Kinetochore-associated Mps1 regulates the strength of kinetochore-microtubule attachments via Ndc80 phosphorylation

Dividing cells detect and correct erroneous kinetochore-microtubule attachments during mitosis, thereby avoiding chromosome mis-segregation. Most studies of this process have focused on the Aurora B kinase, which phosphorylates microtubule-binding elements specifically at incorrectly attached kinetochores, promoting their release and providing another chance for proper attachments to form. However, growing evidence suggests additional mechanisms, potentially involving Mps1 kinase, that also underlie error correction. Because these mechanisms overlap in vivo, and because both Mps1 and Aurora B function in numerous other vital processes, their contributions to the correction of erroneous kinetochore attachments have been difficult to disentangle. Here we directly examine how Mps1 activity affects kinetochore-microtubule attachments using a reconstitution-based approach that allowed us to separate its effects from Aurora B activity. When endogenous Mps1 that co-purifies with isolated kinetochores is activated in vitro, it weakens their attachments to microtubules via phosphorylation of Ndc80, a major microtubule-binding element of the outer kinetochore. Mps1 phosphorylation of Ndc80 appears to contribute to error correction because phospho-deficient Ndc80 mutants exhibit genetic interactions and segregation defects when combined with mutants in an intrinsic error correction pathway. In addition, Mps1 phosphorylation of Ndc80 is stimulated on kinetochores lacking tension. These data suggest that Mps1 provides an additional mechanism for correcting erroneous kinetochore-microtubule attachments, complementing the well-known activity of Aurora B.

chTOG is a conserved mitotic error correction factor

Accurate chromosome segregation requires kinetochores on duplicated chromatids to biorient by attaching to dynamic microtubules from opposite spindle poles, which exerts forces to bring kinetochores under tension. However, kinetochores initially bind to microtubules indiscriminately, resulting in errors that must be corrected. While the Aurora B protein kinase destabilizes low-tension attachments by phosphorylating kinetochores, low-tension attachments are intrinsically less stable than those under higher tension in vitro independent of Aurora activity. Intrinsic tension-sensitive behavior requires the microtubule regulator Stu2 (budding yeast Dis1/XMAP215 ortholog), which we demonstrate here is likely a conserved function for the TOG protein family. The human TOG protein, chTOG, localizes to kinetochores independent of microtubules by interacting with Hec1. We identify a chTOG mutant that regulates microtubule dynamics but accumulates erroneous kinetochore-microtubule attachments that are not destabilized by Aurora B. Thus, TOG proteins confer a unique, intrinsic error correction activity to kinetochores that ensures accurate chromosome segregation.

Oncogene Concatenated Enriched Amplicon Nanopore Sequencing for Rapid, Accurate, and Affordable Somatic Mutation Detection

Nanopore sequencing is more than 10-fold faster than sequencing-by-synthesis and provides reads that are roughly 100-fold longer. However, nanopore sequencing’s 7.5% intrinsic error rate renders it difficult to call somatic mutations with low variant allele frequencies (VAFs) without significant false positives. Here, we introduce the Oncogene Concatenated Enriched Amplicon Nanopore Sequencing (OCEANS) method, in which variants with low VAFs are selectively amplified and subsequently concatenated for nanopore sequencing. OCEANS allows accurate detection of somatic mutations with VAF limits of detection between 0.05% and ≤ 1%. We constructed 4 distinct multi-gene OCEANS panels targeting recurrent mutations in acute myeloid leukemia, melanoma, non-small-cell lung cancer, and hepatocellular carcinoma. Comparison experiments against Illumina NGS showed 99.79% to 99.99% area under the receiver-operator curve for these panels on clinical FFPE tumor samples. Furthermore, we identified a significant number of mutations below the standard NGS limit of detection in clinical tissue samples using each OCEANS panel. Comparison against digital PCR on 10 of putative mutations at ≤1% VAF showed 9 concordant positive calls with VAFs between 0.02% and 0.66%. By overcoming the primary challenge of nanopore sequencing on detecting low VAF single nucleotide variant mutations, OCEANS is poised to enable same-day clinical sequencing panels.

chTOG is a conserved mitotic error correction factor

Accurate chromosome segregation requires kinetochores on duplicated chromatids to biorient by attaching to dynamic microtubules from opposite spindle poles, which exerts forces to bring kinetochores under tension. However, kinetochores initially bind to MTs indiscriminately, resulting in errors that must be corrected. While the Aurora B protein kinase destabilizes low-tension attachments by phosphorylating kinetochores, low-tension attachments are intrinsically less stable than those under higher tension in vitro independent of Aurora activity. Intrinsic tensionsensitive behavior requires the microtubule regulator Stu2 (budding yeast Dis1/XMAP215 ortholog), which we demonstrate here is likely a conserved function for the TOG protein family. The human TOG protein, chTOG, localizes to kinetochores independent of microtubules by interacting with Hec1. We identify a chTOG mutant that regulates microtubule dynamics but accumulates erroneous kinetochore-microtubule attachments that Aurora B fails to destabilize. Thus, TOG proteins confer a unique, intrinsic error correction activity to kinetochores that ensures accurate chromosome segregation.

