Biology subjects

Sharp, L.

Publications and source records attributed to Sharp, L..

5 recordsLinked to original sources

bioRxiv2025

RainBar: Optical Barcoding for Pooled Live-Cell Imaging with Single-Cell Resolution

High-throughput pooled screening has advanced functional genomics, but most existing methods rely on endpoint sequencing and are blind to dynamic, time-resolved phenotypes. We developed RainBar (Rainbow Barcodes), an optical barcoding system that supports pooled live-cell imaging with single-cell resolution. RainBar uses lentiviral co-delivery of spectrally distinct nuclear and cytoplasmic fluorescent proteins to encode up to 64 unique perturbations per well. To mitigate barcode recombination and improve decoding accuracy, we employed single-template viral production, codon diversification, and a ratio-based spectral unmixing pipeline tailored to overlapping fluorophores. An inverted cytoplasmic segmentation approach and multilayer perceptron classifier enabled accurate barcode identification in both arrayed and pooled formats. As a proof of concept, we applied RainBar to dissect NF-{kappa}B signaling dynamics in epithelial cells. Live imaging of RelA translocation uncovered stimulus-specific kinetics: IL-1{beta} triggered rapid recovery, while TNF induced prolonged nuclear localization. In pooled CRISPRi screens, RainBar recovered known NF-{kappa}B regulators (e.g., IL1R1, MYD88, TNFRSF1A) and highlighted additional modulators, including the Ino80 chromatin remodeling complex subunits and KAT2A acetyltransferase. Together, these results position RainBar as a flexible platform for multiplexed, image-based functional genomics, with potential to reveal dynamic signaling architectures across diverse cellular contexts in live cells.

genomics↗

bioRxiv2023

Varying the position of phospholipid acyl chain unsaturation modulates hopanoid and sterol ordering

The cell membrane must balance mechanical stability with fluidity to function as both a barrier and an organizational platform. Key to this balance is the thermodynamic ordering of lipids. Most Eukaryotes employ sterols, which are uniquely capable of modulating lipid order to decouple membrane stability from fluidity. Ancient sterol analogues known as hopanoids are found in many bacteria and are proposed as ancestral ordering lipids. The juxtaposition of sterols and hopanoids in extant organisms prompts us to ask why both pathways persist, especially in light of their convergent ability to order lipids. We reveal that both hopanoids and sterols order unsaturated phospholipids differently based on the position of double bonds in the phospholipids acyl chain. We find that cholesterol and diplopterols methyl group distributions lead to distinct effects on unsaturated lipids. In Mesoplasma florum, diplopterols constrained ordering capacity reduces membrane resistance to osmotic stress, unlike cholesterol. These findings suggest cholesterols broader lipid ordering ability may have facilitated the exploration of a more diverse lipidomic landscape in eukaryotic membranes.

biophysics↗

bioRxiv2023

Anionic Phospholipids Control Mechanisms of GPCR-G Protein Recognition

G protein-coupled receptors (GPCRs) are embedded in phospholipids that strongly influence drug-stimulated signaling. Anionic lipids are particularly important for GPCR signaling complex formation, but a mechanism for this role is not understood. Using NMR spectroscopy, we visualized the impact of anionic lipids on the function-related conformational equilibria of the human A2A adenosine receptor (A2AAR) in bilayers containing defined mixtures of zwitterionic and anionic phospholipids. Anionic lipids primed the receptor to form complexes with G proteins through a conformational selection process. Without anionic lipids, signaling complex formation proceeded through a less favorable induced fit mechanism. In computational models, anionic lipids mimicked interactions between a G protein and positively charged residues in A2AAR at the receptor intracellular surface, stabilizing a pre-activated receptor conformation. Replacing these residues strikingly altered the receptor response to anionic lipids in experiments. High sequence conservation of the same residues among all GPCRs supports a general role for lipid-receptor charge complementarity in signaling.

biophysics↗

bioRxiv2020

A multilayered post-GWAS assessment on genetic susceptibility to pancreatic cancer

Pancreatic cancer (PC) is a complex disease in which both non-genetic and genetic factors interplay. To-date, 40 GWAS hits have been associated with PC risk in individuals of European descent, explaining 4.1% of the phenotypic variance. Here, we complemented a classical new PC GWAS (1D) with spatial autocorrelation analysis (2D) and Hi-C maps (3D) to gain additional insight into the inherited basis of PC. In-silico functional analysis of public genomic information allowed prioritization of potentially relevant candidate variants. We replicated 17/40 previous PC-GWAS hits and identified novel variants with potential biological functions. The spatial autocorrelation approach prioritized low MAF variants not detected by GWAS. These were further expanded via 3D interactions to 54 target regions with high functional relevance. This multi-step strategy, combined with an in-depth in-silico functional analysis, offers a comprehensive approach to advance the study of PC genetic susceptibility and could be applied to other diseases.

genetics↗

bioRxiv2019

Direct Binding of Phosphatidylglycerol at Specific Sites Modulates Desensitization of a Pentameric Ligand-Gated Ion Channel

Pentameric ligand-gated ion channels (pLGICs) are essential determinants of synaptic transmission, and are modulated by specific lipids including anionic phospholipids. The exact modulatory effect of anionic phospholipids in pLGICs and the mechanism of this effect are not well understood. Using native mass spectrometry, coarse-grained molecular dynamics simulations and functional assays, we show that the anionic phospholipid, 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), preferentially binds to and stabilizes the pLGIC, Erwinia ligand-gated ion channel (ELIC), and decreases ELIC desensitization. Mutations of five arginines located in the interfacial regions of the transmembrane domain (TMD) reduce POPG binding, and a subset of these mutations increase ELIC desensitization. In contrast, the L240A mutant known to decrease ELIC desensitization, increases POPG binding. The results support a mechanism by which POPG stabilizes the open state of ELIC relative to the desensitized state by direct binding at specific sites.

biophysics↗