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Quantitative live cell imaging reveals influenza virus manipulation of Rab11A transport through reduced dynein association.

ABSTRACT: Assembly of infectious influenza A viruses (IAV) is a complex process involving transport from the nucleus to the plasma membrane. Rab11A-containing recycling endosomes have been identified as a platform for intracellular transport of viral RNA (vRNA). Here, using high spatiotemporal resolution light-sheet microscopy (~1.4 volumes/second, 330 nm isotropic resolution), we quantify Rab11A and vRNA movement in live cells during IAV infection and report that IAV infection decreases speed and increases arrest of Rab11A. Unexpectedly, infection with respiratory syncytial virus alters Rab11A motion in a manner opposite to IAV, suggesting that Rab11A is a common host component that is differentially manipulated by respiratory RNA viruses. Using two-color imaging we demonstrate co-transport of Rab11A and IAV vRNA in infected cells and provide direct evidence that vRNA-associated Rab11A have altered transport. The mechanism of altered Rab11A movement is likely related to a decrease in dynein motors bound to Rab11A vesicles during IAV infection.

SUBMITTER: Bhagwat AR

PROVIDER: S-EPMC6946661 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Quantitative Live Cell Imaging Reveals Differential Manipulation of Rab11A Transport Pathways by Influenza virus and RSV

Project description:Assembly of infectious influenza A viruses (IAV) is a complex process involving transport from the nucleus to the plasma membrane. Rab11A containing recycling endosomes have been identified as a platform for intracellular transport of viral RNA (vRNA). Using high spatiotemporal resolution light-sheet microscopy (~1.4 volumes/s, 330 nm isotropic spatial resolution), we quantify Rab11A and vRNA movement in live cells during IAV infection. Here we report that IAV infection decreases speed and increases arrest of Rab11A. Unexpectedly, infection with RSV alters Rab11A motion in a manner opposite to IAV, suggesting that Rab11A is a common host component that is differentially manipulated by respiratory RNA viruses. Using two-color imaging we demonstrate co-transport between Rab11A and IAV vRNA in infected cells and provide direct evidence that vRNA-associated Rab11A have altered transport. The mechanism of altered Rab11A movement is likely related to a decrease in dynein motors bound to Rab11A vesicles during IAV infection.

2020-01-14 | PXD016331 | Pride

Quantitative live-cell imaging of secretion activity reveals dynamic immune responses

Project description:Quantification of cytokine secretion has facilitated advances in the field of immunology, yet the dynamic and varied secretion profiles of individual cells, particularly those obtained from limited human samples, remain obscure. Herein, we introduce a new technology for quantitative live-cell imaging of secretion activity (qLCI-S) that enables high-throughput and dual-color monitoring of secretion activity at the single-cell level over several days, followed by transcriptome analysis of individual cells based on their phenotype. The efficacy of qLCI-S was demonstrated by visualizing the characteristic temporal pattern of cytokine secretion of group 2 innate lymphoid cells, which constitute less than 0.01% of human peripheral blood mononuclear cells, and by revealing minor subpopulations with enhanced cytokine production. The underlying mechanism of this feature was linked to the gene expression of stimuli receptors. This new technology paves the way for exploring gene expression signatures linked to the spatiotemporal dynamic nature of various secretory functions.

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Quantitative microspectroscopic imaging reveals viral and cellular RNA helicase interactions in live cells.

Project description:Human cells detect RNA viruses through a set of helicases called RIG-I-like receptors (RLRs) that initiate the interferon response via a mitochondrial signaling complex. Many RNA viruses also encode helicases, which are sometimes covalently linked to proteases that cleave signaling proteins. One unresolved question is how RLRs interact with each other and with viral proteins in cells. This study examined the interactions among the hepatitis C virus (HCV) helicase and RLR helicases in live cells with quantitative microspectroscopic imaging (Q-MSI), a technique that determines FRET efficiency and subcellular donor and acceptor concentrations. HEK293T cells were transfected with various vector combinations to express cyan fluorescent protein (CFP) or YFP fused to either biologically active HCV helicase or one RLR (i.e. RIG-I, MDA5, or LGP2), expressed in the presence or absence of polyinosinic-polycytidylic acid (poly(I:C)), which elicits RLR accumulation at mitochondria. Q-MSI confirmed previously reported RLR interactions and revealed an interaction between HCV helicase and LGP2. Mitochondria in CFP-RIG-I:YFP-RIG-I cells, CFP-MDA5:YFP-MDA5 cells, and CFP-MDA5:YFP-LGP2 cells had higher FRET efficiencies in the presence of poly(I:C), indicating that RNA causes these proteins to accumulate at mitochondria in higher-order complexes than those formed in the absence of poly(I:C). However, mitochondria in CFP-LGP2:YFP-LGP2 cells had lower FRET signal in the presence of poly(I:C), suggesting that LGP2 oligomers disperse so that LGP2 can bind MDA5. Data support a new model where an LGP2-MDA5 oligomer shuttles NS3 to the mitochondria to block antiviral signaling.

