Biology subjects

Carrington, J.

Publications and source records attributed to Carrington, J..

8 recordsLinked to original sources

bioRxiv2026

Sequence adaptations satisfy the constraints of mitochondrial membrane protein evolution

Inner mitochondrial membrane proteins must be sufficiently hydrophilic to withstand aqueous exposure during translation and transit to the mitochondria. Meanwhile, their transmembrane segments must be sufficiently hydrophobic to stably embed in the lipid membrane. We hypothesized that sequence-level adaptations evolved to balance these constraints. Here, we integrate structure-informed evolutionary analyses of mitochondrial proteins with atomistic simulations and cell-based experiments to identify aliphatic-to-threonine substitutions (ATS) as a potential solution to these constraints. With high statistical confidence, this transmembrane segment-specific adaptation is recurrently and convergently observed throughout mitochondrial evolution. Conformational analyses show that threonine interacts with both water and the transmembrane helix backbone, thereby lowering hydrophobicity without destabilizing secondary structure. In the extremely hydrophobic ATP6 protein, reverting threonines to aliphatic residues disrupts mitochondrial targeting, while introducing threonines into a poorly targeted variant improves its mitochondrial localization. These findings have implications for mitochondrial genome evolution, the rational design of membrane proteins, and potentially mitochondrial gene therapy.

biochemistry↗

bioRxiv2025

Targeted and random mutagenesis of cassava brown streak disease susceptibility factors reveal molecular determinants of disease severity

Cassava brown streak disease (CBSD) is caused by cassava brown streak viruses (CBSVs) from the family Potyviridae. Potyvirid viral genome-linked protein (VPg) recruitment of host eukaryotic translation initiation factor 4E (eIF4E) proteins is a critical step in the viral life cycle. CBSV VPg interacts with all five cassava eIF4E-family members. Simultaneously knocking out eIF4E-family genes nCBP-1 and nCBP-2, in cultivar 60444, strongly reduces CBSD root symptoms and viral titer but does not result in complete resistance, likely due to gene family redundancy. To test for redundancy, we generated single and double mutants for each clade of the eIF4E gene family in farmer preferred cultivar TME419. Double mutants for the eIF(iso)4E and nCBP clades both exhibited reduced symptom severity, with ncbp-1 ncbp-2 having the strongest phenotype. A yeast two-hybrid screen for nCBP-2 mutants that lose VPg affinity identified fifty-one mutants, including an L51F mutant. This finding is consistent with one of the recovered cassava mutants that had a 6 amino acid deletion, including L51, in nCBP-2 and showed a reduction in symptoms relative to wild type. The data presented here suggest that generating mutations corresponding to L51F of nCBP-2 in multiple or all five cassava eIF4E proteins may lead to stronger resistance to CBSD while avoiding pleiotropic effects.

plant biology↗

bioRxiv2024

Direct mitochondrial import of lactate supports resilient carbohydrate oxidation

Lactate is the highest turnover circulating metabolite in mammals. While traditionally viewed as a waste product, lactate is an important energy source for many organs, but first must be oxidized to pyruvate for entry into the tricarboxylic acid cycle (TCA cycle). This reaction is thought to occur in the cytosol, with pyruvate subsequently transported into mitochondria via the mitochondrial pyruvate carrier (MPC). Using 13C stable isotope tracing, we demonstrated that lactate is oxidized in the myocardial tissue of mice even when the MPC is genetically deleted. This MPC-independent lactate import and mitochondrial oxidation is dependent upon the monocarboxylate transporter 1 (MCT1/Slc16a1). Mitochondria isolated from the myocardium without MCT1 exhibit a specific defect in mitochondrial lactate, but not pyruvate, metabolism. The import and subsequent mitochondrial oxidation of lactate by mitochondrial lactate dehydrogenase (LDH) acts as an electron shuttle, generating sufficient NADH to support respiration even when the TCA cycle is disrupted. In response to diverse cardiac insults, animals with hearts lacking MCT1 undergo rapid progression to heart failure with reduced ejection fraction. Thus, the mitochondrial import and oxidation of lactate enables carbohydrate entry into the TCA cycle to sustain cardiac energetics and maintain myocardial structure and function under stress conditions.

biochemistry↗

bioRxiv2022

Focal adhesion-based cell migration is differentially regulated in vivo versus in vitro by Paxillin phosphorylation

Focal adhesions are important subcellular structures that physically link the cell to the extracellular matrix (ECM), thus facilitating efficient cell migration. Although in vitro cell culture studies have provided a wealth of information regarding focal adhesion biology, it is critical to understand how focal adhesions are dynamically regulated in their native environment. We developed a zebrafish transplantation system in which we could efficiently visualize focal adhesion structures during single cell migration in vivo with high-resolution live cell imaging. By comparing focal adhesions between this in vivo system and the traditional in vitro cell culture model, we show differential regulation of a core focal adhesion protein, Paxillin. We find that a key site of phosphoregulation on Paxillin, tyrosine 118 (Y118), exhibits reduced phosphorylation in migrating cells in vivo in both zebrafish and mouse melanoma models, contrary to the pivotal role for this phosphorylation event in cell culture studies. Furthermore, direct modulation of this residue by site directed mutagenesis leads to opposite cell migration phenotypes in vivo versus in vitro in both migrating cancer cells and macrophages. Unexpectedly, expression of a non-phosphorylatable version of Y118-Paxillin promotes cell migration in vivo, despite inhibiting cell migration in the in vitro cell culture conditions. To further understand the mechanism of this regulation, we find that the upstream kinase, focal adhesion kinase (FAK), is downregulated in cells in vivo, and that cells expressing non-phosphorylatable Y118-Paxillin exhibit increased interactions between Paxillin and CRKII, an adaptor protein known to promote cell migration signaling. Collectively, our findings provide significant new insight into how focal adhesions are regulated in cells migrating in their native environment.

