SummaryA global concern has emerged with the pandemic spread of Zika virus (ZIKV) infections that can cause severe neurological symptoms in adults and newborns. ZIKV is a positive-strand RNA virus replicating in virus-induced membranous replication factories (RFs). Here we used various imaging techniques to investigate the ultrastructural details of ZIKV RFs and their relationship with host cell organelles. Analyses of human hepatic cells and neural progenitor cells infected with ZIKV revealed endoplasmic reticulum (ER) membrane invaginations containing pore-like openings toward the cytosol, reminiscent to RFs in Dengue virus-infected cells. Both the MR766 African strain and the H/PF/2013 Asian strain, the latter linked to neurological diseases, induce RFs of similar architecture. Importantly, ZIKV infection causes a drastic reorganization of microtubules and intermediate filaments forming cage-like structures surrounding the viral RF. Consistently, ZIKV replication is suppressed by cytoskeleton-targeting drugs. Thus, ZIKV RFs are tightly linked to rearrangements of the host cell cytoskeleton.

Assembly and release of human immunodeficiency virus (HIV) occur at the plasma membrane of infected cells and are driven by the Gag polyprotein. Previous studies analyzed viral morphogenesis using biochemical methods and static images, while dynamic and kinetic information has been lacking until very recently. Using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated the assembly and release of fluorescently labeled HIV-1 at the plasma membrane of living cells with high time resolution. Gag assembled into discrete clusters corresponding to single virions. Formation of multiple particles from the same site was rarely observed. Using a photoconvertible fluorescent protein fused to Gag, we determined that assembly was nucleated preferentially by Gag molecules that had recently attached to the plasma membrane or arrived directly from the cytosol. Both membrane-bound and cytosol derived Gag polyproteins contributed to the growing bud. After their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over 1–2 hours. Assembly kinetics were rapid: the number of Gag molecules at a budding site increased, following a saturating exponential with a rate constant of ∼5×10−3 s−1, corresponding to 8–9 min for 90% completion of assembly for a single virion. Release of extracellular particles was observed at ∼1,500±700 s after the onset of assembly. The ability of the virus to recruit components of the cellular ESCRT machinery or to undergo proteolytic maturation, or the absence of Vpu did not significantly alter the assembly kinetics.

Plasmodium falciparum parasites in the merozoite stage invade human erythrocytes and cause malaria. Invasion requires multiple interactions between merozoite ligands and erythrocyte receptors. P. falciparum reticulocyte binding homolog 5 (PfRh5) forms a complex with the PfRh5-interacting protein (PfRipr) and Cysteine-rich protective antigen (CyRPA) and binds erythrocytes via the host receptor basigin. However, the specific role that PfRipr and CyRPA play during invasion is unclear. Using P. falciparum lines conditionally expressing PfRipr and CyRPA, we show that loss of PfRipr or CyRPA function blocks growth due to the inability of merozoites to invade erythrocytes. Super-resolution microscopy revealed that PfRipr, CyRPA, and PfRh5 colocalize at the junction between merozoites and erythrocytes during invasion. PfRipr, CyRPA, and PfRipr/CyRPA/PfRh5-basigin complex is required for triggering the Ca(2+) release and establishing the tight junction. Together, these results establish that the PfRh5/PfRipr/CyRPA complex is essential in the sequential molecular events leading to parasite invasion of human erythrocytes.

Walther et al. systematically fluorescently tag endogenous Condensin subunits and map their abundance, physical spacing, and mitotic dynamics by fluorescence correlation spectroscopy–calibrated live-cell imaging and superresolution microscopy. They propose a three-step hierarchical loop model of mitotic chromosome compaction.

A live-cell imaging study reveals that a positive feedback loop between dynamin and actin contributes to efficient endocytic membrane scission.

Human immunodeficiency virus (HIV) delivers its genome to a host cell through fusion of the viral envelope with a cellular membrane. While the viral and cellular proteins involved in entry have been analyzed in detail, the dynamics of virus-cell fusion are largely unknown. Single virus tracing (SVT) provides the unique opportunity to visualize viral particles in real time allowing direct observation of the dynamics of this stochastic process. For this purpose, we developed a double-coloured HIV derivative carrying a green fluorescent label attached to the viral matrix protein combined with a red label fused to the viral Vpr protein designed to distinguish between complete virions and subviral particles lacking MA after membrane fusion. We present here a detailed characterization of this novel tool together with exemplary live cell imaging studies, demonstrating its suitability for real-time analyses of HIV-cell interaction.

We demonstrate stimulated emission depletion (STED) microscopy of whole bacterial and eukaryotic cells using fluorogenic labels that reversibly bind to their target structure. A constant exchange of labels guarantees the removal of photobleached fluorophores and their replacement by intact fluorophores, thereby circumventing bleaching-related limitations of STED super-resolution imaging. We achieve a constant labeling density and demonstrate a fluorescence signal for long and theoretically unlimited acquisition times. Using this concept, we demonstrate whole-cell, 3D, multicolor, and live-cell STED microscopy.

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