Biology subjects

Cutler, S.

Publications and source records attributed to Cutler, S..

8 recordsLinked to original sources

bioRxiv2026

Liverwort MpSDR4 regulates abscisic acid sensitivity and gemma cup growth

Complex traits often evolve by co-opting pre-existing genetic components. Orthologs of the transcriptional co-factor SEED DORMANCY 4 (SDR4) are found across seed-bearing and seed-free land plants. In flowering plants, loss-of-function mutants of sdr4 orthologs in rice and Arabidopsis exhibit opposite phenotypes in seed germination, a trait regulated by the phytohormone abscisic acid (ABA). We uncovered a major gene duplication event that closely preceded the origin of seed plants, by using a structure-informed molecular phylogenetic analysis. This duplication gave rise to the paralogous SDR4 and DIG/DIL clades, which are broadly retained across the seed plants. Investigating the role of SDR4 homologs in the common liverwort Marchantia polymorpha, a seed-free model organism with only a single SDR4-like homolog (MpSDR4), may provide insights into how SDR4 evolved to regulate dormancy and germination in other land plants. Loss-of-function mutants of Mpsdr4 and transcriptome profiling showed that MpSDR4 modulates ABA sensitivity, consistent with the idea that SDR4 subsequently evolved to regulate hormone responses related to seed germination, with additional functional divergence between monocots and eudicots. Furthermore, Mpsdr4 mutants exhibit elevated growth of gemma cups, contrasting the roles of SDR4 orthologs in asexual reproduction in liverwort with the regulation of seed traits in flowering plants.

plant biology↗

bioRxiv2025

Computational design of dynamic biosensors for emerging synthetic opioids

Nitazenes are an emergent class of synthetic opioids that rival or exceed fentanyl in their potency. These compounds have been detected internationally in illicit drugs and are the cause of increasing numbers of hospitalizations and overdoses. New analogs are consistently released, making detection challenging - new ways of testing a wide range of nitazenes and their metabolic products are urgently needed. Here, we developed a computational protocol to redesign the plant abscisic acid receptor PYR1 to bind diverse nitazenes and maintain its dynamic transduction mechanism. The best design has a low nanomolar limit of detection in vitro against nitazene and menitazene. Deep mutational scanning yielded sensors able to recognize a range of clinically relevant nitazenes and the common metabolic byproduct in complex biological matrices with limited cross-specificity against unrelated opioids. Application of protein design tools on privileged receptors like PYR1 may yield general sensors for a wide range of applications in vitro and in vivo.

biochemistry↗

bioRxiv2024

Robotic Imaging and Machine Learning Analysis of Seed Germination: Dissecting the Influence of ABA and DOG1 on Germination Uniformity

Seed germination research has evolved over the years, increasingly incorporating technology. Recent advances in phenotyping platforms have increased the accessibility of high throughput phenotyping technologies to more labs, leading to valuable insights into germination biology. These platforms benefit researchers by limiting manual labor and increasing the temporal resolution of imaging. Each of the platforms developed presents unique benefits and challenges, from scalability to price to computing resources. Performing experiments involving thousands of seeds remains a daunting task due to the limitations of current phenotyping platforms and image analysis pipelines. To overcome these challenges, we introduce SPENCER (Seed Phenotype Evaluation and Germination Curve Estimation Robot), a high-throughput phenotyping platform. SPENCER accommodates 32 rectangular petri plates, capable of assessing up to 8000 Arabidopsis seeds per experiment. Our design allows for high quality images while maintaining optimal humidity, crucial for precise germination assessment over longer experiments. The image analysis workflow incorporates advanced image analysis using semantic segmentation models trained for Arabidopsis and lettuce, providing researchers with accessible, reproducible, and efficient tools. We applied SPENCER to investigate the relative roles of DELAY OF GERMINATION 1 (DOG1) and abscisic acid (ABA) in Arabidopsis dormancy. DOG1 mutants exhibited rapid germination, whereas ANT application had a greater impact on the slower-germinating Ler ecotype. Our findings suggest that DOG1 plays a significant role in dormancy, particularly in non-dormant accessions, while ABAs influence is more pronounced under stress conditions. Additionally, we explored germination uniformity, another agriculurally relevant trait, observing parallels with germination timing. SPENCER offers a powerful and accessible tool for dissecting complex biological traits in conjunction with chemical and genetic manipulations. Its scalability and versatility make it suitable for large-scale genetic and chemical germination screens.