Bed-Sediment Transport Conditions along the Sagavanirktok River in Northern Alaska, USA

This manuscript presents a study in predicting bed-sediment transport rates along the Sagavanirktok River in Alaska. Extensive field activities took place to accomplish this goal: four hydro-meteorological stations were installed in a 150 km reach along the river in summer 2015. During the same year, pits were excavated near the stations, and in subsequent summers, the pits were surveyed multiple times in conjunction with taking discharge measurements. Water slope was measured and bed sediment was characterized. Site-specific relationships between water levels and cross-section water depths were developed. Volume change between consecutive surveys was calculated, and main flood events between surveys were identified. Finally, the first bed-sediment transport equations valid for the Sagavanirktok River were developed. Considering the intrinsic error in sediment transport predictions, the agreement between predicted and measured sediment transport values is good. These equations could be used by resource managers when predicting the expected time for an excavated material site in the Sagavanirktok River to refill.

The Role of Stress Granules and the Nonsense-mediated mRNA Decay Pathway in Antiviral Defence

Eukaryotic cells have evolved a number of survival tactics and quality control pathways that are able to counter intrinsic error-prone mechanisms and allow for maintenance of cellular homeostasis in the face of external stresses. This review will discuss the role of two cellular eukaryotic processes that are vital for maintenance of cellular homeostasis – 1) the nonsense-mediated mRNA decay (NMD) pathway and 2) the transient formation of stress granules (SG) – and explore the current literature on their roles in antiviral defence. Within the NCCR RNA & Disease, the laboratories of Proffs. O. Mühlemann and Volker Thiel teamed up to unravel the roles of NMD and SGs, and their interconnections in defending cells from alphavirus and coronavirus infections.

A new and homogeneous metallicity scale for Galactic classical Cepheids

We gathered more than 1130 high-resolution optical spectra for more than 250 Galactic classical Cepheids. The spectra were collected with the optical spectrographs UVES at VLT, HARPS at 3.6 m, FEROS at 2.2 m MPG/ESO, and STELLA. To improve the effective temperature estimates, we present more than 150 new line depth ratio (LDR) calibrations that together with similar calibrations already available in the literature allowed us to cover a broad range in wavelength (5348 ≤ λ ≤ 8427 Å) and in effective temperature (3500 ≤ Teff ≤ 7700 K). This gives us the unique opportunity to cover both the hottest and coolest phases along the Cepheid pulsation cycle and to limit the intrinsic error on individual measurements at the level of ~100 K. As a consequence of the high signal-to-noise ratio of individual spectra, we identified and measured hundreds of neutral and ionized lines of heavy elements, and in turn, have the opportunity to trace the variation of both surface gravity and microturbulent velocity along the pulsation cycle. The accuracy of the physical parameters and the number of Fe I (more than one hundred) and Fe II (more than ten) lines measured allowed us to estimate mean iron abundances with a precision better than 0.1 dex. We focus on 14 calibrating Cepheids for which the current spectra cover either the entire or a significant portion of the pulsation cycle. The current estimates of the variation of the physical parameters along the pulsation cycle and of the iron abundances agree very well with similar estimates available in the literature. Independent homogeneous estimates of both physical parameters and metal abundances based on different approaches that can constrain possible systematics are highly encouraged.

Translesion and Repair DNA Polymerases: Diverse Structure and Mechanism

The number of DNA polymerases identified in each organism has mushroomed in the past two decades. Most newly found DNA polymerases specialize in translesion synthesis and DNA repair instead of replication. Although intrinsic error rates are higher for translesion and repair polymerases than for replicative polymerases, the specialized polymerases increase genome stability and reduce tumorigenesis. Reflecting the numerous types of DNA lesions and variations of broken DNA ends, translesion and repair polymerases differ in structure, mechanism, and function. Here, we review the unique and general features of polymerases specialized in lesion bypass, as well as in gap-filling and end-joining synthesis.