Biology subjects

Cooper, A. M.

Publications and source records attributed to Cooper, A. M..

5 recordsLinked to original sources

bioRxiv2026

Quantifying interleaflet coupling of phase behavior and observing anti-registered phases in asymmetric lipid bilayers

Model asymmetric lipid bilayers provide a powerful platform for probing how lateral phase behavior in one leaflet is coupled to that of the opposing leaflet. Here, we use calcium-induced hemifusion to generate asymmetric giant unilamellar vesicles (aGUVs) and investigate how lipid composition modulates interleaflet coupling of liquid-liquid phase separation. Symmetric GUVs composed of cholesterol, the high-melting lipid DPPC, and a low-melting phosphatidylcholine (either 14:1-PC or 16:1-PC) were prepared at compositions exhibiting coexisting liquid-ordered (Lo) and liquid-disordered (Ld) phases. Hemifusion with a uniformly mixed supported lipid bilayer composed of the low-melting lipid and cholesterol selectively altered the outer leaflet composition, producing aGUVs with controlled but variable asymmetry. Quantification of outer leaflet exchange using both probe-exit and probe-entry fluorescence measurements revealed substantial vesicle-to-vesicle variability within a given preparation, resulting in overlapping populations of phase-separated and uniformly mixed aGUVs. To account for this variability, we developed a population-based, coupled-distributions framework that enables robust determination of the asymmetric miscibility boundary, defined as the outer leaflet composition at which macroscopic phase separation is suppressed. Independent analyses of probe-exit and probe-entry data yielded consistent boundary locations. Comparing the two lipid systems, we find that aGUVs containing 14:1-PC require significantly greater outer leaflet exchange to abolish phase separation than those containing 16:1-PC. Only in the 14:1-PC system do we observe vesicles exhibiting coexistence of distinct anti-registered phases, a theoretically predicted but rarely observed regime consistent with large hydrophobic mismatch. By expressing both symmetric and asymmetric miscibility boundaries in a common fractional-coordinate framework, we introduce a phenomenological parameter, {Delta}*, that quantifies the direction and strength of interleaflet coupling of phase behavior. Together, these results demonstrate that modest changes in lipid chain length can markedly alter asymmetric miscibility boundaries and provide a quantitative link between experimental observations, leaflet dominance concepts, and coupled-leaflet theories of membrane organization. Statement of SignificanceMembrane asymmetry is a defining feature of eukaryotic cells whose influence on lateral membrane organization remains unclear. Using asymmetric giant vesicles, we find that coexisting liquid-ordered and liquid-disordered domains transition to a uniform appearance as saturated lipid in the outer leaflet is replaced with unsaturated lipid. The extent of exchange required to disrupt phase separation increased with acyl-chain length mismatch, revealing a compositional dependence of interleaflet coupling. In mixtures with greater hydrophobic mismatch, we also observe coexisting anti-registered phases predicted by theory but rarely observed experimentally, providing new constraints for models of coupled-leaflet behavior. By accounting for vesicle-to-vesicle compositional variability, these results provide a framework for measuring asymmetric miscibility boundaries and for connecting asymmetric membrane organization to lipid raft phenomena.

biophysics↗

bioRxiv2025

Nanodomain formation in lipid bilayers II: The influence of mixed-chain saturated lipids

An important class of lipids found in biological membranes are composed of two structurally different hydrocarbon chains. Among these, low-melting lipids possessing both a saturated and unsaturated chain have been intensely studied because of their biological abundance and influence on lipid rafts. In contrast, much less is known about the biophysical effects of mixed chains in high-melting lipids. Here, we investigated two such lipids--MSPC (14:0-18:0 PC) and SMPC (18:0-14:0 PC)--to determine how chain length mismatch and acyl chain position on the glycerol backbone influence lateral organization. We studied the temperature-and composition-dependent phase behavior of liposomes composed of either mixed-chain or symmetric-chain high-melting lipids plus DOPC and cholesterol, using techniques sensitive to domain formation at both microscopic and nanoscopic length scales. All studied mixtures exhibited liquid-ordered (Lo) + liquid-disordered (Ld) phase coexistence with domains that were visible in confocal microscopy experiments. FRET measurements showed that all mixtures also exhibited nanoscopic heterogeneity at temperatures above the microscopic miscibility transition temperature, and cryo-EM imaging further revealed bilayer thickness variation consistent with coexisting Ld and Lo phases. Both the microscopic miscibility transition temperature, m-Tmix, and its nanoscopic counterpart, nm-Tmix, were strongly correlated with the melting transition temperature of the saturated lipid; the sole exception was SMPC/DOPC/Chol, whose m-Tmix showed a significant negative deviation from the expected value, implying an enhanced propensity for nanoscopic phase separation in mixtures containing this high-melting species. These results point to strong effects of acyl chain position within mixed-chain high-TM lipids on the microscopic phase behavior of ternary mixtures.

