Biology subjects

Lowe, N.

Publications and source records attributed to Lowe, N..

5 recordsLinked to original sources

bioRxiv2026

Fasciclin 2 Cooperates with Discs Large to Maintain Epithelial Architecture

Cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) coordinate adhesive interactions with intracellular organization during tissue morphogenesis. In the Drosophila follicular epithelium, epithelial maintenance depends on reintegration, a process in which mitotically displaced cells reincorporate into the epithelial monolayer. Previous work identified the IgCAMs Fasciclin 2 (Fas2) and Neuroglian (Nrg) as parallel, partially redundant regulators of reintegration, but the intracellular mechanisms linking adhesion to reintegration remained unclear. Here, we show that Fas2 supports reintegration through two mechanistically distinct modes: a transmembrane mode and a GPI-linked mode. Although both contribute to reintegration, the transmembrane mechanism is more effective and depends on stabilization of a cortical Fas2 pool through intracellular coupling. Using yeast two-hybrid screening, genetics, and fluorescence recovery after photobleaching (FRAP), we identify the scaffold protein Discs large (Dlg1) as a functional intracellular partner of transmembrane Fas2. Partial disruption of Dlg1 preferentially sensitizes epithelia in which the parallel Nrg-dependent reintegration mechanism is compromised, consistent with Dlg1 functioning primarily within the Fas2-dependent reintegration arm. While Dlg1 is not required for Fas2 membrane localization, Dlg1 disruption increases the mobile fraction of transmembrane Fas2, indicating that Dlg1 promotes retention of a stabilized cortical Fas2 pool. Together, these findings support a model in which epithelial reintegration depends on coordinated adhesion-scaffold coupling and reveal mechanistic parallels between epithelial reintegration and IgCAM-dependent processes in the developing nervous system.

cell biology↗

bioRxiv2024

Plasma-derived extracellular vesicles (EVs) as biomarkers of sepsis in burn patients via label-free Raman spectroscopy

Sepsis following burn trauma is a global complication with high mortality, with [~]60% of burn patient deaths resulting from infectious complications. Sepsis diagnosis is complicated by confounding clinical manifestations of the burn injury, and current biomarkers markers lack the sensitivity and specificity required for prompt treatment. Circulating extracellular vesicles (EVs) from patient liquid biopsy as biomarkers of sepsis due to their release by pathogens from bacterial biofilms and roles in subsequent immune response. This study applies Raman spectroscopy to patient plasma derived EVs for rapid, sensitive, and specific detection of sepsis in burn patients, achieving 97.5% sensitivity and 90.0% specificity. Furthermore, spectral differences between septic and non-septic burn patient EVs could be traced to specific glycoconjugates of bacterial strains associated with sepsis morbidity. This work illustrates the potential application of EVs as biomarkers in clinical burn trauma care, and establishes Raman analysis as a fast, label-free method to specifically identify features of bacterial EVs relevant to infection amongst the host background.

biophysics↗

bioRxiv2022

Repurposing the CRISPR-Cas9 System for Targeted Chromatin O-linked β-N- acetylglucosamine Editing

