Biology subjects

Vermeersch, M.

Publications and source records attributed to Vermeersch, M..

10 recordsLinked to original sources

bioRxiv2026

BackBone Builder (B3): A modular Golden Gate standard with a compatible Agrobacterium parts library

Agrobacterium-mediated transformation relies on binary vectors in which T-DNA and virulence genes are maintained on separate replicons. While Golden Gate cloning has become standard for T-DNA assembly, no modular framework exists for systematic construction of Agrobacterium vector backbones. Here, we present BackBone Builder (B3), a Golden Gate-based platform for combinatorial backbone assembly. B3 uses the Type IIS enzyme PaqCI to minimize domestication and enables one-pot assembly of nine backbone modules plus a selectable cloning cassette. The system is compatible with GreenGate and remains independent of downstream cloning strategies. We generated a library of 42 backbone components, supporting a theoretical design space exceeding 370,000 constructs. A 4 x 4 origin-of-replication (ORI) matrix combining four Escherichia coli and four Agrobacterium ORIs assembled with 100% efficiency and functioned robustly in bacterial and plant contexts. Reporter expression reflected expected ORI-dependent patterns in E. coli, Agrobacterium, and Nicotiana benthamiana. A B3-derived maize transformation backbone achieved stable transformation efficiencies comparable to established vectors. B3 establishes a standardized and extensible framework for rational engineering of Agrobacterium binary vector architecture.

plant biology↗

bioRxiv2025

Secretory carrier membrane proteins assist with aquaporin trafficking in Arabidopsis.

Aquaporins belonging to the plasma membrane intrinsic protein (PIP) subfamily are channel proteins that control water flow across cells, allowing plants to rapidly adjust hydraulic conductivity and thereby sustain growth, gas exchange, and recovery from drought. We show that secretory carrier membrane proteins (SCAMP) regulate the abundance of the aquaporins and thereby water transport in Arabidopsis root cells. SCAMPs are evolutionarily conserved multi-spanning transmembrane proteins. In animal cells, they function in secretion, endocytosis and autophagy. Knowledge on their role in plants is restricted to localization and trafficking experiments in heterologous systems and few genetic perturbation experiments. Here, we analysed all five members of the Arabidopsis SCAMP family. We identified conserved tyrosine motifs assisting in transport to the plasma membrane and N-terminally located NPF motifs that are required for internalization. SCAMPs dimerize both at the plasma membrane and endosomes, and dimerization is required for their internalization. Functionally, several PIPs were identified as common targets of multiple SCAMP isoforms. Triple and quintuple scamp mutants show mild developmental delay under standard growth conditions, but they are less sensitive to drought-induced leaf wilting. This observation cannot be explained by altered stomatal dynamics or densities, nor by differential soil water content. However, scamp mutant root protoplasts contain less PIPs and swell less under hypotonic conditions compared to wild type, indicating reduced PIP plasma membrane levels. In conclusion, our research identifies the SCAMP membrane trafficking proteins as regulators of PIP abundance at the plasma membrane in root cells. We propose that the reduced PIP levels in the scamp mutants act as a priming mechanism, allowing them to respond better to conditions of reduced water availability.

plant biology↗

bioRxiv2024

Duodenal organoids from metabolic dysfunction-associated steatohepatitis patients exhibit altered digestive homeostasis

Background and AimsMetabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease that can lead to fibrosis, cirrhosis, and hepatocellular carcinoma. Though MASH is closely tied to metabolic risk factors, the underlying pathogenic mechanisms remain scarcely understood. Recent research underscores the importance of the gut-liver axis in its pathogenesis, an aspect less explored in human studies. Here, we investigated whether the duodenal epithelium of MASH patients, could exhibit intrinsic dysfunctions. MethodsDuodenal epithelial organoids were generated from 16 MASH patients and 14 healthy controls. Biopsies and patient-derived organoid transcriptomes were then analyzed to evaluate if specific intestinal pathways were differentially modulated in MASH subjects. Functional assays were performed to assess the duodenal epithelial digestive potential and barrier functionality. ResultsOrganoid formation efficiency was similar between control-derived epithelial organoids (CDEOs) and MASH-derived epithelial organoids (MDEOs) (71% and 69%, respectively). Despite global heterogeneity in growth patterns, MDEOs frequently exhibited cystic spheroid morphology. MDEOs displayed altered digestive homeostasis associated with reduced mature absorptive cell fate, but they retained their lipid metabolic capacity, possibly mediated by lipid oxidation in stem/progenitor cells. Additionally, MDEOs misexpressed components of tight and adherens junctions and desmosomes compared to controls. However, MDEOs maintained pore and leak pathway integrity, indicating that the duodenal epithelial barrier remained functionally preserved under tested conditions. ConclusionsThis study provides evidence that the duodenal epithelium of MASH patients exhibits significant alterations in its digestive and barrier functions. This study sheds light on the intricate dynamics of duodenal epithelial alterations in MASH, highlighting potential therapeutic avenues for restoring intestinal homeostasis.

