Biology subjects

Vu, M. H.

Publications and source records attributed to Vu, M. H..

6 recordsLinked to original sources

bioRxiv2025

Comparative Proteomic Profiling of Receptor Kinase Signaling Reveals Key Trafficking Components Enforcing Plant Stomatal Development

Receptor kinases are pivotal for growth, development, and environmental response of plants. Yet, their regulatory mechanisms and spatial dynamics are still underexplored. The ERECTA-family receptor kinases coordinate diverse developmental processes, including stomatal development. To understand the proteomic landscape of the ERECTA-mediated signaling pathways, we here report comparative analyses of the ERECTA interactome and proximitome by epitope-tagged affinity-purification (ET-AP) and TurboID-based proximity labeling (TbID-PL) mass-spectrometry, respectively. While ET-AP successfully recovered receptor complex components (e.g., TOO MANY MOUTHS), TbID-PL effectively captured transient associations with the components of endosomal trafficking, i.e., clathrin-mediated endocytosis (CME) machinery. We further identify that specific subfamily members of phosphatidylinositol-binding clathrin assembly proteins (PICALMs) interact with and synergistically regulate ERECTA internalization. Mutations of these PICALMs impair ERECTA endocytosis and lead to excessive stomatal clustering by dampening the downstream signaling output. Taken together, our work provides a proteomic atlas of the ERECTA signaling network and suggests that timely removal of receptor kinase by the endocytosis machinery is essential for active signal transduction enforcing stomatal patterning.

plant biology↗

bioRxiv2025

Ligand-induced ubiquitination regulates endocytosis and homeostasis of the ERECTA receptor kinase for stomatal development

Stomata, valves on the plant epidermis, control gas and water vapor exchange. The patterning and spacing of stomata are primarily regulated by the ERECTA leucine-rich repeat receptor kinase (LRR-RK). Ubiquitination is a crucial mechanism to regulate the homeostasis of RKs by impacting their protein stability and localization. It has been shown that plant U-box ubiquitin E3 ligases, PUB30 and PUB31, act as key attenuators of the ERECTA signaling pathway. However, the molecular link between ubiquitination and function of ERECTA remains unclear. Here, we reveal that perception of the peptide ligand EPIDERMAL PATTERNING FACTOR2 (EPF2) by ERECTA induces the K63-linked ubiquitination of ERECTA mediated by PUB30 and PUB31. We further identify the specific ubiquitination sites within the juxtamembrane and kinase domains of ERECTA. Importantly, the site-directed mutagenesis of these K63-linked ubiquitination sites overly inhibited stomatal development, indicating that the ubiquitination-deficient ERECTA is hyperactive. The ubiquitination-deficient ERECTA stably accumulates at the plasma membrane and fails to internalize upon EPF2 application. Our findings thus uncover a mechanism by which ligand-induced ubiquitination orchestrates receptor homeostasis and endocytosis for proper stomatal patterning and differentiation.

plant biology↗

bioRxiv2024

Redirecting DNA repair for efficient CRISPR-Cas-based gene targeting in tomato

The CRISPR-Cas-based gene targeting (GT) method has enabled precise modifications of genomic DNA ranging from single base to several kilobase scales through homologous recombination (HR). In plant somatic cells, canonical nonhomologous end-joining (cNHEJ) is the predominant mechanism for repairing double-stranded breaks (DSBs), thus limiting the HR-mediated GT. In this study, we implemented various approaches to shift the repair pathway preference toward HR by using a dominant-negative KU80 mutant protein (KUDN) to disrupt the initiation of cNHEJ and enhance DSB end resection through nucleases. Our results show from 1.71- to 3.55-fold improvement of the GT efficiency at the callus stage and a more remarkable, up to 9.84-fold, increase in GT efficiency at two specific tomato loci, SlHKT1;2 and SlEPSPS1, when we screened transformants obtained from the KUDN-mediated cNHEJ suppression approach. With practical levels of efficiency, this enhanced KUDN-based GT tool successfully facilitated GT at an additional locus, SlCAB13. These findings provide a promising method for more efficient and precise plant breeding in the future.

plant biology↗

bioRxiv2021

A billion synthetic 3D-antibody-antigen complexes enable unconstrained machine-learning formalized investigation of antibody specificity prediction