biophysics↗

bioRxiv2025

The 3' Region of the ZPA Regulatory Sequence (ZRS) is required for activity and contains a critical E-box

1BackgroundDuring development, Hand2 and Hoxd13 transcription factors (TFs) regulate Sonic hedgehog (Shh) expression in the zone of polarizing activity (ZPA) in the distal posterior limb mesoderm. The ZPA regulatory sequence (ZRS) is a conserved, limb-specific enhancer that controls Shh expression. The ZRS can be divided into 5, central, and 3 subdomains, each with an E-box site that can bind basic helix-loop-helix (bHLH) TFs like Hand2. In addition, two Hoxd13 sites are present in the 5 and central subdomains. Hand2 purportedly binds the ZRS through the central E-box, and both Hand2 and Hoxd13 have been shown to activate the ZRS in vitro. We hypothesized that the central E-box was required for activity, while the other E-boxes and Hoxd13 sites localize ZRS activity to the distal posterior limb mesoderm. MethodsTo identify the functional role of each subdomain, we generated three ZRS fragments (5, central, and 3) and combined fragment constructs to test subdomain collective contributions. Additionally, we disrupted the five binding sites, alone or in concert, using site-directed mutagenesis. All ZRS constructs were cloned into a GFP reporter and evaluated in an in vivo chicken limb bioassay. We validated our findings using select ZRS constructs in transgenic mice. ResultsWe found that the 3 fragment was necessary for ZRS activity, while the 5 and central fragments had no activity alone or when combined. However, combining the 3 fragment with the 5 fragment restored robust activity. Further, mutation of all five binding sites markedly reduced ZRS activity. Reinstating each of the Hoxd13 sites restored focal activity, while restoring the 5 and central E-boxes had little effect. However, the 3 E-box proved sufficient for robust activity even in the absence of the other four binding sites. ConclusionsOur data indicate that the ZRS 3, not the central, subdomain is necessary for activity and contains the 3 E-box that Hand2 likely uses to induce Shh expression, while the 5 and central E-boxes appear to be inhibitory. Our data also suggest that the Hoxd13 binding sites promote localized activity within the ZPA.

developmental biology↗

bioRxiv2025

An Open-Source Image Analysis Method for Quantifying Reporter Fluorescence

Image analysis is a rapidly developing field that provides unique opportunities to characterize and quantify spatial information in images. We study cis-regulatory modules (CRMs), non-coding DNA regions that regulate gene expression, during development using fluorescent reporters in vivo. Characterizing CRM activity during development presents challenges including image segmentation into biologically relevant regions of interest that are not easily distinguishable via common segmentation methods, fluorophore band passing, and variable transfection undermine standardized analysis. To quantify and compare CRM activity levels, we compiled an open-source computer vision tool stack in the form of a Python-based Jupyter notebook and tested four analysis methods to assess their efficacy in quantifying CRM expression in limb development. This Jupyter notebook provides a reproducible, standardized workflow that can be adapted to numerous image analysis applications.

developmental biology↗

bioRxiv2018

Differential expression of an alternative splice variant of IL-12Rβ1 impacts early dissemination in the mouse and associates with disease outcome in both mouse and humans exposed to tuberculosis

Experimental mouse models of TB suggest that early events in the lung impact immunity. Early events in the human lung in response to TB are difficult to probe and their impact on disease outcome is unknown. We have shown in mouse that a secreted alternatively-spliced variant of IL-12R{beta}1, lacking the transmembrane domain and termed {Delta}TM-IL-12R{beta}1, promotes dendritic cell migration to the draining lymph node, augments T cell activation and limits dissemination of M. tuberculosis (Mtb). We show here that CBA/J and C3H/HeJ mice (both highly susceptible to Mtb) express higher levels of {Delta}TM-IL-12R{beta}1 than resistant C57BL6 mice and limit early dissemination of Mtb from the lungs. Both CD11c+ cells and T cells express {Delta}TM-IL-12R{beta}1 in humans, and mice unable to make {Delta}TM-IL-12R{beta}1 in either CD4 or CD11c expressing cells permit early dissemination from the lung. Analysis of publically available blood transcriptomes indicates that pulmonary TB is associated with high {Delta}TM-IL-12R{beta}1 expression and that of all IL-12 related signals, the {Delta}TM-IL-12R{beta}1 signal best predicts active disease. {Delta}TM-IL-12R{beta}1 expression reflects the heterogeneity of latent TB infection and has the capacity to discriminate between latent and active disease. In a new Chinese TB patient cohort, {Delta}TM-IL-12R{beta}1 effectively differentiates TB from latent TB, healthy controls and pneumonia patients. Finally, {Delta}TM-IL-12R{beta}1 expression drops in drug-treated individuals in the UK and China where infection pressure is low. We propose that {Delta}TM-IL-12R{beta}1 regulates early dissemination from the lung and that it has diagnostic potential and provides mechanistic insights into human TB.

immunology↗