Viruses have a dual nature: particles are “passive substances” lacking chemical energy transformation, whereas infected cells are “active substances” turning-over energy. How passive viral substances convert to active substances, comprising viral replication and assembly compartments has been of intense interest to virologists, cell and molecular biologists and immunologists. Infection starts with virus entry into a susceptible cell and delivers the viral genome to the replication site. This is a multi-step process, and involves the cytoskeleton and associated motor proteins. Likewise, the egress of progeny virus particles from the replication site to the extracellular space is enhanced by the cytoskeleton and associated motor proteins. This overcomes the limitation of thermal diffusion, and transports virions and virion components, often in association with cellular organelles. This review explores how the analysis of viral trajectories informs about mechanisms of infection. We discuss the methodology enabling researchers to visualize single virions in cells by fluorescence imaging and tracking. Virus visualization and tracking are increasingly enhanced by computational analyses of virus trajectories as well as in silico modeling. Combined approaches reveal previously unrecognized features of virus-infected cells. Using select examples of complementary methodology, we highlight the role of actin filaments and microtubules, and their associated motors in virus infections. In-depth studies of single virion dynamics at high temporal and spatial resolutions thereby provide deep insight into virus infection processes, and are a basis for uncovering underlying mechanisms of how cells function.

Co-immunoprecipitation (co-IP) of protein complexes from cell lysates is widely used to study protein-protein interactions. However, establishing robust co-IP assays often involves considerable optimization. Moreover, co-IP results are frequently presented in non-quantitative ways. This protocol presents an optimized co-IP workflow with an analysis based on semi-quantitative immunoblot densitometry to increase reliability and reproducibility.

Dynamin GTPases are best studied for their role in the terminal membrane fission process of clathrin-mediated endocytosis (CME); but, they have also been proposed to regulate earlier stages of CME. Although highly enriched in neurons, dynamin-1 (Dyn1) is, in fact, widely expressed along with dynamin-2 (Dyn2), but inactivated in non-neuronal cells via phosphorylation by GSK3β kinase. Here, we study the differential, isoform-specific functions of Dyn1 and Dyn2 as regulators of CME. Endogenously expressed Dyn1 and Dyn2 were fluorescently-tagged either separately or together in two cell lines with contrasting Dyn1 expression levels. By quantitative live cell dual and triple-channel total internal reflection fluorescence microscopy we find that Dyn2 is more efficiently recruited to clathrin-coated pits (CCPs) than Dyn1, and that Dyn2, but not Dyn1 exhibits a burst of assembly prior to CCV formation. Activation of Dyn1 by acute inhibition of GSK3β results in more rapid endocytosis of transferrin receptors, increased rates of CCP initiation and decreased CCP lifetimes, but did not significantly affect the extent of Dyn1 recruitment to CCPs. Thus, activated Dyn1 can regulate early stages of CME even when present at low, substoichiometric levels relative to Dyn2, and apparently without assembly into supramolecular collar-like structures. Under physiological conditions Dyn1 is activated downstream of EGF-receptor signaling to alter CCP dynamics. We identify sorting nexin 9 (SNX9) as a preferred binding partner to activated Dyn1 that is partially required for Dyn1-dependent effects on early stages of CCP maturation. Together, we decouple regulatory and scission functions of dynamins and report a scission-independent, isoformspecific regulatory role for Dyn1 in clathrin-mediated endocytosis.