Biology subjects

Borchers, C.

Publications and source records attributed to Borchers, C..

8 recordsLinked to original sources

bioRxiv2025

eIF4E assembly into C. elegans germ granules is essential for its repressive function

Metazoan germ cells form intracellular germ granules, cytoplasmic RNA-protein condensates that contain a variety of RNAs and proteins essential for germline identity, maintenance, and fertility. P granules are a type of C. elegans germ granule proposed to be sites of mRNA repression. Proper P granule assembly is dependent on PGL-1 and its granule-forming protein relatives. Numerous RNA-binding proteins localize to P granules, like the eIF4E mRNA cap binding homolog, IFE-1. IFE-1 directly interacts with PGL-1 in vivo and in vitro. The molecular function of P granules remains enigmatic. Here, PGL-1 was molecularly dissected in vivo to determine protein regions required for P granule assembly, binding partner recruitment, and germ cell development. A specific region in the PGL-1 C-terminus was necessary and sufficient for IFE-1 recruitment to P granules and for fertility. IFE-1 RNA targets were identified, and reporters of top gene targets were repressed in the adult germline. This repression was dependent on PGL-1 and its IFE-1 binding peptide. These findings provide evidence that IFE-1 and P granules are a factor and site of mRNA repression, respectively. This repression required IFE-1 assembly into P granules, supporting the model that RNA-protein condensate assembly is necessary for its biological and biochemical functions.

developmental biology↗

bioRxiv2025

Exogenous expression of a histone H3.3 isoform causes extranuclear divisions in the C. elegans intestine

Histone proteins condense DNA into chromatin and play significant roles in gene regulation. Mutations that alter histone function can disrupt critical gene expression programs and are implicated in driving cancer and other human diseases. Most mechanistic research on histones has been conducted in cell culture, mitotic tissues, or animal disease models rather than in normal tissue. Caenorhabditis elegans is a model organism with a well-established developmental program and conserved histone mechanisms shared with other metazoans. Prior work generated several single-copy histone reporters expressing exogenous histone protein in the intestine. Surprisingly, the histone H3.3 reporter worms possessed extra nuclei in the anterior intestine. This phenotype was incompletely penetrant and arose early during larval development. Mutations predicted to perturb histone H3.3 reporter expression or deposition into chromatin ameliorated the phenotype. DNA Fluorescence In situ Hybridization (FISH) was used to measure chromosome copy number and revealed approximately half the DNA content in the extra nuclei versus undivided controls, suggesting aberrant nuclear division during normal rounds of intestinal endoreduplication. Thus, these reporters serendipitously identified a distinct function of histone H3.3 in triggering aberrant nuclear division during animal development. Other histone isoforms may have previously unrecognized biological functions in terminally differentiated tissue alongside their roles in mitotic tissue.

developmental biology↗

bioRxiv2025

In vivo pulse-chase in C. elegans reveals intestinal histone turnover changes upon starvation

The ability to study protein dynamics and function in the authentic context of a multicellular organism is paramount to better understand biological phenomena in animal health and disease. Pulse-chase of self-labeling fusion protein tags provide the opportunity to label proteins of interest and track those proteins over time. There are currently several challenges associated with performing in vivo protein pulse-chase in animals, such as cost, reproducibility, and accurate detection methods. The C. elegans model organism has attributes that alleviate many of these challenges. This work tests the feasibility of applying the Halo modified enzyme (HaloTag) for in vivo protein pulse-chase in C. elegans. HaloTag intestinal histone reporters were created in the worm and used to demonstrate that reporter protein could be efficiently pulse-labeled by soaking animals in ligand. Labeled protein stability could be monitored over time by fluorescent confocal microscopy. Further investigation revealed reporter protein stability was dependent on the animals nutritional state. Chromatin Immunoprecipitation and sequencing (ChIP-seq) of the reporters showed incorporation in chromatin with little change hours into starvation, implying a lack of chromatin regulation at the time point tested. Collectively, this work presents a straightforward method to label and track proteins of interest in C. elegans that can address a multitude of biological questions surrounding protein stability and dynamics in this animal model.

biochemistry↗

bioRxiv2023

An effective C. elegans CRISPR training module for high school and undergraduate summer research experiences in molecular biology

Engaging in research experiences as a high school or undergraduate student interested in science, technology, engineering, and mathematics (STEM) is pivotal for their academic and professional development. A structured teaching framework can help cultivate a students curiosity and passion for learning and research. In this study, an effective eight-week training program has been created that encompasses fundamental molecular biology principles and hands-on laboratory activities. This curriculum focuses on using clustered regularly interspaced short palindromic repeats (CRISPR) gene editing in the Caenorhabditis elegans model organism. Through pre- and post-program assessments, substantial enhancements in students molecular biology proficiency and enthusiasm for scientific exploration was observed. Overall, this diligently crafted training module that employs C. elegans as an educational tool to instruct inexperienced students has demonstrated its accessibility and ability to engage students in molecular biology and gene editing methodologies.

