Biology subjects

Rawle, R. J.

Publications and source records attributed to Rawle, R. J..

8 recordsLinked to original sources

bioRxiv2025

Surface-attached model lipid membranes derived from human red blood cells

Red blood cells (RBCs) are the most abundant human cell type and interface interactions with the RBC membrane are at the heart of many processes relevant for human health, such as immune system modulation, interactions with foreign pathogens and with pharmacological drugs. To better study such membrane interface interactions, it would be useful to employ surface - attached model lipid membranes derived from RBCs to enable surface-sensitive biophysical and biochemical measurements. Here, we present approaches to prepare two such types of RBC-derived model lipid membranes - supported lipid bilayers (RBC-SLBs) and tethered RBC liposomes. We present data characterizing and validating these model membranes, including assessing lipid mobility, the distribution and mobility of the glycophorin A membrane protein, the functionality of the acetylcholinesterase enzyme, and the utility of the RBC-SLBs as binding targets for viral pathogens. We anticipate that our results and methodologies will be of interest to researchers studying molecular interactions with RBC membranes, as well as those interested in the engineering of model membrane platforms derived from other physiological membranes.

biochemistry↗

bioRxiv2025

Single virus lipid mixing study of Sendai virus provides insight into fusion mechanism

Single virus studies have proven useful to interrogate the entry mechanism for several viral families. Here, we employ a fluorescence microscopy-based single virus assay to study the fusion (lipid mixing) of Sendai virus to model membranes, the first for any paramyxovirus to our knowledge. We find that fusion wait times following binding are exponentially distributed, suggesting a single rate-limiting step. Compared to previously studied viruses, fusion is relatively slow (tens of minutes) and inefficient (only a small fraction of virions undergo fusion). Trypsin treatment of the virus or different viral receptors in the target alter the efficiency, although the wait time distribution remains unchanged in both cases. This provides constraints on the fusion mechanism and the identity of the rate-limiting step. Together, our data paints a picture of Sendai virus as a comparatively inefficient and slow fusion "machine" and sets the stage for investigation of other paramyxoviruses.

biophysics↗

bioRxiv2025

Single VLP lipid-mixing measurements confirm off-pathway state in dengue virus fusion mechanism

Dengue virus (DENV) is the causative agent of dengue fever and exerts a substantial healthcare burden worldwide. Like other flaviviruses, DENV must undergo membrane fusion with the host cell in order to initiate infection. This membrane fusion occurs following acidification during endocytosis and is pH dependent. Here, we interrogate whether the mechanism of DENV fusion contains an off-pathway state, such has been reported previously for two other flaviviruses - Zika virus and West Nile virus. To do this, we utilize single particle lipid mixing measurements of DENV virus-like particles (VLPs) to tethered liposomes, together with computational modeling inspired by chemical kinetics. By observing and then modeling the pH dependence of single VLP fusion kinetics, we provide evidence that the DENV fusion mechanism must contain an off-pathway state. Measuring the proportion of VLPs undergoing hemi-fusion over time, we also demonstrate that the off-pathway state appears to be slowly reversible over tens of minutes, at least for some virions. Additionally, we find that late endosomal anionic lipids do not appear to influence the off-pathway mechanism to any great extent. In conjunction with the prior reports on Zika virus and West Nile virus, this work indicates that an off-pathway fusion state may be a feature of flavivirus fusion more broadly. We also note that the platform and mechanistic model described in this study may be useful in elucidating the mechanism of action of small molecule inhibitors of flavivirus fusion developed by our group and others. Statement of SignificanceDengue virus (DENV) causes dengue fever and infects an estimated hundreds of millions of people annually. To date, there are no specific antiviral drugs for DENV and limited vaccination options, highlighting the need to better understand this important pathogen. In this report, we investigate the mechanism of DENV membrane fusion, an early step in the viral infectious cycle, using a mix of experimental techniques and computer simulations. We find strong evidence that the DENV fusion mechanism contains an off-pathway state, in which it can get stalled prior to membrane fusion. Understanding this off-pathway state could be an avenue to develop antiviral strategies against DENV and other related viruses.

biophysics↗

bioRxiv2024

Lipid loss and compositional change during preparation of liposomes by common biophysical methods

