Fusion of cell membranes is a ubiquitous biological process involved in vesicle fusion to membrane synapses, fertilization between sperm and egg cells, the merging of intracellular lysosomes, and membrane-enveloped virus fusion to endosomes1
. The fusion step is critical to the delivery of material across membranes. For example, in virus infection, membrane-enveloped viruses, such as influenza, infect cells via the endocytotic pathway, which necessitates the merging of the viral membrane with the endosomal membrane to pass viral genetic material into the cytosol. For many enveloped viruses, a drop in endosomal pH triggers conformational changes in the viral coat proteins required to initiate fusion between the viral and endosomal membranes2
. Characterization of virus fusion kinetics is important for a number of reasons beyond understanding fundamental fusion processes, such as classification of viral strain virulence and in the development of anti-fusogenic drugs3
. Yet, directly studying fusion in vivo
is difficult because it occurs inside intracellular compartments that are cumbersome to control and assay. Therefore, much of what is known about virus fusion has been determined using bulk or ensemble in vitro
fusion assays that report on the collective fusion behavior of many viruses to model membranes4–14
In bulk fusion assays, virus fusion is typically reported by a collective change in intensity resulting from fluorescence dequenching upon fusion of an ensemble of fluorescently-labeled viruses to model membranes4–14
. Fusion is initiated by acidification of the bulk solution. From the resulting temporal change in the fluorescence signal, some information about the kinetics of virus fusion can be obtained. Many studies of virus fusion to date have been conducted using this type of assay4–14
, but there are significant limitations with this approach. First, because individual events cannot be observed in this assay, viral binding and fusion cannot be distinguished from each other; this constraint impedes the separation of transport limitations from the fusion kinetics. Second, as the output signal is an aggregate of fluorescence changes resulting from many stochastic fusion events, only averaged information can be obtained from these assays; this drawback can obscure processes that occur at shorter timescales. Third, temporal limitations in uniformly acidifying the solution can spread initiation times of individual events, impacting signal response and its analysis. This limitation can reduce the temporal resolution of the measurements and obscure the sensitivity of initiating pH on kinetics15
Direct observation of individual virus fusion events circumvents many of the drawbacks of ensemble methods. Single particle virus fusion methods were first developed around the early 1990's16–18
, and have improved significantly since then with modern electronics and optics capable of single molecule fluorescence detection, microfluidic approaches for fluid handling, and new strategies for creating robust membranes. More recent work has provided information on the kinetics of intermediate steps of the fusion mechanism by employing total internal reflection fluorescence microscopy (TIRF)19
to detect individual virus fusion events to solid-supported lipid bilayers (SLBs) adsorbed to the walls of microfluidic devices20–21
. Although today's single particle virus fusion studies are easier to implement and can provide more insight into virus fusion than previously possible, two significant limitations of this approach remain: the rate at which acidification can be achieved in the confined space of the microfluidic device via acidic buffer exchange and the subsequent shearing that is imposed in the channel due to the flow.
Here we describe a method to achieve rapid acidification under quiescent conditions by integrating a photoisomerizable compound, o
-NBA) into our single particle fusion assay. o
-NBA donates a proton to the surrounding solution when illuminated with a 355 nm long wave ultraviolet laser22
with release times on the order of microseconds23
. This acidification method will hereafter be referred to as “proton uncaging”. The photolysis of o
-NBA to create a pH jump has been used in the investigation of the mechanisms of biological systems because it offers very high time resolution for kinetic measurements. In a review by McCray et al
, applications for uncaging are highlighted that include the study of active transport of proteins in muscle fibers, mechanistic studies of ion channels, and time-resolved responses of bacterial flagella motors to rapid changes in extracellular pH. Abbruzzetti et al
used it to examine the dissociation kinetics of histidines in Gu HCl-unfolded Fe(III) cytochrome C to increase the temporal resolution of data acquisition and allow for investigation over a wider temperature range. Saxena et al
studied the kinetics of proton transfer in green fluorescent protein (GFP) using o
-NBA, as a model system for characterizing the correlation between dynamics and function of proteins in general. Each of these examples illustrates the advantages of using a rapid pH jump to study pH-dependent kinetic processes. To the best of our knowledge, however, uncaging has not yet been employed for the study of pH-dependent fusion kinetics of enveloped viruses to host membranes.
There are several advantages of an uncaging strategy that are particularly beneficial for kinetic studies of viral fusion. First, the rate of release of the effector molecule (a proton) is much faster than rapid exchange of solution. Second, the effector molecule can be released close to the target, i.e., the fusion protein. Increasing the certainty of when the acidification occurs and ensuring the coordinated initiation of fusion events improves the resolution of fusion kinetics. Uncaging times are much faster than the protein conformational change for influenza hemagglutinin (HA) X:31 at the optimum triggering pH of ~ 5.026
. A third advantage is that the environment in which the dynamics are studied is unperturbed by external forces resulting from hydrodynamic flow. The quiescent surroundings more closely mimic the endosomal environment and eliminate the possibility of hydrodynamic deformation of protein structures, which could (slightly) change the conformation of the protein-receptor complex and impact fusion kinetics. Fourth, the absence of flow makes it possible to follow multiple processes (e.g., binding, hemifusion, pore formation) within an individual virion without it leaving the field of view.
By adjusting the concentration of o-NBA, the triggering pH immediately following uncaging can be tuned to achieve pH values within the range of physiological fusion pH for influenza. The characterization of the pH change in nanoliter volumes is a challenge, however, as the UV irradiation triggering proton uncaging also typically bleaches pH sensing reporter dyes. In order to be able to quantify pH a more UV resistant sensor probe thus had to be developed.