Biology subjects

Neal, A.

Publications and source records attributed to Neal, A..

8 recordsLinked to original sources

bioRxiv2026

Nuclear SUN2 coordinates endothelial cell-matrix interactions to regulate blood vessel homeostasis and barrier function

Vascular endothelial cells respond to environmental forces to remodel vessels during development and to achieve homeostasis, and mis-regulated responses lead to vascular dysfunction and disease. The nucleus participates in force transduction to cell-matrix junctions via the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex that resides in the nuclear envelope, but how these forces are regulated and relayed is incompletely understood. We found that the LINC complex protein SUN2 is required for proper endothelial cell-matrix interactions that occur far from the nucleus and affect angiogenic expansion, vascular responses to flow, and barrier integrity. Endothelial cells lacking SUN2 had inappropriate flow responses and reduced expression of flow-mediated transcription factors in vitro and in vivo. Expression of several matrix and adhesion genes was reduced in SUN2-depleted cells, leading to defective extracellular matrix, dysmorphic focal adhesions resistant to dynamic turnover, and disturbed cell-matrix force distribution. Mechanistically, nuclear SUN2 affected dynamic regulation of the microtubule cytoskeleton that correlated with matrix metalloprotease-dependent barrier dysfunction. These findings indicate that nuclear SUN2 establishes and maintains blood vessel homeostasis by controlling microtubule-mediated effects on focal adhesion turnover and extracellular matrix properties, with implications for cardiovascular aging and diseases such as Marfan syndrome that affect vessel wall integrity.

cell biology↗

bioRxiv2026

Hypoxia differentially affects coronary vessel formation during heart development

BACKGROUNDThe coronary vessel system is a dense and diverse network of arteries, veins and capillaries formed by endothelial cells from a variety of sources. While hypoxia is a known stimulus for angiogenic sprouting generally, the exact mechanisms by which hypoxia, and resultant increased VEGFA, influences vessel growth in the heart are not clearly delineated. METHODSWe used a genetic model to mimic hypoxia through ectopic stabilisation of myocardial HIF. This enabled us to study the consequences of hypoxia without vascular depletion. Changes in coronary ECs in these hearts relative to littermate controls were assessed by single cell RNA-sequencing, and by examining the activity of enhancer:reporter transgenes active in different coronary vessel beds downstream of distinct vascular regulatory pathways. RESULTSAnalysis of hypoxia-mimic hearts found increased angiogenic gene expression alongside expanded activity of the VEGFA-MEF2-driven angiogenic regulatory pathway in a pattern that indicated increased endocardial-derived angiogenic sprouting. Conversely, regulatory pathways specifically active in the sinus venosus (SV)-derived plexus showed little variance in response to stabilized HIF, and sprouting from the SV was not expanded. Although hypoxia and increased VEGFA levels have been previously linked to increased arterial differentiation, we saw little change in initial arterial EC differentiation in the experimental hearts. However, mature coronary arterial formation was delayed. CONCLUSIONSThese observations further emphasize a direct and specific link between hypoxia and endocardial coronary vessel sprouting and suggest a role of hypoxia/VEGFA in guiding coronary arterial coalescence.

developmental biology↗

bioRxiv2026

A transcriptional code controlling fluid shear stress-induced gene expression

The formation and health of the vascular system is dependent on fluid shear stress (FSS), a hemodynamic force exerted onto endothelium by flowing blood. FSS strongly induces the endothelial expression of Kruppel-like factor 2 (KLF2), an atheroprotective TF essential for vascular development and homeostasis. Despite its early and crucial role in the cascade of cardiovascular events triggered by FSS, the transcriptional mechanisms by which FSS regulates KLF2 expression remain unclear, although they are known to involve the widely expressed MEF2 proteins. Here, we identified and characterized two FSS-dependent enhancers for KLF2 which collectively recapitulate endogenous endothelial KLF2 expression, and determined the TFs contributing to their regulation. This analysis identified an essential and precisely spaced MEF2-TBP double motif also shared by the FSS-sensitive KLF2 promoter. MEF-TBP double motifs are extremely rare across the genome but were also found within regulatory elements of three other FSS-induced KLF genes, including KLF4. Although normally part of the basal transcriptional machinery, TBP specifically bound all KLF elements at the MEF-TBP double motifs in a FSS-dependent manner. Collectively, this work demonstrates a specific and targetable requirement for combined MEF2-TBP binding during FSS-induced gene activation. Significance statementBlood flow induces a force known as fluid shear stress (FSS) which is required for vascular development and for the health of the mature arterial system. One of the first endothelial responses to FSS is the induction of Kruppel-like transcription factors (KLFs). However, the mechanisms by which FSS activates KLF gene expression are incompletely understood. In this paper, we characterized all regulatory elements involved in driving FSS-induced expression of KLF2. This identified an essential MEF2-TBP double motif that was extremely rare across the genome, yet found within regulatory elements for multiple FSS-responsive KLF genes including KLF2 and KLF4. This MEF2- and TBP-bound motif therefore enables blood flow to specifically activate the cascade of cardiovascular responses necessary for atheroprotective gene expression.