Liposomes are widely used as model lipid membrane platforms in many fields, ranging from basic biophysical studies to drug delivery and biotechnology applications. Various methods exist to prepare liposomes, but common procedures include thin-film hydration followed by extrusion, freeze-thaw, and/or sonication. These procedures have the potential to produce liposomes at specific concentrations and membrane compositions, and researchers often assume that the concentration and composition of their liposomes are similar to, if not identical, to what would be expected if no lipid loss occurred during preparation. However, lipid loss and concomitant biasing of lipid composition can in principle occur at any preparation step due to nonideal mixing, lipid-surface interactions, etc. Here, we report a straightforward method using HPLC-ELSD to quantify the lipid concentration and membrane composition of liposomes, and apply that method to study the preparation of simple POPC/cholesterol liposomes. We examine many common steps in liposome formation, including vortexing during re-suspension, hydration of the lipid film, extrusion, freeze-thaw, sonication, and the percentage of cholesterol in the starting mixture. We found that the resuspension step can play an outsized role in determining the overall lipid loss (up to [~]50% under seemingly rigorous procedures). The extrusion step yielded smaller lipid losses ([~]10-20%). Freeze-thaw and sonication could both be employed to improve lipid yields. Hydration times up to 60 minutes and increasing cholesterol concentrations up to 50 mole% had little influence on lipid recovery. Fortunately, even conditions with large lipid loss did not substantially influence the target membrane composition more than [~]5% under the conditions we tested. From our results, we identify best practices for producing maximum levels of lipid recovery and minimal changes to lipid composition during liposome preparation protocols. We expect our results can be leveraged for improved preparation of model membranes by researchers in many fields. Statement of SignificanceLiposomes are spherical lipid membranes that can be prepared by a variety of biophysical techniques. Researchers use liposomes in a variety of ways, including fundamental biophysical studies of lipid membranes, in drug delivery, drug formulation, and other biotechnology applications. In this report, we study the process to prepare liposomes by several common techniques and validate how reliable each technique is at producing consistent liposome concentrations and lipid compositions. We identify best practices for researchers to produce reliable liposome preparations.

biophysics↗

bioRxiv2021

Single virus assay reveals membrane determinants and mechanistic features of Sendai virus binding

Sendai virus (SeV, formally murine respirovirus) is a membrane-enveloped, negative sense RNA virus in the Paramyxoviridae family, and is closely related to human parainfluenza viruses. SeV has long been utilized as a model paramyxovirus and has recently gained attention as a viral vector candidate for both laboratory and clinical applications. To infect host cells, SeV must first bind to sialic-acid glycolipid or glycoprotein receptors on the host cell surface via its hemagglutinin-neuraminidase (HN) protein. Receptor binding induces a conformational change in HN, which allosterically triggers the viral fusion (F) protein to catalyze membrane fusion. While it is known that SeV binds to 2,3-linked sialic acid receptors, and there has been some study into the chemical requirements of those receptors, key mechanistic features of SeV binding remain unknown, in part because traditional approaches often convolve binding and fusion. Here, we develop and employ a fluorescence microscopy-based assay to observe SeV binding to supported lipid bilayers (SLBs) at the single particle level, which easily disentangles binding from fusion. Using this assay, we investigate mechanistic questions of SeV binding. We identify chemical structural features of ganglioside receptors that influence viral binding and demonstrate that binding is cooperative with respect to receptor density. We measure the characteristic decay time of unbinding and provide evidence supporting a "rolling" mechanism of viral mobility following receptor binding. We also study the dependence of binding on target cholesterol concentration. Interestingly, we find that while SeV binding shows striking parallels in cooperative binding with a prior report of Influenza A virus, it does not demonstrate a similar sensitivity to cholesterol concentration and receptor nano-cluster formation. STATEMENT OF SIGNIFICANCEParamyxoviruses are a family of membrane-enveloped viruses with many notable human and animal pathogens. In this study, we develop and use an assay to observe the initial step of infection - virus binding to the host membrane - for Sendai virus, the prototypical paramyxovirus, at the single virus level. This assay uses cell membrane mimics - supported lipid bilayers - as targets for virus binding to enable easy control of the membrane components with which the virus interacts. Using our assay, we gain insight into basic biophysical questions about Sendai virus binding, including the chemical characteristics of the receptor, the cooperative nature of binding, the influence of cholesterol, and the mechanism of viral mobility following binding.