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Quantitative live-imaging reveals the dynamics of apical cells during gametophyte development in ferns.

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Project description:Quantitative live-cell imaging of secretion activity reveals dynamic immune responses

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In vivo analysis of outer arm dynein transport reveals cargo-specific intraflagellar transport properties.

Project description:Outer dynein arms (ODAs) are multiprotein complexes that drive flagellar beating. Based on genetic and biochemical analyses, ODAs preassemble in the cell body and then move into the flagellum by intraflagellar transport (IFT). To study ODA transport in vivo, we expressed the essential intermediate chain 2 tagged with mNeonGreen (IC2-NG) to rescue the corresponding Chlamydomonas reinhardtii mutant oda6. IC2-NG moved by IFT; the transport was of low processivity and increased in frequency during flagellar growth. As expected, IFT of IC2-NG was diminished in oda16, lacking an ODA-specific IFT adapter, and in ift46 IFT46ΔN lacking the ODA16-interacting portion of IFT46. IFT loading appears to involve ODA16-dependent recruitment of ODAs to basal bodies followed by handover to IFT. Upon unloading from IFT, ODAs rapidly docked to the axoneme. Transient docking still occurred in the docking complex mutant oda3 indicating that the docking complex stabilizes rather than initiates ODA-microtubule interactions. In full-length flagella, ODAs continued to enter and move inside cilia by short-term bidirectional IFT and diffusion and the newly imported complexes frequently replaced axoneme-bound ODAs. We propose that the low processivity of ODA-IFT contributes to flagellar maintenance by ensuring the availability of replacement ODAs along the length of flagella.

| S-EPMC6254574 | biostudies-literature

Rapid and quantitative imaging of excitation polarized fluorescence reveals ordered septin dynamics in live yeast.

Project description:We report an imaging method for fast, sensitive analysis of the orientation of fluorescent molecules by employing a liquid-crystal based universal polarizer in the optical path of a wide-field light microscope. We developed specific acquisition and processing algorithms for measuring the anisotropy and for correcting artifacts caused by fluorescence bleaching, background light, and differential transmission of optical components. We call this approach the Fluorescence LC-PolScope and we used it to analyze the architectural dynamics of septin-green fluorescent protein (septin-GFP) constructs in the neck region of budding yeast. We describe three different states of highly anisotropic septin arrays in which the prevailing orientation of GFP dipoles was either parallel or perpendicular to the mother-bud axis. The transitions between these ordered states were characterized by transient isotropic states. To analyze the patterns of polarized fluorescence, we modeled the alignment of septin-GFP constructs in different stages of septin ring formation. Based on our model, our experimental data are consistent with the formation of paired rather than single filaments and the axis of the ?-helical septin terminus linked to a GFP molecule is likely oriented normal to the cell surface. The Fluorescence LC-PolScope combines the molecular specificity of fluorescence tagging with the structural specificity of polarized light analysis.

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Dynlrb1 is Essential for Dynein Mediated Transport and Neuronal Survival

Project description:We identified Dynlrb1 as an essential gene for sensory neuron survival and performed RNA sequencing to identify the molecular mechanism mediating the role of Dynlrb1 in sensory neuron survival.

2020-03-04 | GSE131455 | GEO

Dynein and intraflagellar transport.

Project description: Not available

| S-EPMC4433774 | biostudies-literature

In vivo optical trapping indicates kinesin's stall force is reduced by dynein during intracellular transport.

Project description:Kinesin and dynein are fundamental components of intracellular transport, but their interactions when simultaneously present on cargos are unknown. We built an optical trap that can be calibrated in vivo during data acquisition for each individual cargo to measure forces in living cells. Comparing directional stall forces in vivo and in vitro, we found evidence that cytoplasmic dynein is active during minus- and plus-end directed motion, whereas kinesin is only active in the plus direction. In vivo, we found outward (?plus-end) stall forces range from 2 to 7 pN, which is significantly less than the 5- to 7-pN stall force measured in vitro for single kinesin molecules. In vitro measurements on beads with kinesin-1 and dynein bound revealed a similar distribution, implying that an interaction between opposite polarity motors causes this difference. Finally, inward (?minus-end) stalls in vivo were 2-3 pN, which is higher than the 1.1-pN stall force of a single dynein, implying multiple active dynein.

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