Biology subjects

Nelson, C. R.

Publications and source records attributed to Nelson, C. R..

7 recordsLinked to original sources

bioRxiv2024

Architecture and flexibility of native kinetochores revealed by structural studies utilizing a thermophilic yeast

Eukaryotic chromosome segregation requires kinetochores, multi-megadalton protein machines that assemble on the centromeres of chromosomes and mediate attachments to dynamic spindle microtubules. Kinetochores are built from numerous complexes, and understanding how they are arranged is key to understanding how kinetochores perform their multiple functions. However, an integrated understanding of kinetochore architecture has not yet been established. To address this, we purified functional, native kinetochores from Kluyveromyces marxianus and examined them by electron microscopy, cryo-electron tomography and atomic force microscopy. The kinetochores are extremely large, flexible assemblies that exhibit features consistent with prior models. We assigned kinetochore polarity by visualizing their interactions with microtubules and locating the microtubule binder Ndc80c. This work shows that isolated kinetochores are more dynamic and complex than what might be anticipated based on the known structures of recombinant subassemblies, and provides the foundation to study the global architecture and functions of kinetochores at a structural level.

biophysics↗

bioRxiv2023

Phylogenomics recovers multiple origins of portable case-making in caddisflies (Insecta: Trichoptera), the world's most common underwater architects

Caddisflies (Trichoptera) are among the most diverse groups of freshwater animals with more than 16,000 described species. They play an outsized role in freshwater ecology and environmental engineering in streams, rivers, and lakes. Because of this, they are frequently used as indicator organisms in biomonitoring programs. Despite their importance, key questions concerning the evolutionary history of caddisflies, such as the timing and origin of larval case-making, have been unanswered due to the lack of a well-resolved phylogenetic tree. To shed light on these questions in Trichoptera, we estimated a phylogenetic tree using a combination of transcriptomes and targeted enrichment data for 206 species, representing 48 of 52 extant families and 174 genera. We calibrated and dated the tree with a set of 33 carefully selected fossils. The first caddisflies originated in the Permian and the major suborders began to diversify in the Triassic. Ancestral state reconstruction and diversification analysis revealed that portable case-making evolved in three separate lineages and shifts in diversification occurred in concert with key evolutionary innovations other than case-making.

evolutionary biology↗

bioRxiv2022

Cyclization studies of Japanese encephalitis virus non-coding RNA terminal regions.

Numerous viruses utilize essential long-range RNA-RNA genome interactions, specifically flaviviruses. Using Japanese encephalitis virus (JEV) as a model system, we computationally predicted and then biophysically validated and characterized its long-range RNA-RNA genomic interaction. Using multiple RNA computation assessment programs, we determine the primary RNA-RNA interacting site among JEV isolates and numerous related viruses. Following in vitro transcription of RNA, we provide, for the first time, characterization of an RNA-RNA interaction using multi-angle light scattering (SEC-MALS) and analytical ultra-centrifugation (AUC). Next, we report the first RNA-RNA interaction study quantified by microscale thermophoresis (MST), demonstrating that the 5 and 3 TR of JEV interact with nM affinity, which is significantly reduced when the conserved cyclization sequence is not present. Furthermore, we perform computational kinetic analyses validating the cyclization sequence as the primary driver of this RNA-RNA interaction. Finally, we examined the 3-dimensional structure of the interaction using small-angle X-ray scattering, revealing a flexible yet stable interaction. This pathway can be adapted and utilized to study various viral and human long-non-coding RNA-RNA interactions, and determine their binding affinities, a critical pharmacological property of designing potential therapeutics. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biochemistry↗

bioRxiv2021

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.

cell biology↗

bioRxiv2019

Genome sequence of the model rice variety KitaakeX

Here, we report the de novo genome sequencing and analysis of Oryza sativa ssp. japonica variety KitaakeX, a Kitaake plant carrying the rice XA21 immune receptor. Our KitaakeX sequence assembly contains 377.6 Mb, consisting of 33 scaffolds (476 contigs) with a contig N50 of 1.4 Mb. Complementing the assembly are detailed gene annotations of 35,594 protein coding genes. We identified 331,335 genomic variations between KitaakeX and Nipponbare (ssp. japonica), and 2,785,991 variations between KitaakeX and Zhenshan97 (ssp. indica). We also compared Kitaake resequencing reads to the KitaakeX assembly and identified 219 small variations. The high-quality genome of the model rice plant KitaakeX will accelerate rice functional genomics.

genomics↗

bioRxiv2018

PCH-2TRIP13 regulates spindle checkpoint strength

Spindle checkpoint strength is dictated by three criteria: the number of unattached kinetochores, cell volume and cell fate. We show that the conserved AAA-ATPase, PCH-2/TRIP13, which remodels the checkpoint effector Mad2 from an active conformation to an inactive one, controls checkpoint strength in C. elegans. When we manipulate embryos to decrease cell volume, PCH-2 is no longer required for the spindle checkpoint or recruitment of Mad2 at unattached kinetochores. This role in checkpoint strength is not limited to large cells: the stronger checkpoint in germline precursor cells also depends on PCH-2. PCH-2 is enriched in germline precursor cells and this enrichment relies on conserved factors that induce asymmetry in the early embryo. Finally, the stronger checkpoint in germline precursor cells is regulated by CMT-1, the ortholog of p31comet, which is required for both PCH-2s localization to unattached kinetochores and its enrichment in germline precursor cells. Thus, PCH-2, likely by regulating the availability of inactive Mad2 at and near unattached kinetochores, governs checkpoint strength. This role may be specifically relevant in scenarios where maintaining genomic stability is particularly challenging, such as in oocytes and early embryos enlarged for developmental competence and germline cells that maintain immortality.

cell biology↗

bioRxiv2018

Shugoshin is essential for meiotic prophase checkpoints in C. elegans

The conserved factor Shugoshin is dispensable in C. elegans for the two-step loss of sister chromatid cohesion that directs the proper segregation of meiotic chromosomes. We show that the C. elegans ortholog of Shugoshin, SGO-1, is required for checkpoint activity in meiotic prophase. This role in checkpoint function is similar to that of the meiotic chromosomal protein, HTP-3. Null sgo-1 mutants exhibit additional phenotypes similar to that of a partial loss of function allele of HTP-3: premature synaptonemal complex disassembly, the activation of alternate DNA repair pathways and an inability to recruit a conserved effector of the DNA damage pathway, HUS-1. SGO-1 localizes to pre-meiotic nuclei, when HTP-3 is present but not yet loaded onto chromosome axes, suggesting an early role in regulating meiotic chromosome metabolism. We propose that SGO-1 acts during pre-meiotic replication to ensure fully functional meiotic chromosome architecture, rendering these chromosomes competent for checkpoint activity and normal progression of meiotic recombination. Given that most research on Shugoshin has been focused on its regulation of sister chromatid cohesion in meiosis, this novel role may be conserved but previously uncharacterized in other organisms. Further, our findings expand the repertoire of Shugoshins functions beyond coordinating regulatory activities at the centromere.

cell biology↗