plant biology↗

bioRxiv2024

The ABA Receptor Antagonist Antabactin Restores Germination of Thermoinhibited Lettuce

Germination is critical to agricultural productivity because low germination rates and/or asynchronous germination negatively affect stand establishment and subsequent yields. Exposure to high temperatures during seed imbibition can decrease both germination synchrony and rates through an ABA-mediated process called thermoinhibition. Methods to reduce thermoinhibition would be agriculturally valuable, particularly with increasing global mean temperatures. Lettuce seed germination is particularly sensitive to high temperatures and is a classic system for studying thermoinhibition. Extensive evidence using mutants and carotenoid biosynthetic inhibitors (e.g. fluridone) has demonstrated that endogenous abscisic acid (ABA) biosynthesis is required for thermoinhibition in lettuce and Arabidopsis. In principle, ABA biosynthetic inhibitors could be used to block thermoinhibition, but carotenoid biosynthetic inhibitors are not well-suited for this application due to their herbicidal effects and current hydroxamate ABA biosynthetic inhibitors lack potency. Here we explore the potential of ABA receptor antagonism to disrupt thermoinhibition using Antabactin (ANT), a broad spectrum high-affinity receptor antagonist. We show low {micro}M ANT treatments (10{micro}M) during seed imbibition reduce thermoinhibition at temperatures of up to 40{degrees}C, demonstrating that ABA signaling is required for thermoinhibition. We further explored interactions between ANT and seed priming, which is used commercially to improve seed germination and reduce thermoinhibition and is achieved by partial hydration and subsequent desiccation of seeds. We show that primed lettuce seeds have improved germination rates at high temperatures, consistent with previous observations; moreover, seeds primed in the presence of ANT show further increases in germination at elevated temperatures, showing that the two treatments can be combined to improve lettuce seed germination. Our data demonstrate that ABA antagonists are candidate agrochemicals for mitigating the effects of high temperature on seed germination and stand establishment that may be of increasing importance due to climate change.

plant biology↗

bioRxiv2024

Functional genomic screening in Komagataella phaffii enabled by high-activity CRISPR-Cas9 library

CRISPR-based high-throughput genome-wide loss-of-function screens are a valuable approach to functional genetics and strain engineering. The yeast Komagataella phaffii is a host of particular interest in the biopharmaceutical industry and as a metabolic engineering host for proteins and metabolites. Here, we design and validate a highly active 6-fold coverage genome-wide sgRNA library for this biotechnologically important yeast containing 30,848 active sgRNAs targeting over 99% of its coding sequences. Conducting fitness screens in the absence of functional non-homologous end joining (NHEJ), the dominant DNA repair mechanism in K. phaffii, provides a quantitative means to assess the activity of each sgRNA in the library. This approach allows for the experimental validation of each guides targeting activity, leading to more precise screening outcomes. We used this approach to conduct growth screens with glucose as the sole carbon source and identify essential genes. Comparative analysis of the called gene sets identified a core set of K. phaffii essential genes, many of which relate to protein production, secretion, and glycosylation. The high activity, genome-wide CRISPR library developed here enables functional genomic screening in K. phaffii, applied here to gene essentiality classification, and promises to enable other genetic screens. HighlightsO_LIDesigned and validated a high activity genome-wide CRISPR-Cas9 library for K. phaffii C_LIO_LIDisabling NHEJ DNA repair enables the generation of genome-wide guide activity profiles C_LIO_LIActivity-corrected fitness screens identify a high confidence set of essential genes in K. phaffii C_LIO_LIProtein production, secretion, and glycosylation pathways are essential in K. phaffii but not in other yeasts C_LI

systems biology↗

bioRxiv2024

GMMA can stabilize proteins across different functional constraints

Stabilizing proteins without otherwise hampering their function is a central task in protein engineering and design. PYR1 is a plant hormone receptor that has been engineered to bind diverse small molecule ligands. We sought a set of generalized mutations that would provide stability without affecting functionality for PYR1 variants with diverse ligand-binding capabilities. To do this we used a global multi-mutant analysis (GMMA) approach, which can identify substitutions that have stabilizing effects and do not lower function. GMMA has the added benefit of finding substitutions that are stabilizing in different sequence contexts and we hypothesized that applying GMMA to PYR1 with different functionalities would identify this set of generalized mutations. Indeed, conducting FACS and deep sequencing of libraries for PYR1 variants with two different functionalities and applying a GMMA analysis identified 5 substitutions that, when inserted into four PYR1 variants that each bind a unique ligand, provided 2-6{degrees}C in increased thermal stability and no decrease in functionality.

