Imaging Early Steps of Sindbis Virus Infection by Total Internal Reflection Fluorescence Microscopy

Sindbis virus (SINV) is an alphavirus that has a broad host range and has been widely used as a vector for recombinant gene transduction, DNA-based vaccine production, and oncolytic cancer therapy. The mechanism of SINV entry into host cells has yet to be fully understood. In this paper, we used single virus tracking under total internal reflection fluorescence microscopy (TIRFM) to investigate SINV attachment to cell surface. Biotinylated viral particles were labeled with quantum dots, which retained viral viability and infectivity. By time-lapse imaging, we showed that the SINV exhibited a heterogeneous dynamics on the surface of the host cells. Analysis of SINV motility demonstrated a two-step attachment reaction. Moreover, dual color TIRFM of GFP-Rab5 and SINV suggested that the virus was targeted to the early endosomes after endocytosis. These findings demonstrate the utility of quantum dot labeling in studying the early steps and behavior of SINV infection

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology

Intercropping Improves the Yield by Increasing Nutrient Metabolism Capacity and Crucial Microbial Abundance in Root of Camellia oleifera in Purple Soil

Intercropping system influences the endophytic microbial abundance, hormone balance, nutrient metabolism and yield, but the molecular mechanism of yield advantage in Camellia oleifera intercropping with peanut is not clear. In this study, the C. oleifera monoculture (CK) and C. oleifera-peanut intercropping (CP) treatments in purple soil were conducted, and the physicochemical properties, gene expressions, signal pathways and crucial microbial abundances were investigated to reveal the molecular mechanism of the yield advantage of intercropped C. oleifera. The results showed that the intercropping system increased in contents of pigment, carbohydrate, available nitrogen and phosphorus in leaf and root, as well as the abundances of Burkholderia, Ralstonia, Delftia, Pseudoalteromonas and Caulobacter, enhanced the relative expression levels of CoSPS, CoGBE, CoGlgP, CoGBSS/GlgA genes to promote sugar metabolism, decreased the relative expression levels of CoASA, CoTSB, CoPAI, CoTDC and CoCYP71A13 genes for inhibiting IAA biosynthesis and signal transduction, as well as microbial diversity, Fusarium, Albifimbria and Coniosporium abundances in root, ultimately improved the fruit yield of C. oleifera. These findings indicate that intercropping system improves the fruit yield by enhancing the nutrient metabolism capability and crucial microbial abundances in root of C. oleifera in purple soil.https://opus.govst.edu/fac/1022/thumbnail.jp

Dynamics of Kv1 Channel Transport in Axons

Concerted actions of various ion channels that are precisely targeted along axons are crucial for action potential initiation and propagation, and neurotransmitter release. However, the dynamics of channel protein transport in axons remain unknown. Here, using time-lapse imaging, we found fluorescently tagged Kv1.2 voltage-gated K+ channels (YFP-Kv1.2) moved bi-directionally in discrete puncta along hippocampal axons. Expressing Kvβ2, a Kv1 accessory subunit, markedly increased the velocity, the travel distance, and the percentage of moving time of these puncta in both anterograde and retrograde directions. Suppressing the Kvβ2-associated protein, plus-end binding protein EB1 or kinesin II/KIF3A, by siRNA, significantly decreased the velocity of YFP-Kv1.2 moving puncta in both directions. Kvβ2 mutants with disrupted either Kv1.2-Kvβ2 binding or Kvβ2-EB1 binding failed to increase the velocity of YFP-Kv1.2 puncta, confirming a central role of Kvβ2. Furthermore, fluorescently tagged Kv1.2 and Kvβ2 co-moved along axons. Surprisingly, when co-moving with Kv1.2 and Kvβ2, EB1 appeared to travel markedly faster than its plus-end tracking. Finally, using fission yeast S. pombe expressing YFP-fusion proteins as reference standards to calibrate our microscope, we estimated the numbers of YFP-Kv1.2 tetramers in axonal puncta. Taken together, our results suggest that proper amounts of Kv1 channels and their associated proteins are required for efficient transport of Kv1 channel proteins along axons