developmental biology↗

bioRxiv2025

A divergent Plasmodium NEK4 acts as a key regulator driving the early events of meiosis

Meiosis is a conserved yet evolutionarily varied process underpinning sexual reproduction in eukaryotes. In the malaria parasite Plasmodium, meiosis is unconventional: it occurs immediately after fertilisation (post-zygotic) and must be coordinated with the transformation of the zygote into a motile ookinete. The mechanisms synchronising these meiotic and morphogenetic programmes remain unknown. Here, we identify the Plasmodium berghei NIMA-related kinase NEK4 as a key regulator that couples meiotic initiation with zygote morphogenesis. Using ultrastructure expansion microscopy, we show that NEK4 accumulates at the microtubule-organising centre (MTOC) and the apical polar complex (APC) shortly after fertilisation, preceding the assembly of perinuclear and cortical microtubules. We reveal that Plasmodium zygotes undergo MTOC-associated nuclear migration, analogous to the meiotic nuclear movement in fission yeast. Deletion of the Pbnek4 gene results in complete developmental arrest: MTOC duplication and microtubule formation are blocked, chromatin remains uncondensed, and nuclear migration and cell polarity fail to establish. Transcriptomic and phosphoproteomic analyses reveal that absence of NEK4 causes a collapse in transcriptional and phosphoregulatory networks governing meiosis and cytoskeletal organisation, leading to reduced expression and phosphorylation of important players, including HOP1, REC8, and AP2-O. These findings establish NEK4 as a key regulator driving meiotic entry and zygote maturation.

microbiology↗

bioRxiv2025

Dynamics and Regulation of mRNA Cap Recognition by Human eIF4F

Efficient eukaryotic messenger RNA translation requires dynamic collaboration between the three subunits of initiation factor 4F (eIF4F, eIF4E*G*A), which recognises and activates mRNA at its 5' cap structure for ribosome recruitment. Despite its high biological and pharmacological importance, the dynamics of full human eIF4F-mRNA engagement remain largely uncharacterised, hindering mechanistic understanding of translation initiation and its regulation. Here we observed human eIF4F activity with single-molecule fluorescence assays that directly visualise mRNA cap recognition by its eIF4E subunit. Unexpectedly, we find that inherently transient eIF4E-cap binding is repressed by full-length human eIF4G, predominantly through its C-terminus, representing an unanticipated role for eIF4G as a central rate-limiting factor in the eIF4F complex. This repression is relieved by nucleotide-bound eIF4A in the eIF4F heterotrimer, placing eIF4A as a crucial determinant of efficient cap recognition for translation. Molecular dynamics simulations reveal that electrostatic modulation of eIF4E-mRNA interaction allows eIF4G to control the cap-recognition frequency. Our findings also indicate that intrinsic eIF4F- mRNA dynamics are insufficient to support cap-tethered ribosomal scanning to locate translation start sites. They illuminate fundamental design-principle differences for the overall mechanism and division of labour among eIF4F subunits during mRNA recognition in humans and yeast.

biophysics↗

bioRxiv2024

Automated segmentation of epilepsy surgical resection cavities: comparison of four methods to manual segmentation