biochemistry↗

bioRxiv2020

Systematic characterization of gene function in a photosynthetic organism

Photosynthetic organisms are essential for human life, yet most of their genes remain functionally uncharacterized. Single-celled photosynthetic model systems have the potential to accelerate our ability to connect genes to functions. Here, using a barcoded mutant library of the model eukaryotic alga Chlamydomonas reinhardtii, we determined the phenotypes of more than 58,000 mutants under more than 121 different environmental growth conditions and chemical treatments. 78% of genes are represented by at least one mutant that showed a phenotype, providing clues to the functions of thousands of genes. Mutant phenotypic profiles allow us to place known and previously uncharacterized genes into functional pathways such as DNA repair, photosynthesis, the CO2-concentrating mechanism, and ciliogenesis. We illustrate the value of this resource by validating novel phenotypes and gene functions, including the discovery of three novel components of a defense pathway that counteracts actin cytoskeleton inhibitors released by other organisms. The data also inform phenotype discovery in land plants: mutants in Arabidopsis thaliana genes exhibit similar phenotypes to those we observed in their Chlamydomonas homologs. We anticipate that this resource will guide the functional characterization of genes across the tree of life.

systems biology↗

bioRxiv2018

Isw2 and Ino80 chromatin remodeling factors regulate chromatin, replication, and copy number at the yeast ribosomal DNA locus

In the budding yeast Saccharomyces cerevisiae, ribosomal RNA genes are encoded in a highly repetitive tandem array referred to as the ribosomal DNA (rDNA) locus. The yeast rDNA is the site of a diverse set of DNA-dependent processes, including transcription of ribosomal RNAs by RNA Polymerases I and III, transcription of non-coding RNAs by RNA Polymerase II, DNA replication initiation, replication fork blocking, and recombination-mediated regulation of rDNA repeat copy number. All of this takes place in the context of chromatin, but relatively little is known about the roles played by ATP-dependent chromatin remodeling factors at the yeast rDNA. In this work, we report that the Isw2 and Ino80 chromatin remodeling factors are targeted to this highly repetitive locus. We characterize for the first time their function in modifying local chromatin structure, finding that loss of these factors affects the occupancy of nucleosomes in the 35S ribosomal RNA gene and the positioning of nucleosomes flanking the ribosomal origin of replication. In addition, we report that Isw2 and Ino80 promote efficient firing of the ribosomal origin of replication and facilitate the regulated increase of rDNA repeat copy number. This work significantly expands our understanding of the importance of ATP-dependent chromatin remodeling for rDNA biology.\n\nAuthor SummaryTo satisfy high cellular demand for ribosomes, genomes contain many copies of the genes encoding the RNA components of ribosomes. In the budding yeast Saccharomyces cerevisiae, these ribosomal RNA genes are located in the \"ribosomal DNA locus\", a highly repetitive array that contains approximately 150 copies of the same unit, in contrast to the single copies that suffice for most genes. This repetitive quality creates unique regulatory needs. Chromatin structure, the packaging and organization of DNA, is a critical determinant of DNA-dependent processes throughout the genome. ATP-dependent chromatin remodeling factors are important regulators of chromatin structure, and yet relatively little is known about how members of this class of protein affect DNA organization or behavior at the rDNA. In this work, we show that the Isw2 and Ino80 chromatin remodeling factors regulate two features of chromatin structure at the rDNA, the occupancy and the positioning of nucleosomes. In addition, we find that these factors regulate two critical processes that function uniquely at this locus: DNA replication originating from within the rDNA array, and the regulated increase of rDNA repeat copy number.

molecular biology↗