights into how this fascinating machine moves genomes

Stable Kinetochore-Microtubule Attachment Constrains Centromere Positioning in Metaphase

With a single microtubule attachment, budding-yeast kinetochores provide an excellent system for understanding the coordinated linkage to dynamic microtubule plus ends for chromosome oscillation and positioning. Fluorescent tagging of kinetochore proteins indicates that, on average, all centromeres are clustered, distinctly separated from their sisters, and positioned equidistant from their respective spindle poles during metaphase. However, individual fluorescent chromosome markers near the centromere transiently reassociate with their sisters and oscillate from one spindle half to the other. To reconcile the apparent disparity between the average centromere position and individual centromere proximal markers, we utilized fluorescence recovery after photobleaching to measure stability of the histone-H3 variant Cse4p/CENP-A. Newly synthesized Cse4p replaces old protein during DNA replication. Once assembled, Cse4-GFP is a physically stable component of centromeres during mitosis. This allowed us to follow centromere dynamics within each spindle half. Kinetochores remain stably attached to dynamic microtubules and exhibit a low incidence of switching orientation or position between the spindle halves. Switching of sister chromatid attachment may be contemporaneous with Cse4p exchange and early kinetochore assembly during S phase; this would promote mixing of chromosome attachment to each spindle pole. Once biorientation is attained, centromeres rarely make excursions beyond their proximal half spindle

SeaWiFS Technical Report Series

For Earth-observing satellite instruments, it was standard to consider each instrument band to have a spectral response that is infinitely narrow, i.e., to have a response from a single wavelength. The Sea-viewing Wide Field-of-view Sensor (SeaWiFS) bands, however, have nominal spectral bandwidths of 20 and 40nm. These bandwidths affect the SeaWiFS measurements on orbit. The effects are also linked to the manner in which the instrument was calibrated and to the spectral shape of the radiance that SeaWiFS views. Currently, SeaWiFS is calibrated such that the digital counts from each instrument band are linked to the Earth-exiting radiance at an individual center wavelength. Before launch, SeaWiFS will be recalibrated so that the digital counts from each band will be linked to the Earth-exiting radiance integrated over the spectral response of that band. In this technical memorandum, the effects of the instrument calibration and the source spectral shape on SeaWiFS measurements, including the in-band and out-of-band responses, and the center wavelengths are discussed

Leveraging Geospatial Information to address Space Epidemiology through Multi\unicode{x2013}omics \unicode{x2013} Report of an Interdisciplinary Workshop

This article will summarize the workshop proceedings of a workshop conducted at the University of Missouri that addressed the use of multi-omics fused with geospatial information to assess and improve the precision and environmental analysis of indicators of crew space health. The workshop addressed the state of the art of multi-omics research and practice and the potential future use of multi-omics platforms in extreme environments. The workshop also focused on potential new strategies for data collection, analysis, and fusion with crosstalk with the field of environmental health, biosecurity, and radiation safety, addressing gaps and shortfalls and potential new approaches to enhancing astronaut health safety and security. Ultimately, the panel proceedings resulted in a synthesis of new research and translational opportunities to improve space and terrestrial epidemiology. In the future, early disease prevention that employs new and expanded data sources enhanced by the analytic precision of geospatial information and artificial intelligence algorithms.Comment: 9 pages, 1 figur

Bub1 Kinase and Sgo1 Modulate Pericentric Chromatin in Response to Altered Microtubule Dynamics

Tension sensing of bi-oriented chromosomes is essential for the fidelity of chromosome segregation. The spindle assembly checkpoint (SAC) conveys lack of tension or attachment to the anaphase promoting complex. Components of the SAC (Bub1) phosphorylate histone H2A (S121) and recruit the protector of cohesin, Shugoshin (Sgo1) to the inner centromere. How the chromatin structural modifications of the inner centromere are integrated into the tension sensing mechanisms and the checkpoint are not known

The role of the proteins Kar9 and Myo2 in orienting the mitotic spindle of budding yeast

AbstractBackground: Two genetic ‘pathways' contribute to the fidelity of nuclear segregation during the process of budding in the yeast Saccharomyces cerevisiae. An early pathway, involving Kar9p and other proteins, orients the mitotic spindle along the mother–bud axis. Upon the onset of anaphase, cytoplasmic dynein provides the motive force for nuclear movement into the bud. Loss of either pathway results in nuclear-migration defects; loss of both is lethal. Here, to visualize the functional steps leading to correct spindle orientation along the mother–bud axis, we imaged live yeast cells expressing Kar9p and dynein as green fluorescent protein fusions.Results: Transport of Kar9p into the bud was found to require the myosin Myo2p. Kar9p interacted with microtubules through the microtubule-binding protein Bim1p and facilitated microtubule penetration into the bud. Once microtubules entered the bud, Kar9p provided a platform for microtubule capture at the bud cortex. Kar9p was also observed at sites of microtubule shortening in the bud, suggesting that Kar9p couples microtubule shortening to nuclear migration.Conclusions: Thus, Kar9p provides a key link between the actin cytoskeleton and microtubules early in the cell cycle. A cooperative mechanism between Kar9p and Myo2p facilitates the pre-anaphase orientation of the spindle. Later, Kar9p couples microtubule disassembly with nuclear migration