cell biology↗

bioRxiv2022

Systematic optimization of Cas12a base editors in wheat and maize using the ITER platform

The ever-increasing number of CRISPR components creates a significant burden when developing new genome engineering tools. Plant biotechnology in particular has few high-throughput options to perform iterative design-build-test-learn cycles when creating new gene-editing reagents. We have established ITER (Iterative Testing of Editing Reagents) based on arrayed protoplast transfections and high-content imaging, allowing one optimization cycle - from design to results- within three weeks. We validated ITER in wheat and maize protoplasts using Cas9 cytosine and adenine base editors. Given that previous LbCas12a-ABEs have low or no activity in plants, we used ITER to develop an optimized LbCas12a-ABE. We show that the sequential improvement of five components -NLS, crRNA, LbCas12a, adenine deaminase and linker- led to a remarkable increase in ABE activity from almost undetectable levels to 40% on an extrachromosomal GFP reporter. We confirmed the activity of LbCas12a-ABE at endogenous targets and in stable wheat transformants and leveraged these improvements to develop a highly mutagenic LbCas12a nuclease and LbCas12a-CBE. Our data show that ITER is a sensitive, versatile, and high-throughput platform that can be harnessed to accelerate the development of genome editing technologies in plants.

synthetic biology↗

bioRxiv2022

Simple and efficient modification of Golden Gate design standards and parts using oligo stitching

The assembly of DNA parts is a critical aspect of contemporary biological research. Gibson assembly and Golden Gate cloning are two popular options. Here, we explore the use of single stranded DNA oligos with Gibson assembly to augment Golden Gate cloning workflows in a process called "oligo stitching". Our results show that oligo stitching can efficiently convert Golden Gate parts between different assembly standards and directly assemble incompatible Golden Gate parts without PCR amplification. Building on previous reports, we show that it can also be used to assemble de novo sequences. As a final application, we show that restriction enzyme recognition sites can be removed from plasmids and utilize the same concept to perform saturation mutagenesis. Given oligo stitchings versatility and high efficiency, we expect that it will be a useful addition to the molecular biologists toolbox. Abstract Graphic O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

synthetic biology↗

bioRxiv2021

Keratin dynamics govern the establishment of the maternal-fetal interface.

After implantation, the mouse embryo undergoes gastrulation and forms mesoderm and endoderm. Mesoderm participates in embryonic and extra-embryonic tissues including the amnion, yolk sac, chorion and allantois, the umbilical cord precursor. Extra-embryonic mesoderm is rich in intermediate filaments. Two-photon live imaging of keratin 8-eYFP knock-in embryos allowed recording nucleation and elongation of keratin filaments, which formed apical cables coordinated across multiple cells in amnion, allantois, and blood islands. Embryos lacking all keratins displayed a deflated exocoelomic cavity, a narrow thick amnion, and a short allantois, indicating a hitherto unknown role for keratin filaments in post-implantation extra-embryonic membranes morphogenesis. Single-cell RNA sequencing of mesoderm cells, microdissected amnion, chorion, and allantois provided an interactive atlas of transcriptomes with germ layer and regional information. Keratin 8high mesenchymal cells in contact with the exocoelom shared a cytoskeleton and adhesion expression profile that might explain the adaptation of extra-embryonic structures to the increasing mechanical pressure. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/438772v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@30dfe4org.highwire.dtl.DTLVardef@bb19d6org.highwire.dtl.DTLVardef@19d6758org.highwire.dtl.DTLVardef@ebd3a5_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2020

Distinct Cellular Strategies Determine Sensitivity to Mild Drought of Arabidopsis Natural Accessions

The world-wide distribution of Arabidopsis thaliana (Arabidopsis) accessions imposes different types of evolutionary pressures, which contributes to various responses of these accessions to environmental stresses. Drought stress responses have been well studied, particularly in Columbia, a common Arabidopsis accession. However, the reactions to drought stress are complex and our understanding of which of these responses contribute to the plants tolerance to mild drought is very limited. Here, we studied the mechanisms by which natural accessions react to mild drought at a physiological and molecular level during early leaf development. We documented variations in mild drought tolerance among natural accessions and used transcriptome sequencing of a drought-sensitive accession, ICE163, and a drought-tolerant accession, Yeg-1, to get insights into the mechanisms underlying this tolerance. This revealed that ICE163 preferentially induces jasmonates and anthocyanin-related pathways, which are beneficial in biotic stress defense, while Yeg-1 has a more pronounced activation of abscisic acid signaling, the classical abiotic stress response. Related physiological traits, including content of proline, anthocyanins and ROS, stomatal closure and cellular leaf parameters, were investigated and linked to the transcriptional responses. We conclude that most of these processes constitute general drought response mechanisms that are regulated similarly in drought-tolerant and -sensitive accessions. However, the capacity to close stomata and maintain cell expansion under mild drought appeared to be major factors that contribute to a better leaf growth under mild drought. One-sentence summaryThis paper demonstrates that an efficient closure of stomata and maintenance of cell expansion during drought conditions are crucial to maximally preserve plant growth during water deficit.