Accurate resection cavity segmentation on MRI is important for neuroimaging research involving epilepsy surgical outcomes. Manual segmentation, the gold standard, is highly labour intensive. Automated pipelines are an efficient potential solution; however, most have been developed for use following temporal epilepsy surgery. Our aim was to compare the accuracy of four automated segmentation pipelines following surgical resection in a mixed cohort of subjects following temporal or extra temporal epilepsy surgery. We identified 4 open-source automated segmentation pipelines. Epic-CHOP and ResectVol utilise SPM-12 within MATLAB, while Resseg and Deep Resection utilise 3D U-net convolutional neural networks. We manually segmented the resection cavity of 50 consecutive subjects who underwent epilepsy surgery (30 temporal, 20 extratemporal). We calculated Dice similarity coefficient (DSC) for each algorithm compared to the manual segmentation. No algorithm identified all resection cavities. ResectVol (n=44, 88%) and Epic-CHOP (n=43, 86%) were able to detect more resection cavities than Resseg (n=22, 44%, P<0.001) and Deep Resection (n=21, 42%, P<0.001). The SPM-based pipelines (Epic-CHOP and ResectVol) performed better than the deep learning-based pipelines in the overall and extratemporal surgery cohorts, however there was no difference between methods in the temporal surgery cohort. These pipelines could be applied to machine learning studies of outcome prediction to improve efficiency in pre-processing data, however human quality control is still required.

neuroscience↗

bioRxiv2024

Differential endothelial cell cycle status in postnatal retinal vessels revealed using a novel PIP-FUCCI reporter and zonation analysis

Cell cycle regulation is critical to blood vessel formation and function, but how the endothelial cell cycle integrates with vascular regulation is not well-understood, and available dynamic cell cycle reporters do not precisely distinguish all cell cycle stage transitions in vivo. Here we characterized a recently developed improved cell cycle reporter (PIP-FUCCI) that precisely delineates S phase and the S/G2 transition. Live image analysis of primary endothelial cells revealed predicted temporal changes and well-defined stage transitions. A new inducible mouse cell cycle reporter allele was selectively expressed in postnatal retinal endothelial cells upon Cre-mediated activation and predicted endothelial cell cycle status. We developed a semi-automated zonation program to define endothelial cell cycle status in spatially defined and developmentally distinct retinal areas and found predicted cell cycle stage differences in arteries, veins, and remodeled and angiogenic capillaries. Surprisingly, the predicted dearth of proliferative tip cells at the vascular front was accompanied by an unexpected enrichment for endothelial tip cells in G2, suggesting G2 stalling as a contribution to tip-cell arrest. Thus, this improved reporter precisely defines endothelial cell cycle status in vivo and reveals novel G2 regulation that may contribute to unique aspects of blood vessel network expansion.

cell biology↗

bioRxiv2022

Endothelial Caspase-9 Promotes Glial Changes, Inflammation, and Contrast Sensitivity Decline in Retinal Vascular Injury

Retinal glial cells-- microglia, astrocytes, and Muller glia--provide homeostatic support, regulate vascular blood flow, and react to injury by releasing inflammatory cytokines. Glial reactivity has been shown to be relevant for retinal vascular pathology and neuronal death. Non-apoptotic expression of endothelial caspase-9 (EC Casp9) was recently identified as a key mediator of retinal edema, hypoxic-ischemic injury, and neurodegeneration in retinal vein occlusion (RVO). In the current study we aimed to determine the glial responses that are modulated by EC Casp9 as a means to identify relevant neuro-immune mechanisms for the development of retinal edema and neurodegeneration. To this end we used a mouse model of RVO and a tamoxifen inducible EC Casp9 KO mouse line. We show that EC Casp9 leads to an increase in reactive microglia and to macrogliosis in a time-dependent manner. RVO induced an EC Casp9 dependent astroglial caspase-6 and cleavage of GFAP. Cytokine array analysis revealed that RVO increases expression of inflammatory cytokines out of which CX3CL1, IGF-1, IL-4, LIX, IL-1, M-CSF, TNF-, IL-1{beta}, IL-10, and VEGF-A, were regulated by EC Casp9. Moreover, we found that EC Casp9 deletion resulted in protection from contrast sensitivity decline one day post-RVO. These results demonstrate that caspase-9 in hypoxic endothelial cells regulates retinal inflammatory signaling in microglia, astrocytes and Muller cells and changes in visual function.

neuroscience↗