Visualizing Influenza Virus Membrane Fusion: Inhibition and Kinetics

The influenza virus hemagglutinin (HA) surface protein is a primary antigenic target for neutralization of viral infection. HA also mediates membrane fusion between the virus and a cell, which is the first critical step during infection. Traditional techniques to study infection neutralization by antibodies or the membrane fusion process rely on ensemble measurements, confounding the precise mechanism of infection neutralization and obscuring transient conformational intermediates. This dissertation describes advances made in a fluorescence microscopy-based single-particle fusion assay to overcome the limitations of ensemble measurements in these types of studies. Virus particles are labeled to visualize lipid mixing between a virus and a target membrane formed upon a glass or polymer support. Optionally, the viral lumen can be labeled to visualize the subsequent release of viral contents

Technologies bringing young Zebrafish from a niche field to the limelight

Fundamental life science and pharmaceutical research are continually striving to provide physiologically relevant context for their biological studies. Zebrafish present an opportunity for high-content screening (HCS) to bring a true in vivo model system to screening studies. Zebrafish embryos and young larvae are an economical, human-relevant model organism that are amenable to both genetic engineering and modification, and direct inspection via microscopy. The use of these organisms entails unique challenges that new technologies are overcoming, including artificial intelligence (AI). In this perspective article, we describe the state-of-the-art in terms of automated sample handling, imaging, and data analysis with zebrafish during early developmental stages. We highlight advances in orienting the embryos, including the use of robots, microfluidics, and creative multi-well plate solutions. Analyzing the micrographs in a fast, reliable fashion that maintains the anatomical context of the fluorescently labeled cells is a crucial step. Existing software solutions range from AI-driven commercial solutions to bespoke analysis algorithms. Deep learning appears to be a critical tool that researchers are only beginning to apply, but already facilitates many automated steps in the experimental workflow. Currently, such work has permitted the cellular quantification of multiple cell types in vivo, including stem cell responses to stress and drugs, neuronal myelination and macrophage behavior during inflammation and infection. We evaluate pro and cons of proprietary versus open-source methodologies for combining technologies into fully automated workflows of zebrafish studies. Zebrafish are poised to charge into HCS with ever-greater presence, bringing a new level of physiological context

Eph-ephrin signaling modulated by polymerization and condensation of receptors

Eph receptor signaling plays key roles in vertebrate tissue boundary formation, axonal pathfinding, and stem cell regeneration by steering cells to positions defined by its ligand ephrin. Some of the key events in Eph-ephrin signaling are understood: ephrin binding triggers the clustering of the Eph receptor, fostering transphosphorylation and signal transduction into the cell. However, a quantitative and mechanistic understanding of how the signal is processed by the recipient cell into precise and proportional responses is largely lacking. Studying Eph activation kinetics requires spatiotemporal data on the number and distribution of receptor oligomers, which is beyond the quantitative power offered by prevalent imaging methods. Here we describe an enhanced fluorescence fluctuation imaging analysis, which employs statistical resampling to measure the Eph receptor aggregation distribution within each pixel of an image. By performing this analysis over time courses extending tens of minutes, the information-rich 4D space (x, y, oligomerization, time) results were coupled to straightforward biophysical models of protein aggregation. This analysis reveals that Eph clustering can be explained by the combined contribution of polymerization of receptors into clusters, followed by their condensation into far larger aggregates. The modeling reveals that these two competing oligomerization mechanisms play distinct roles: polymerization mediates the activation of the receptor by assembling monomers into 6- to 8-mer oligomers; condensation of the preassembled oligomers into large clusters containing hundreds of monomers dampens the signaling. We propose that the polymerization–condensation dynamics creates mechanistic explanation for how cells properly respond to variable ligand concentrations and gradients

Using enhanced number and brightness to measure protein oligomerization dynamics in live cells