plant biology↗

bioRxiv2020

Long-term homeostatic T cell proliferation is driven by AMPK-dependent regulation of oxygen reactive species

The AMP-activated kinase (AMPK) is a major energy sensor metabolic enzyme that is activated early during T cell immune responses but its role in the generation of effector T cells is still controversial.Using both in vitro and in vivo models of T cell proliferation, we show herein that AMPK is dispensable for early TCR signaling and short-term proliferation but required for sustained long-term T cell proliferation and effector / memory T cell survival. In particular, AMPK promoted accumulation of effector / memory T cells in competitive homeostatic proliferation settings. Transplantation of AMPK-deficient hematopoïetic cells into allogeneic host recipients led to a reduced graft-versus-host disease, further bolstering a role for AMPK in the expansion and pathogenicity of effector T cells.Mechanistically, AMPK expression enhances the mitochondrial membrane potential of T cells, limits reactive oxygen species (ROS) production, and resolves ROS-mediated toxicity. Moreover, dampening ROS production alleviates the proliferative defect of AMPK-deficient T cells, therefore indicating a role for an AMPK-mediated ROS control of T cell fitness.Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗

bioRxiv2019

The deubiquitinating enzymes UBP12 and UBP13 negatively regulate the activity of the ubiquitin-dependent peptidases DA1, DAR1 and DAR2

Protein ubiquitination is a very diverse post-translational modification leading to protein degradation or delocalization, or altering protein activity. In Arabidopsis thaliana, two E3 ligases, BIG BROTHER (BB) and DA2, activate the latent peptidases DA1, DAR1 and DAR2 by mono-ubiquitination at multiple sites. Subsequently, these activated peptidases destabilize various positive regulators of growth. Here, we show that two ubiquitin-specific proteases, UBP12 and UBP13, deubiquitinate DA1, DAR1 and DAR2, hence reducing their peptidase activity. Overexpression of UBP12 or UBP13 strongly decreased leaf size and cell area, and resulted in lower ploidy levels. Mutants in which UBP12 and UBP13 were downregulated produced smaller leaves that contained fewer and smaller cells. Remarkably, neither UBP12 nor UBP13 were found to be cleavage substrates of the activated DA1. Our results therefore suggest that UBP12 and UBP13 work upstream of DA1, DAR1 and DAR2 to restrict their protease activity and hence fine-tune plant growth and development.

plant biology↗

bioRxiv2018

Mutations in nucleoporin NUP88 cause lethal fetal akinesia deformation sequence

Nucleoporins build the nuclear pore complex (NPC), which, as sole gate for nuclear-cytoplasmic exchange, are of outmost importance for normal cell function. Defects in the process of nucleocytoplasmic transport or in its machinery have been frequently described in human diseases, such as cancer and neurodegenerative disorders, but only in a few cases of developmental disorders. Here we report biallelic mutations in the nucleoporin NUP88 as a novel cause of lethal fetal akinesia deformation sequence (FADS) in two families. FADS comprises a spectrum of clinically and genetically heterogeneous disorders with congenital malformations related to impaired fetal movement. We show that genetic disruption of nup88 in zebrafish results in pleiotropic developmental defects reminiscent of those seen in affected human fetuses, including locomotor defects as well as defects at neuromuscular junctions. Phenotypic alterations become visible at distinct developmental stages, both in affected human fetuses and in zebrafish, whereas early stages of development are apparently normal. The zebrafish phenotypes caused by nup88 deficiency are only rescued by expressing wild-type nup88 and not the disease-linked mutant forms of nup88. Furthermore, using human and mouse cell lines as well as immunohistochemistry on fetal muscle tissue, we demonstrate that NUP88 depletion affects rapsyn, a key regulator of the muscle nicotinic acetylcholine receptor at the neuromuscular junction. Together, our studies provide the first characterization of NUP88 in vertebrate development, expand our understanding of the molecular events causing FADS, and suggest that variants in NUP88 should be investigated in cases of FADS.

cell biology↗