Chromosome integrity at a double-strand break requires exonuclease 1 and MRX

The continuity of duplex DNA is generally considered a prerequisite for chromosome continuity. However, as previously shown in yeast as well as human cells, the introduction of a double-strand break (DSB) does not generate a chromosome break (CRB) in yeast or human cells. The transition from DSB to CRB was found to be under limited control by the tethering function of the RAD50/MRE11/XRS2 (MRX) complex. Using a system for differential fluorescent marking of both sides of an endonuclease-induced DSB in single cells, we found that nearly all DSBs are converted to CRBs in cells lacking both exonuclease 1 (EXO1) activity and MRX complex. Thus, it appears that some feature of exonuclease processing or resection at a DSB is critical for maintaining broken chromosome ends in close proximity. In addition, we discovered a thermal sensitive (cold) component to CRB formation in an MRX mutant that has implications for chromosome end mobility and/or end-processing

Enrichment of dynamic chromosomal crosslinks drive phase separation of the nucleolus

Regions of highly repetitive DNA, such as those found in the nucleolus, show a self-organization that is marked by spatial segregation and frequent self-interaction. The mechanisms that underlie the sequestration of these sub-domains are largely unknown. Using a stochastic, bead-spring representation of chromatin in budding yeast, we find enrichment of protein-mediated, dynamic chromosomal cross-links recapitulates the segregation, morphology and self-interaction of the nucleolus. Rates and enrichment of dynamic crosslinking have profound consequences on domain morphology. Our model demonstrates the nucleolus is phase separated from other chromatin in the nucleus and predicts that multiple rDNA loci will form a single nucleolus independent of their location within the genome. Fluorescent labeling of budding yeast nucleoli with CDC14-GFP revealed that a split rDNA locus indeed forms a single nucleolus. We propose that nuclear sub-domains, such as the nucleolus, result from phase separations within the nucleus, which are driven by the enrichment of protein-mediated, dynamic chromosomal crosslinks

Tension-dependent nucleosome remodeling at the pericentromere in yeast

Dynamics of histones under tension in the pericentromere depends on RSC and ISW2 chromatin remodeling. The underlying pericentromeric chromatin forms a platform that is required to maintain kinetochore structure when under spindle-based tension.Nucleosome positioning is important for the structural integrity of chromosomes. During metaphase the mitotic spindle exerts physical force on pericentromeric chromatin. The cell must adjust the pericentromeric chromatin to accommodate the changing tension resulting from microtubule dynamics to maintain a stable metaphase spindle. Here we examine the effects of spindle-based tension on nucleosome dynamics by measuring the histone turnover of the chromosome arm and the pericentromere during metaphase in the budding yeast Saccharomyces cerevisiae. We find that both histones H2B and H4 exhibit greater turnover in the pericentromere during metaphase. Loss of spindle-based tension by treatment with the microtubule-depolymerizing drug nocodazole or compromising kinetochore function results in reduced histone turnover in the pericentromere. Pericentromeric histone dynamics are influenced by the chromatin-remodeling activities of STH1/NPS1 and ISW2. Sth1p is the ATPase component of the Remodels the Structure of Chromatin (RSC) complex, and Isw2p is an ATP-dependent DNA translocase member of the Imitation Switch (ISWI) subfamily of chromatin-remodeling factors. The balance between displacement and insertion of pericentromeric histones provides a mechanism to accommodate spindle-based tension while maintaining proper chromatin packaging during mitosis

SeaWiFS technical report series. Volume 20: The SeaWiFS bio-optical archive and storage system (SeaBASS), part 1

This document provides an overview of the Sea-viewing Wide Field-of-view Sensor (SeaWiFS) Bio-Optical Archive and Storage System (SeaBASS), which will serve as a repository for numerous data sets of interest to the SeaWiFS Science Team and other approved investigators in the oceanographic community. The data collected will be those data sets suitable for the development and evaluation of bio-optical algorithms which include results from SeaWiFS Intercalibration Round-Robin Experiments (SIRREXs), prelaunch characterization of the SeaWiFS instrument by its manufacturer -- Hughes/Santa Barbara Research Center (SBRC), Marine Optical Characterization Experiment (MOCE) cruises, Marine Optical Buoy (MOBY) deployments and refurbishments, and field studies of other scientists outside of NASA. The primary goal of the data system is to provide a simple mechanism for querying the available archive and requesting specific items, while assuring that the data is made available only to authorized users. The design, construction, and maintenance of SeaBASS is the responsibility of the SeaWiFS Calibration and Validation Team (CVT). This report is concerned with documenting the execution of this task by the CVT and consists of a series of chapters detailing the various data sets involved. The topics presented are as follows: 1) overview of the SeaBASS file architecture, 2) the bio-optical data system, 3) the historical pigment database, 4) the SIRREX database, and 5) the SBRC database