Protein dimerization and oligomerization are essential to most cellular functions, yet measurement of the size of these oligomers in live cells, especially when their size changes over time and space, remains a challenge. A commonly used approach for studying protein aggregates in cells is number and brightness (N&B), a fluorescence microscopy method that is capable of measuring the apparent average number of molecules and their oligomerization (brightness) in each pixel from a series of fluorescence microscopy images. We have recently expanded this approach in order to allow resampling of the raw data to resolve the statistical weighting of coexisting species within each pixel. This feature makes enhanced N&B (eN&B) optimal for capturing the temporal aspects of protein oligomerization when a distribution of oligomers shifts toward a larger central size over time. In this protocol, we demonstrate the application of eN&B by quantifying receptor clustering dynamics using electron-multiplying charge-coupled device (EMCCD)-based total internal reflection microscopy (TIRF) imaging. TIRF provides a superior signal-to-noise ratio, but we also provide guidelines for implementing eN&B in confocal microscopes. For each time point, eN&B requires the acquisition of 200 frames, and it takes a few seconds up to 2 min to complete a single time point. We provide an eN&B (and standard N&B) MATLAB software package amenable to any standard confocal or TIRF microscope. The software requires a high-RAM computer (64 Gb) to run and includes a photobleaching detrending algorithm, which allows extension of the live imaging for more than an hour

Visualizing influenza virus membrane fusion: Inhibition and kinetics

The influenza virus hemagglutinin (HA) surface protein is a primary antigenic target for neutralization of viral infection. HA also mediates membrane fusion between the virus and a cell, which is the first critical step during infection. Traditional techniques to study infection neutralization by antibodies or the membrane fusion process rely on ensemble measurements, confounding the precise mechanism of infection neutralization and obscuring transient conformational intermediates. This dissertation describes advances made in a fluorescence microscopy-based single-particle fusion assay to overcome the limitations of ensemble measurements in these types of studies. Virus particles are labeled to visualize lipid mixing between a virus and a target membrane formed upon a glass or polymer support. Optionally, the viral lumen can be labeled to visualize the subsequent release of viral contents. Recently isolated antibodies recognizing highly conserved epitopes on the HA protein that are critical for the protein's fusogenic capacity are able to neutralize infection from a broad range of influenza subtypes. Binding of these antibodies to a virus prior to inducing fusion with a target membrane resulted in inhibition of the fusion process, directly confirming one mechanism of infection neutralization. Fluorescently labeling the antibodies allowed for functional stoichiometric measurements that indicate a virion can be rendered non-fusogenic without the need for antibodies to bind and inactivate every HA present on the viral surface. A molecular model of fusion inhibition is proposed wherein coordination between neighboring HA is disrupted that leads to neutralization of the entire particle. Though polyunsaturated lipids are critical components of biological membranes that frequently undergo membrane fusion, their precise effects on the fusion process are poorly characterized. A fully automated data extraction program was designed and implemented to enhance the reproducibility of selecting fusing and non-fusing virions. Analysis of the resulting fusion kinetics revealed that the rate of fusion pore expansion is enhanced by high degrees of lipid unsaturation. During fusion at low pH, lower degrees of saturation resulted in the onset of a restricted hemifusion state. It is postulated that cholesterol and the HA fusion peptide create a molecular fence that confines lipid diffusion between the virus and target membranes

Visualization of Membrane Fusion, One Particle at a Time

<p>Protein-mediated fusion between phospholipid bilayers is a fundamental and necessary mechanism for many cellular processes. The short-lived nature of the intermediate states visited during fusion makes it challenging to capture precise kinetic information using classical, ensemble-averaging biophysical techniques. Recently, a number of single-particle fluorescence microscopy-based assays that allow researchers to obtain highly quantitative data about the fusion process by observing individual fusion events in real time have been developed. These assays depend upon changes in the acquired fluorescence signal to provide a direct readout for transitions between the various fusion intermediates. The resulting data yield meaningful and detailed kinetic information about the transitory states en route to productive membrane fusion. In this review, we highlight recent in vitro and in vivo studies of membrane fusion at the single-particle level in the contexts of viral membrane fusion and SNARE mediated synaptic vesicle fusion. These studies afford insight into mechanisms of coordination between fusion mediating proteins as well as coordination of the overall fusion process with other cellular processes. The development of single-particle approaches to investigate membrane fusion and their successful application to a number of model systems have resulted in a new experimental paradigm and open up considerable opportunities to extend these methods to other biological processes that involve membrane fusion.</p>