Machine learning (ML) is a key technology for accurate prediction of antibody-antigen binding. Two orthogonal problems hinder the application of ML to antibody-specificity prediction and the benchmarking thereof: The lack of a unified ML formalization of immunological antibody specificity prediction problems and the unavailability of large-scale synthetic benchmarking datasets of real-world relevance. Here, we developed the Absolut! software suite that enables parameter-based unconstrained generation of synthetic lattice-based 3D-antibody-antigen binding structures with ground-truth access to conformational paratope, epitope, and affinity. We formalized common immunological antibody specificity prediction problems as ML tasks and confirmed that for both sequence and structure-based tasks, accuracy-based rankings of ML methods trained on experimental data hold for ML methods trained on Absolut!-generated data. The Absolut! framework thus enables real-world relevant development and benchmarking of ML strategies for biotherapeutics design. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/451258v3_ufig1.gif" ALT="Figure 1"> View larger version (86K): org.highwire.dtl.DTLVardef@4c3a6aorg.highwire.dtl.DTLVardef@8f1deborg.highwire.dtl.DTLVardef@1f55ec6org.highwire.dtl.DTLVardef@2135dd_HPS_FORMAT_FIGEXP M_FIG The software framework Absolut! enables (A,B) the generation of virtually arbitrarily large numbers of synthetic 3D-antibody-antigen structures, (C,D) the formalization of antibody specificity as machine learning (ML) tasks as well as the exploration of ML strategies for real-world antibody-antigen binding or paratope-epitope prediction. C_FIG HighlightsO_LISoftware framework Absolut! to generate an arbitrarily large number of synthetic 3D-antibody-antigen structures that contain biological layers of antibody-antigen binding complexity that render ML predictions challenging C_LIO_LIImmunological antibody specificity prediction problems formalized as machine learning tasks for which the in silico complexes are immediately usable as benchmark datasets C_LIO_LIExploration of machine learning prediction accuracy as a function of architecture, dataset size, choice of negatives, and sequence-structure encoding C_LIO_LIRelative ML performance learnt on Absolut! datasets transfers to experimental datasets C_LI

immunology↗

bioRxiv2021

α1-COP delivers sphingolipid modifiers and controls plasmodesmal callose deposition in Arabidopsis

Callose is a plant cell wall polymer in the form of {beta}-1,3-glucan, which regulates symplasmic channel size at plasmodesmata (PD). It plays a crucial role in a variety of processes in plants through the regulation of intercelluar symplasmic continuity. However, how to maintain callose homeostasis at PD in the molecular levels is poorly understood. To further elucidate the mechanism of PD callose homeostasis, we screened and identified an Arabidopsis mutant plant that exhibited excessive callose deposition at PD. Based on the Next-generation sequencing (NGS)-based mapping, other mutant allele analysis, and complementation assay, the mutated gene was shown to be 1-COP, which encodes a member of the COPI coatomer complex comprised of , {beta}, {beta}', {gamma}, {delta}, {varepsilon}, and {zeta} subunits. Since there is no report on the link between COPI and callose/PD, it was extremely curious to know the roles of 1-COP or COPI in PD regulation through callose deposition. Here, we report that loss-of-function of 1-COP directly elevates the callose accumulation at PD by affecting subcellular protein localization of callose degradation enzyme PdBG2. This process is linked to ERH1, an inositol phosphoryl ceramide synthase (IPCS), and glucosylceramide synthase (GCS) functions through physical interactions with the 1-COP protein. In addition, the loss-of-function of 1-COP also alters the subcellular localization of ERH1 and GCS proteins, results in a reduction of GlcCers and GlcHCers molecules, which are the key SL species for lipid raft formation. According to our findings, we propose that 1-COP protein, together with the SL modifiers controlling lipid raft compositions, regulates the function of GPI-anchored PD proteins and hence the callose turnover at PD and symplastic movement of biomolecules. Our findings provide the first key clue to link the COPI-mediated intracellular trafficking pathway to the callose-mediated intercellular signaling pathway through PD. One-sentence summaryPlant-specific coatomer protein functions as a negative regulator of callose accumulation by regulating the translocation of sphingolipid enzymes.

cell biology↗

bioRxiv2020

Glucosylhydroxyceramides Modulate Secretion Machinery of a Subset of Plasmodesmata Proteins and a Change in the Callose Accumulation

The plasma membranes encapsulated in the plasmodesmata (PDs) with symplasmic nano-channels contain abundant lipid rafts, which are enriched by sphingolipids and sterols. The attenuation of sterol compositions has demonstrated the role played by lipid raft integrity in the intercellular trafficking of glycosylphosphatidylinositol (GPI)-anchored PD proteins, particularly affecting in the callose enhancement. The presence of callose at PD is tightly attributed to the callose metabolic enzymes, callose synthases (CalSs) and {beta}-1,3-glucanases (BGs) in regulating callose accumulation and callose degradation, respectively. Sphingolipids have been implicated in signaling and membrane protein trafficking, however the underlying processes linking sphingolipid compositions to the control of symplasmic apertures remain unknown. A wide variety of sphingolipids in plants prompts us to investigate which sphingolipid molecules are important in regulating symplasmic apertures. Here, we demonstrate that perturbations of sphingolipid metabolism by introducing several potential sphingolipid (SL) pathway inhibitors and genetically modifying SL contents from two independent SL pathway mutants are able to modulate callose deposition to control symplasmic connectivity. Our data from pharmacological and genetic approaches show that the alteration in glucosylhydroxyceramides (GlcHCers) particularly disturb the secretory machinery for GPI-anchored PdBG2 protein, resulting in an over accumulated callose. Moreover, our results reveal that SL-enriched lipid rafts link symplasmic channeling to PD callose homeostasis by controlling the targeting of GPI-anchored PdBG2. This study elevates our understanding of the molecular linkage underlying intracellular trafficking and precise targeting to specific destination of GPI-anchored PD proteins incorporated with GlcHCers contents.

plant biology↗