biophysics↗

bioRxiv2020

Kinetic Modeling of West Nile Virus Fusion Indicates an Off-pathway State

West Nile virus (WNV) is a prominent mosquito-borne flavivirus that causes febrile illness in humans. To infect host cells, WNV virions first bind to plasma membrane receptors, then initiate membrane fusion following endocytosis. The viral transmembrane E protein, triggered by endosomal pH, catalyzes fusion while undergoing a dimer-to-trimer transition. Previously, single-particle WNV fusion data was interrogated with a stochastic cellular automaton simulation, which modeled the E proteins during the fusion process. The results supported a linear fusion mechanism, with E protein trimerization being rate-limiting. Here, we present corrections to the previous simulation, and apply them to the WNV fusion data. We observe that a linear mechanism is no longer sufficient to fit the data. Instead, an off-pathway state is necessary; these results are corroborated by chemical kinetics modeling. When compared with a similar Zika virus fusion model, this suggests that off-pathway fusion mechanisms may characterize flaviviruses more broadly.

biophysics↗

bioRxiv2020

Influenza hemagglutinin drives viral entry via two sequential intramembrane mechanisms

Enveloped viruses enter cells via a process of membrane fusion between the viral envelope and a cellular membrane. For influenza virus, mutational data have shown that the membrane-inserted portions of the hemagglutinin protein play a critical role in achieving fusion. In contrast to the relatively well-understood ectodomain, a predictive mechanistic understanding of the intramembrane mechanisms by which influenza hemagglutinin drives fusion has been elusive. We have used molecular dynamics simulations of fusion between a full-length hemagglutinin proteoliposome and a lipid bilayer to analyze these mechanisms. In our simulations, hemagglutinin first acts within the membrane to increase lipid tail protrusion and promote stalk formation and then acts to engage the distal leaflets of each membrane and promote stalk widening, curvature, and eventual fusion. These two sequential mechanisms, one occurring prior to stalk formation and one after, are consistent with experimental measurements we report of single-virus fusion kinetics to liposomes of different sizes. The resulting model also helps explain and integrate prior mutational and biophysical data, particularly the mutational sensitivity of the fusion peptide N-terminus and the length sensitivity of the transmembrane domain. We hypothesize that entry by other enveloped viruses may also utilize sequential processes of acyl tail exposure followed by membrane curvature and distal leaflet engagement.

biophysics↗

bioRxiv2019

Detecting and controlling dye effects in single-virus fusion experiments

Fluorescent dye-dequenching assays provide a powerful and versatile means to monitor membrane fusion events. They have been used in bulk assays, for measuring single events in live cells, and for detailed analysis of fusion kinetics for liposomal, viral, and cellular fusion processes; however, the dyes used also have the potential to perturb membrane fusion. Here, using single-virus measurements of influenza membrane fusion, we show that fluorescent membrane probes can alter both the efficiency and the kinetics of lipid mixing in a dye- and illumination-dependent manner. R18, a dye that is commonly used to monitor lipid mixing between membranes, is particularly prone to these effects, while Texas Red is somewhat less sensitive. R18 further undergoes photoconjugation to viral proteins in an illumination-dependent manner that correlates with its inactivation of viral fusion. These results demonstrate how fluorescent probes can perturb measurements of biological activity and provide both data and a method for determining minimally perturbative measurement conditions.\n\nStatement of SignificanceFluorescent dyes are powerful tools for labeling membranes and tracking subcellular objects, and fluorescence dequenching has further been used as a sensitive assay for membrane fusion. Here we show how incorporation of membrane dyes can perturb membrane fusion by influenza virus in a light-dependent manner. We provide a strategy to mitigate this by minimizing dye and light exposure. Finally, we show how in some cases these effects can be due to covalent reaction of some dyes with viral proteins upon illumination. These phenomena may be general and should be carefully controlled for in experiments using such labels.

biophysics↗