Eukaryotic gene transcription is controlled by many proteins, including the basal transcription machinery, epigenetic chromatin remodeling complexes, and transcription cofactors. Chromatin and genome-mapping consortia identified O-linked {beta}-N-acetylglucosamine (O-GlcNAc) as an abundant chromatin post-translational modification involved in numerous transcriptional processes, including RNA polymerase function, epigenetic dynamics, and transcription factor activity. Thus, O-GlcNAc regulation of cis-regulatory elements is essential for proper gene expression. O-GlcNAc is a single N-acetylglucosamine sugar attached to serine or threonine residues in nuclear, cytoplasmic, or mitochondrial proteins. Two enzymes cycle O-GlcNAc on or off protein; O-GlcNAc transferase (OGT) adds the modification, and O-GlcNAcase (OGA) removes it. O-GlcNAcylation responds to inputs from multiple metabolic and stress pathways including glucose, amino acid, fatty acid, and nucleotide metabolism. Therefore, O-GlcNAc acts as a sensor of cellular homeostasis able to link environmental conditions with gene transcription; however, decoding the precise function of millions of O-GlcNAc regulated elements remains challenging. Technologies to readily manipulate O-GlcNAcylation at specific cis-regulatory elements for functional analysis without pleiotropic consequences are lacking. We have employed novel CRISPR-based gene targeting tools to probe the function of O-GlcNAc regulated cis-elements. First, we developed a programmable CRISPR-Cas9-based targeting system. This was accomplished by fusing a catalytically dead Cas9 (dCas9) to O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA), which allows for highly specific O-GlcNAc manipulation at chromatin cis-regulatory elements. Previously, we demonstrated that O-GlcNAc plays a role in regulating human A{gamma}-globin gene expression by regulating CHD4 function and the formation of the NuRD (Nucleosome Remodeling and Deacetylase) complex at the -566 GATA repressor-binding site. Thus, as a proof of principle and to further explore the function of O-GlcNAc in{gamma} -globin gene transcription, we targeted both dCas9-OGT and -OGA fusion proteins to the A{gamma}-globin gene promoter. When dCas9-OGT or dCas9-OGA was targeted to the -566 GATA silencer site of the A{gamma}-globin promoter, gene expression decreased or increased, respectively. This data strongly correlates with our previous findings and implicates O-GlcNAc cycling in{gamma} -globin gene regulation. Importantly, this method can be employed to investigate O-GlcNAc events known to exist within the eukaryotic genome in a highly specific manner. Together, this tool will be fundamental in elucidating the function of O-GlcNAc in gene transcription.

biochemistry↗

bioRxiv2022

The spindle orienting machinery requires activation, not just localization

The orientation of the mitotic spindle at metaphase determines the placement of the daughter cells. Spindle orientation in animals typically relies on an evolutionarily conserved biological machine comprised of at least four proteins - called Partner of Inscuteable (Pins), Gi, Mushroom body defective (Mud), and Dynein in flies - that exert a pulling force on astral microtubules and reels the spindle into alignment. The canonical model for spindle orientation holds that the direction of pulling is determined by asymmetric placement of this machinery at the cell cortex. In most cell types, this placement is thought to be mediated by Pins, and a substantial body of literature is therefore devoted to identifying polarized cues that govern localized cortical enrichment of Pins. In Drosophila neuroblasts, for example, this cue is thought to be Inscuteable, which helps recruit Pins to the apical cell surface. In this study we revisit the canonical model. We find that spindle orientation in the follicular epithelium requires not only Pins localization but also activation, which relies on direct interaction between Pins and the multifunctional protein Discs large. This mechanism is distinct from the one mediated by Inscuteable, which we find also has an activating step. Together our results show that the canonical model is incomplete. Local enrichment of Pins is not sufficient to determine spindle orientation; an activation step is also necessary.

cell biology↗

bioRxiv2018

An alternative mode of epithelial polarity in the Drosophila midgut

Apical-basal polarity is essential for the formation and function of epithelial tissues, whereas loss of polarity is a hallmark of tumours. Studies in Drosophila have identified conserved polarity factors that define the apical (Crumbs, Stardust, Par-6, aPKC), junctional (Baz/Par-3) and basolateral (Scribbled, Discs large, Lgl) domains of epithelial cells (1, 2). Because these conserved factors mark equivalent domains in diverse vertebrate and invertebrate epithelial types, it is generally assumed that this system organises polarity in all epithelia. Here we show that this is not the case, as none of these canonical factors are required for the polarisation of the endodermal epithelium of the Drosophila adult midgut. Furthermore, unlike other Drosophila epithelia, the midgut forms occluding junctions above adherens junctions, as in vertebrates, and requires the integrin adhesion complex for polarity (3, 4). Thus, Drosophila contains two types of epithelia that polarise by different mechanisms. Since knock-outs of canonical polarity factors often have little effect on the polarity of vertebrate epithelia, this diversity of polarity mechanisms is likely to be conserved in other animals (5-8).

cell biology↗