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Gaudin, Raphaël

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Gaudin

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Raphaël

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Gaudin, Raphaël
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Now showing 1 - 4 of 4
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    Sorting of small infectious virus particles by flow virometry reveals distinct infectivity profiles
    (2014) Gaudin, Raphaël; Barteneva, Natasha S.
    The nature and concentration of lipids and proteins at the surface of viruses are essential parameters for determining particle infectiveness. Historically, averaged bulk analysis of viral particles has been the primary method to quantitatively investigate these parameters, though this neglects heterogeneity within populations. Here we analyze the properties of Junin virus particles using a sensitive flow virometry assay and further sort virions, while conserving their infectivness. This method allows us to characterize the relationship between infectivity, virus size, and RNA content and to compare particles secreted by Vero cells with those from physiologically relevant human primary macrophages. Our study highlights significant differences in particle infectivity according to its nature, the type of producer cells and the lipid membrane composition at the budding site. Together, our results present the flow virometry assay as a powerful and versatile tool to define virus particle profiles.
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    Asymmetric formation of coated pits on dorsal and ventral surfaces at the leading edges of motile cells and on protrusions of immobile cells
    (The American Society for Cell Biology, 2015) Kural, Comert; Akatay, Ahmet Ata; Gaudin, Raphaël; Chen, Bi-Chang; Legant, Wesley R.; Betzig, Eric; Kirchhausen, Tom
    Clathrin/AP2-coated vesicles are the principal endocytic carriers originating at the plasma membrane. In the experiments reported here, we used spinning-disk confocal and lattice light-sheet microscopy to study the assembly dynamics of coated pits on the dorsal and ventral membranes of migrating U373 glioblastoma cells stably expressing AP2 tagged with enhanced green fluorescence (AP2-EGFP) and on lateral protrusions from immobile SUM159 breast carcinoma cells, gene-edited to express AP2-EGFP. On U373 cells, coated pits initiated on the dorsal membrane at the front of the lamellipodium and at the approximate boundary between the lamellipodium and lamella and continued to grow as they were swept back toward the cell body; coated pits were absent from the corresponding ventral membrane. We observed a similar dorsal/ventral asymmetry on membrane protrusions from SUM159 cells. Stationary coated pits formed and budded on the remainder of the dorsal and ventral surfaces of both types of cells. These observations support a previously proposed model that invokes net membrane deposition at the leading edge due to an imbalance between the endocytic and exocytic membrane flow at the front of a migrating cell.
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    Dynamin recruitment and membrane scission at the neck of a clathrin-coated pit
    (The American Society for Cell Biology, 2014) Cocucci, Emanuele; Gaudin, Raphaël; Kirchhausen, Tom
    Dynamin, the GTPase required for clathrin-mediated endocytosis, is recruited to clathrin-coated pits in two sequential phases. The first is associated with coated pit maturation; the second, with fission of the membrane neck of a coated pit. Using gene-edited cells that express dynamin2-EGFP instead of dynamin2 and live-cell TIRF imaging with single-molecule EGFP sensitivity and high temporal resolution, we detected the arrival of dynamin at coated pits and defined dynamin dimers as the preferred assembly unit. We also used live-cell spinning-disk confocal microscopy calibrated by single-molecule EGFP detection to determine the number of dynamins recruited to the coated pits. A large fraction of budding coated pits recruit between 26 and 40 dynamins (between 1 and 1.5 helical turns of a dynamin collar) during the recruitment phase associated with neck fission; 26 are enough for coated vesicle release in cells partially depleted of dynamin by RNA interference. We discuss how these results restrict models for the mechanism of dynamin-mediated membrane scission.
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    Superinfection exclusion is absent during acute Junin virus infection of Vero and A549 cells
    (Nature Publishing Group, 2015) Gaudin, Raphaël; Kirchhausen, Tomas
    Many viruses have evolved strategies of so-called “superinfection exclusion” to prevent re-infection of a cell that the same virus has already infected. Although Old World arenavirus infection results in down-regulation of its viral receptor and thus superinfection exclusion, whether New World arenaviruses have evolved such a mechanism remains unclear. Here we show that acute infection by the New World Junin virus (JUNV) failed to down-regulate the transferrin receptor and did not induce superinfection exclusion. We observed that Vero cells infected by a first round of JUNV (Candid1 strain) preserve an ability to internalize new incoming JUNV particles that is comparable to that of non-infected cells. Moreover, we developed a dual infection assay with the wild-type Candid1 JUNV and a recombinant JUNV-GFP virus to discriminate between first and second infections at the transcriptional and translational levels. We found that Vero and A549 cells already infected by JUNV were fully competent to transcribe viral RNA from a second round of infection. Furthermore, flow cytometry analysis of viral protein expression indicated that viral translation was normal, regardless of whether cells were previously infected or not. We conclude that in acutely infected cells, Junin virus lacks a superinfection exclusion mechanism.