scientific communication and education↗

bioRxiv2023

The E3 Ubiquitin Ligase Nedd4L Acts as a Checkpoint Against Activation in Quiescent Muscle Stem Cells

Adult stem cells play a critical role in tissue repair and maintenance. In tissues with slow turnover, including skeletal muscle, these cells are maintained in a mitotically quiescent state yet remain poised to re-enter the cell cycle to replenish themselves and regenerate the tissue. Using a multiomics approach we identify the PAX7/NEDD4L axis as a checkpoint against muscle stem cell activation in homeostatic skeletal muscle. Our findings demonstrate that PAX7 transcriptionally activates the E3 ubiquitin ligase Nedd4L and that the conditional genetic deletion of Nedd4L impairs muscle stem cell quiescence, with an upregulation of cell cycle and myogenic differentiation genes. Loss of Nedd4L in muscle stem cells results in the expression of DCX which is only expressed during their in vivo activation. Together, this data establishes that the ubiquitin proteasome system, mediated by Nedd4L, is a key regulator of the muscle stem cell quiescent state in non-injured skeletal muscle. Highlights- General inhibition of the ubiquitin proteasome system with MG132 results in muscle stem cells (MuSCs) breaking quiescence. - The E3 ubiquitin ligase Nedd4L is a transcriptional target of Pax7. - The Pax7/Nedd4l axis restricts MuSC activation in homeostatic skeletal muscle. - Genetic deletion of Nedd4L induces MuSCs transition towards activation.

cell biology↗

bioRxiv2021

Proteomic portraits reveal evolutionarily conserved and divergent responses to spinal cord injury

Despite the emergence of promising therapeutic approaches in preclinical studies, the failure of large-scale clinical trials leaves clinicians without effective treatments for acute spinal cord injury (SCI). These trials are hindered by their reliance on detailed neurological examinations to establish outcomes, which inflate the time and resources required for completion. Moreover, therapeutic development takes place in animal models whose relevance to human injury remains unclear. Here, we address these challenges through targeted proteomic analyses of CSF and serum samples from 111 acute SCI patients and, in parallel, a large animal (porcine) model of SCI. We develop protein biomarkers of injury severity and recovery, including a prognostic model of neurological improvement at six months with an AUC of 0.91, and validate these in an independent cohort. Through cross-species proteomic analyses, we dissect evolutionarily conserved and divergent aspects of the SCI response, and establish the CSF abundance of glial fibrillary acidic protein (GFAP) as a biochemical outcome measure in both humans and pigs. Our work opens up new avenues to catalyze translation by facilitating the evaluation of novel SCI therapies, while also providing a resource from which to direct future preclinical efforts.

neuroscience↗

bioRxiv2018

Cross-linking/Mass Spectrometry: A Community-Wide, Comparative Study Towards Establishing Best Practice Guidelines

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has largely increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats that are generally accepted in the field and that have shown to lead to high-quality results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. From the results obtained, common protocols will be established. Our study serves as basis for establishing best practice guidelines in the field for conducting cross-linking experiments, performing data analysis, and reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.

biochemistry↗

bioRxiv2018

MeCP2-E1 isoform is a dynamically expressed, weakly DNA-bound protein with different protein and DNA interactions compared to MeCP2-E2

MeCP2 - a chromatin-binding protein associated with Rett syndrome - has two main isoforms, MeCP2-E1 and MeCP2-E2, with 96% amino acid identity differing in a few N-terminal amino acid residues. Previous studies have shown brain region-specific expression of these isoforms which, in addition to their different cellular localization and differential expression during brain development, suggest they may also have non-overlapping molecular mechanisms. However, differential functions of MeCP2-E1 and E2 remain largely unexplored. Here, we show that the N-terminal domains (NTD) of MeCP2-E1 and E2 modulate the ability of the methyl binding domain (MBD) to interact with DNA as well as influencing the turnover rates, binding dynamics, response to nuclear depolarization, and circadian oscillations of the two isoforms. Our proteomics data indicate that both isoforms exhibit unique interacting protein partners. Moreover, genome-wide analysis using ChIP-seq provide evidence for a shared as well as a specific regulation of different sets of genes. Our findings provide insight into the functional complexity of MeCP2 by dissecting differential aspects of its two isoforms.\n\nSignificanceWhether the two E1 and E2 isoforms of MeCP2 have different structural and/or functional implications has been highly controversial and is not well known. Here we show that the relatively short N-terminal sequence variation between the two isoforms impinges them with an important DNA binding difference. Moreover, MeCP2-E1 and E2 exhibit a different cellular dynamic behavior and have some distinctive interacting partners. In addition, while sharing genome occupancy they specifically bind to several distinctive genes.

biochemistry↗