cell biology↗

bioRxiv2020

Hyperkalemia, not apoptosis, accurately predicts insect chilling injury

During prolonged or severe chilling, the majority of insects accrue chilling injuries that are typically quantified by scoring neuromuscular function after rewarming. In the cold, these chill susceptible insects, like the migratory locust (Locusta migratoria) suffer a loss of ion and water balance that is hypothesized to initiate cell death. Whether apoptotic or necrotic cell death pathways are responsible for this chilling injury is unclear. Here, we use a caspase-3 specific assay to indirectly quantify apoptosis in three locust tissues (muscle, nerves, and midgut) following prolonged chilling and recovery from an injury-inducing cold exposure. Furthermore, we obtain matching measurements of injury, hemolymph [K+], and muscle caspase-3 activity in individual locusts to gain further insight into mechanistic nature of chilling injury. We hypothesized that apoptotic cell death in both muscle and nerve tissue drives motor defects following cold exposure in insects, and that there would be a strong association between cold- induced injury, hyperkalemia, and muscle caspase-3 activity. We found a significant increase in muscle caspase-3 activity, but no such increase was observed in either nervous or gut tissue from the same animals, suggesting that chill injury primarily relates to apoptotic muscle cell death. However, the levels of chilling injury measured at the whole animal level prior to tissue sampling were strongly correlated with the degree of hemolymph hyperkalemia, but not apoptosis. These results support the notion that cold-induced ion balance disruption triggers cell death but also that apoptosis is not the main cell death pathway driving injury in the cold. Significance StatementTemperature has profound effects on animal fitness and sets limits to animal distribution. To understand and model insect responses to climate, we need to know how temperature sets limits to their survival. There is strong evidence that a collapse of ion and water balance occurs in insects in the cold, and it is generally held that the resulting cold injury is caused by activation of programmed cell death (apoptosis). Here, we directly test this idea and show for the first time that although the loss of ion balance is a strong predictor of individual survival outcomes, apoptosis is not the primary cause of cold-induced injury.

physiology↗

bioRxiv2019

Double UP: efficient in utero electroporation via internal controls

In Utero Electroporation is a powerful tool for testing the role of genes in neuronal migration, but this technique suffers from high degrees of variability due to multiple factors. Therefore, we developed Double UP, a novel approach that combines LoxP-flanked reporters and limiting Cre dosages to generate internal controls. This technique allows for more rigorous quantification of migration, while decreasing the number of animals, reagents and time to complete experiments.

neuroscience↗

bioRxiv2019

Discovery of New Mycoviral Genomes Within Publicly Available Fungal Transcriptomic Datasets

The distribution and diversity of RNA viruses in fungi is incompletely understood due to the often cryptic nature of mycoviral infections and the focused study of primarily pathogenic and/or economically important fungi. As most viruses that are known to infect fungi possess either single-stranded or double-stranded RNA genomes, transcriptomic data provides the opportunity to query for viruses in diverse fungal samples without any a priori knowledge of virus infection. Here we describe a systematic survey of all transcriptomic datasets from fungi belonging to the subphylum Pezizomycotina. Using a simple but effective computational pipeline that uses reads discarded during normal RNA-seq analyses, followed by identification of a viral RNA-dependent RNA polymerase (RdRP) motif in de novo assembled contigs, 59 viruses from 44 different fungi were identified. Among the viruses identified, 89% were determined to be new species and 68% are, to our knowledge, the first virus described from the fungal species. Comprehensive analyses of both nucleotide and inferred protein sequences characterize the phylogenetic relationships between these viruses and the known set of mycoviral sequences and support the classification of up to four new families and two new genera. Thus the results provide a deeper understanding of the scope of mycoviral diversity while also increasing the distribution of fungal hosts. Further, this study demonstrates the suitability of analyzing RNA-seq data to facilitate rapid discovery of new viruses.

genomics↗

bioRxiv2017

Raspberry Pi Powered Imaging for Plant Phenotyping

O_LIPremise of the study: Image-based phenomics is a powerful approach to capture and quantify plant diversity. However, commercial platforms that make consistent image acquisition easy are often cost-prohibitive. To make high-throughput phenotyping methods more accessible, low-cost microcomputers and cameras can be used to acquire plant image data.\nC_LIO_LIMethods and Results: We used low-cost Raspberry Pi computers and cameras to manage and capture plant image data. Detailed here are three different applications of Raspberry Pi controlled imaging platforms for seed and shoot imaging. Images obtained from each platform were suitable for extracting quantifiable plant traits (shape, area, height, color) en masse using open-source image processing software such as PlantCV.\nC_LIO_LIConclusion: This protocol describes three low-cost platforms for image acquisition that are useful for quantifying plant diversity. When coupled with open-source image processing tools, these imaging platforms provide viable low-cost solutions for incorporating high-throughput phenomics into a wide range of research programs.\nC_LI

plant biology↗