Simultaneous tracking of many thousands of individual particles in live cells is possible now with the advent of high-density superresolution imaging methods. We present an approach to extract local biophysical properties of cell-particle interaction from such newly acquired large collection of data. Because classical methods do not keep the spatial localization of individual trajectories, it is not possible to access localized biophysical parameters. In contrast, by combining the high-density superresolution imaging data with the present analysis, we determine the local properties of protein dynamics. We specifically focus on AMPA receptor (AMPAR) trafficking and estimate the strength of their molecular interaction at the subdiffraction level in hippocampal dendrites. These interactions correspond to attracting potential wells of large size, showing that the high density of AMPARs is generated by physical interactions with an ensemble of cooperative membrane surface binding sites, rather than molecular crowding or aggregation, which is the case for the membrane viral glycoprotein VSVG. We further show that AMPARs can either be pushed in or out of dendritic spines. Finally, we characterize the recurrent step of influenza trajectories. To conclude, the present analysis allows the identification of the molecular organization responsible for the heterogeneities of random trajectories in cells.stochastic analysis of trajectories | dendritic spines and synapses | single particle tracking | confined diffusion R egulation of cellular physiological processes such as synaptic transmission, signal transduction relies on molecular interactions (binding and unbinding) at specific places and involves trafficking in confined local microdomains. The efficiency of these regulations crucially depends on the underlying molecular spatial organization, the study of which remains a daunting hurdle in cellular biology. Interestingly, superresolution light optical microscopy techniques for in vivo data (1-3) have allowed monitoring a large number of molecular trajectories at the single molecule level and at nanometer resolution, that can potentially reveal unique cellular organizational features. In the recent years, various techniques based on empirical characterization have emerged to track receptors (4), and estimating the mean square displacement (MSD) along isolated trajectories allowed to differentiate between free and confined diffusion (5, 6). In addition, although a large effort was dedicated to developing single molecule tracking algorithms (5, 7, 8), a general method for the analysis of the massive collection of data and for the extraction of quantitative local information is still lacking.In this article, we derive from the classical stochastic description at a molecular level, a method to extract biophysical features from high throughput superresolution data, associated with AMPA receptor (AMPAR) trafficking on neuronal cells. Indeed, neurons are organized in local microdomains characterized by morphological and functiona...

An efficient synthesis of sialic-acid-terminated glycerol dendron to chemically functionalize 2 nm and 14 nm gold nanoparticles (AuNPs) is described. These nanoparticles are highly stable and show high activity towards the inhibition of influenza virus infection. As the binding of the viral fusion protein hemagglutinin to the host cell surface is mediated by sialic acid receptors, a multivalent interaction with sialic-acid-functionalized AuNPs is expected to competitively inhibit viral infection. Electron microscopy techniques and biochemical analysis show a high binding affinity of the 14 nm AuNPs to hemagglutinin on the virus surface and, less efficiently, to isolated hemagglutinin. The functionalized AuNPs are nontoxic to the cells under the conditions studied. This approach allows a new type of molecular-imaging activity-correlation and is of particular relevance for further application in alternative antiviral therapy.

Influenza A virus strains adopt different host specificities mainly depending on their hemagglutinin (HA) protein. Via HA, the virus binds sialic acid receptors of the host cell and, upon endocytic uptake, HA triggers fusion between the viral envelope bilayer and the endosomal membrane by a low pH-induced conformational change leading to the release of the viral genome into the host cell cytoplasm. Both functions are crucial for viral infection enabling the genesis of new progeny virus. Adaptation to different hosts in vitro was shown to require mutations within HA altering the receptor binding and/or fusion behavior of the respective virus strain. Human adapted influenza virus strains (H1N1, H3N2, H2N2) as well as recent avian influenza virus strains (H5, H7 and H9 subtypes) which gained the ability to infect humans mostly contained mutations in the receptor binding site (RBS) of HA enabling increased binding affinity of these viruses to human type (α-2,6 linked sialic acid) receptors. Thus, the receptor binding specificity seems to be the major requirement for successful adaptation to the human host; however, the RBS is not the only determinant of host specificity. Increased binding to a certain cell type does not always correlate with infection efficiency. Furthermore, viruses carrying mutations in the RBS often resulted in reduced viral fitness and were still unable to transmit between mammals. Recently, the pH stability of HA was reported to affect the transmissibility of influenza viruses. This review summarizes recent findings on the adaptation of influenza A viruses to the human host and related amino acid substitutions resulting in altered receptor binding specificity and/or modulated fusion pH of HA. Furthermore, the role of these properties (receptor specificity and pH stability of HA) for adaptation to and transmissibility in the human host is discussed. This article is part of a Special Issue entitled: Viral Membrane Proteins -- Channels for Cellular Networking.

Membrane vesicle traffic to and from the plasma membrane is essential for cellular homeostasis in all eukaryotes. In plants, constitutive traffic to and from the plasma membrane has been implicated in maintaining the population of integral plasma-membrane proteins and its adjustment to a variety of hormonal and environmental stimuli. However, direct evidence for evoked and selective traffic has been lacking. Here, we report that the hormone abscisic acid (ABA), which controls ion transport and transpiration in plants under water stress, triggers the selective endocytosis of the KAT1 K+ channel protein in epidermal and guard cells. Endocytosis of the K+ channel from the plasma membrane initiates in concert with changes in K+ channel activities evoked by ABA and leads to sequestration of the K+ channel within an endosomal membrane pool that recycles back to the plasma membrane over a period of hours. Selective K+ channel endocytosis, sequestration, and recycling demonstrates a tight and dynamic control of the population of K+ channels at the plasma membrane as part of a key plant signaling and response mechanism, and the observations point to a role for channel traffic in adaptive changes in the capacity for osmotic solute flux of stomatal guard cells.

Lysosomes, enveloped viruses, as well as synaptic and secretory vesicles are all examples of natural nanocontainers (diameter ≈ 100 nm) which specifically rely on their lipid bilayer to protect and exchange their contents with the cell. We have applied methods primarily based on atomic force microscopy and finite element modeling that allow precise investigation of the mechanical properties of the influenza virus lipid envelope. The mechanical properties of small, spherical vesicles made from PR8 influenza lipids were probed by an atomic force microscopy tip applying forces up to 0.2 nN, which led to an elastic deformation up to 20%, on average. The liposome deformation was modeled using finite element methods to extract the lipid bilayer elastic properties. We found that influenza liposomes were softer than what would be expected for a gel phase bilayer and highly deformable: Consistent with previous suggestion that influenza lipids do not undergo a major phase transition, we observe that the stiffness of influenza liposomes increases gradually and weakly (within one order of magnitude) with temperature. Surprisingly, influenza liposomes were, in most cases, able to withstand wall-to-wall deformation, and forces >1 nN were generally required to puncture the influenza envelope, which is similar to viral protein shells. Hence, the choice of a highly flexible lipid envelope may provide as efficient a protection for a viral genome as a stiff protein shell.