Jason J. Otterstrom

Mechanisms of Hemagglutinin Targeted Influenza Virus Neutralization

Human monoclonal antibodies have been identified which neutralize broad spectra of influenza A or B viruses. Here, we dissect the mechanisms by which such antibodies interfere with infectivity. We distinguish four mechanisms that link the conserved hemagglutinin (HA) epitopes of broadly neutralizing antibodies to critical processes in the viral life cycle. HA-stem binding antibodies can act intracellularly by blocking fusion between the viral and endosomal membranes and extracellularly by preventing the proteolytic activation of HA. HA-head binding antibodies prevent viral attachment and release. These insights into newly identified ways by which the human immune system can interfere with influenza virus infection may aid the development of novel universal vaccines and antivirals.

Visualization of membrane fusion, one particle at a time

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.

Relating influenza virus membrane fusion kinetics to stoichiometry of neutralizing antibodies at the single-particle level

Significance We determine the number of broadly neutralizing antibodies required to inhibit influenza virus membrane fusion by simultaneously observing individual viral particles undergoing fusion and counting the number of antibodies bound to them. The viral membrane fusion process is mediated by fusion proteins whose activity is blocked through the binding of these antibodies to evolutionarily conserved epitopes. Surprisingly, the number of antibodies required for inhibition is markedly lower than the number of fusion proteins present, indicating virus neutralization does not require saturation of epitope occupancy. Overall, our results support a model of membrane fusion requiring several fusion proteins working together in a coordinated, stochastic fashion, and the inhibition of this process through disruption of fusion protein coordination. The ability of antibodies binding the influenza hemagglutinin (HA) protein to neutralize viral infectivity is of key importance in the design of next-generation vaccines and for prophylactic and therapeutic use. The two antibodies CR6261 and CR8020 have recently been shown to efficiently neutralize influenza A infection by binding to and inhibiting the influenza A HA protein that is responsible for membrane fusion in the early steps of viral infection. Here, we use single-particle fluorescence microscopy to correlate the number of antibodies or antibody fragments (Fab) bound to an individual virion with the capacity of the same virus particle to undergo membrane fusion. To this end, individual, infectious virus particles bound by fluorescently labeled antibodies/Fab are visualized as they fuse to a planar, supported lipid bilayer. The fluorescence intensity arising from the virus-bound antibodies/Fab is used to determine the number of molecules attached to viral HA while a fluorescent marker in the viral membrane is used to simultaneously obtain kinetic information on the fusion process. We experimentally determine that the stoichiometry required for fusion inhibition by both antibody and Fab leaves large numbers of unbound HA epitopes on the viral surface. Kinetic measurements of the fusion process reveal that those few particles capable of fusion at high antibody/Fab coverage display significantly slower hemifusion kinetics. Overall, our results support a membrane fusion mechanism requiring the stochastic, coordinated action of multiple HA trimers and a model of fusion inhibition by stem-binding antibodies through disruption of this coordinated action.

3D motion of vesicles along microtubules helps them to circumvent obstacles in cells

ABSTRACT Vesicle transport is regulated at multiple levels, including regulation by scaffolding proteins and the cytoskeleton. This tight regulation is essential, since slowing or stoppage of transport can cause accumulation of obstacles and has been linked to diseases. Understanding the mechanisms by which transport is regulated as well as how motor proteins overcome obstacles can give important clues as to how these mechanisms break down in disease states. Here, we describe that the cytoskeleton architecture impacts transport in a vesicle-size-dependent manner, leading to pausing of vesicles larger than the separation of the microtubules. We further develop methods capable of following 3D transport processes in living cells. Using these methods, we show that vesicles move using two different modes along the microtubule. Off-axis motion, which leads to repositioning of the vesicle in 3D along the microtubule, correlates with the presence of steric obstacles and may help in circumventing them. Highlighted Article: We develop a new method to track vesicle dynamics in three dimensions and show that vesicles change their position while actively transporting along microtubules in living cells to overcome obstacles.

Eph-ephrin signaling modulated by polymerization and condensation of receptors

Significance Cell communication is a precisely orchestrated mechanism in which cell receptors translate extracellular cues into intracellular signals. The Eph receptors act as a model guidance system steering cells to defined positions by their ligand ephrin. However, we still lack a mechanistic understanding of how membrane receptors can read a wide range of ligand concentrations and gradients and integrate them into coherent cellular responses. Here we reveal the evolution of Eph aggregation upon ephrin stimulation with unprecedented resolution by extending current imaging methods. The results fit biophysical models of protein aggregation. In these models, two protein oligomerization modes, polymerization and condensation, correlate with the “on/off” switching of the receptor activation, providing a precise, proportional, and dynamic response to variable ephrin inputs. 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.

Quantitative high spatiotemporal imaging of biological processes

Super-resolution microscopy has revolutionized fluorescence imaging providing access to length scales that are much below the diffraction limit. The super-resolution methods have the potential for novel discoveries in biology. However, certain technical limitations must be overcome for this potential to be fulfilled. One of the main challenges is the use of super-resolution to study dynamic events in living cells. In addition, the ability to extract quantitative information from the super-resolution images is confounded by the complex photophysics that the fluorescent probes exhibit during the imaging. Here, we will review recent developments we have been implementing to overcome these challenges and introduce new steps in automated data acquisition towards high-throughput imaging.

Nudging Through a Nucleosome

Single-molecule data suggest that RNA polymerase II moves a small step forward only when its DNA template briefly unwraps from the histone core. Medieval monks feverishly transcribing Latin into Olde English would identify with the struggles that the eukaryotic RNA polymerase II complex must overcome in order to write DNA in the language of RNA. While theirs was a conceptual barrier, that of RNA polymerase II is quite physical and embodied by nucleosomes. Much like string wrapped around a spool, nucleosomes consist of a cylindrical protein core and DNA wrapped tightly around it. On page 626 of this issue, Hodges et al. (1) report single-molecule measurements that help to elucidate how the nucleosomal DNA is transcribed.