Journal: Journal of Virology

Article Title: The ACBD3 protein coordinates ER-Golgi contacts to enable productive TBEV infection

doi: 10.1128/jvi.02224-24

Figure Lengend Snippet: Identification of ACBD3 as a host factor in NS4B-APEX2 proximal protein analysis. ( A ) Confocal fluorescence micrographs of HEK293T cells transiently expressing TBEV NS4B-GFP infected with LGTV (MOI 10, 16 h.p.i.) and stained with anti-calnexin (CANX, ER marker) antibodies and anti-dsRNA antibodies. Scale bar, 10 µm. ( B ) Schematic illustration of the NS4B-APEX2 proximal protein biotinylation screen used in this study. ( C ) Volcano plots of the proteins identified in the NS4B-APEX2 proximal protein biotinylation screen in NS4B-APEX2 cells (left) and NS4B-APEX2 cells infected with LGTV (MOI 10, 16 h.p.i.) (right). Proteins are indicated by dots color-coded based on the FC and P -values. Three biological replicates per condition were analyzed, and the P -value was calculated using unpaired t -test. ( D ) Schematic illustration of ERES-Golgi contacts, and the top hit proteins ACBD3, TFG, SEC23IP, and KTN1 ( E ) Quantification of the percentage of GFP-positive KD HEK293T cells expressing E protein at 24 h after LGTV infection (MOI 1). The KD proteins are indicated. Mean ± SD of 3 independent experiments. One-way ANOVA with Dunnett multiple tests, *** P < 0.005, **** P < 0.0001.

Article Snippet: To detect interactions between NS4B-GFP and ACBD3, the samples were incubated with primary antibodies against GFP (mouse monoclonal, JL-08, Clontech) and ACBD3 (rabbit polyclonal, Atlas Antibodies) at two dilution combinations: anti-GFP 1:200 with anti-ACBD3 1:500 and anti-GFP 1:100 with anti-ACBD3 1:1000.

Techniques: Fluorescence, Expressing, Infection, Staining, Marker

Journal: Journal of Virology

Article Title: The ACBD3 protein coordinates ER-Golgi contacts to enable productive TBEV infection

doi: 10.1128/jvi.02224-24

Figure Lengend Snippet: ACBD3 localization in healthy and infected cells. ( A ) Confocal fluorescence micrographs of HEK293T cells transiently expressing NS4B-mCherry and stained with anti-ACBD3 antibodies. Scale bars, 10 µm. Manders’ coefficient between the ACBD3 and NS4B signals is shown on the right-hand side as mean ± SD. ( B ) Fluorescent SIM images of HEK293T and ACBD3 KO cells transfected with NS4B-GFP and processed using the PLA providing a signal for the close proximity between antibodies against GFP and ACBD3 (ab dilution 1). Quantification of the number of PLA signal events per cell is shown on the right. Ab dilution 1: anti-GFP 1:200, anti-ACBD3 1:500; ab dilution 2: anti-GFP 1:100, anti-ACBD3 1:1000. ( C ) Confocal fluorescence micrographs (single Z frame) of LGTV-infected (MOI 1, 16 h.p.i.) HEK293T cells and stained with antibodies against ACBD3, GM130, and dsRNA. Scale bar, 1 µm. Manders’ coefficient between the indicated signals is shown on the right-hand side as mean ± SD. ( D ) SIM fluorescence micrographs (100 nm resolution) of HEK293T cells transiently expressing GFP-ACBD3 and stained with anti-SEC23IP antibodies. Scale bars, 1 µm. ( E ) SIM fluorescence micrographs (100 nm resolution) of HEK293T cells transiently expressing GFP-ACBD3 at 16 h after LGTV infection (MOI 1) stained with anti-SEC23IP and anti-E antibodies. Scale bars, 1 µm. The left-hand panels show the image stack projected along the Z, Y, and X axes. The right-hand panels show a single Z frame of the inset area.

Article Snippet: To detect interactions between NS4B-GFP and ACBD3, the samples were incubated with primary antibodies against GFP (mouse monoclonal, JL-08, Clontech) and ACBD3 (rabbit polyclonal, Atlas Antibodies) at two dilution combinations: anti-GFP 1:200 with anti-ACBD3 1:500 and anti-GFP 1:100 with anti-ACBD3 1:1000.

Techniques: Infection, Fluorescence, Expressing, Staining, Transfection

Journal: Journal of Virology

Article Title: The ACBD3 protein coordinates ER-Golgi contacts to enable productive TBEV infection

doi: 10.1128/jvi.02224-24

Figure Lengend Snippet: Full-length ACBD3 promotes flavivirus infection independently of PI4KB. ( A ) Quantification of the percentage of A549 cells positive for dsRNA after infection with LGTV, TBEV (24 h.p.i, MOI 0.1), or PV (6 h.p.i., MOI 10) in the absence or presence of T-00127-HEV1 (1.25 µM). Data were normalized to DMSO-treated infected A549 cells. Mean ± SD of at least five biological replicates from three independent experiments, unpaired t -test. ( B ) Schematic illustration of ACBD3 domains and the ACBD3 mutants used in the study. ( C ) Quantification of the percentage of GFP-positive HEK293T and ACBD3 KO cells expressing E protein at 24 h after TBEV infection (MOI 0.1) after transfection with different ACBD3 constructs. Data were normalized to GFP-positive HEK293T cells. Mean ± SD of at least 10 biological replicates from two independent experiments. One-way ANOVA with Dunnett multiple tests. ( D ) Confocal fluorescence micrographs of ACBD3 KO cells transiently over-expressing GFP-ACBD3 or GFP-GOLD stained with anti-GM130 and NS3 (only in the infection experiment) antibodies either without infection (left) or 16 h after LGTV infection at MOI 1 (right). Scale bar, 10 µm. ( E ) Quantification of the percentage of NS3 area overlapping with GFP in GFP-ACBD3 or GFP-GOLD-transfected cells. Data are presented in scatter plots with mean ± SD. Number of NS3 areas analyzed, GFP-ACBD3 ( n = 130), GFP-GOLD ( n = 179). Mann-Whitney test. ( F ) Confocal fluorescence micrographs of ACBD3 KO cells transiently expressing NS4B-mCherry and GFP-GOLD. Scale bar, 10 µm. ( G ) Quantification of the percentage of the fluorescence NS4B area overlapping with the fluorescence of GFP-GOLD. Data are presented in a scatter plot with mean ± SD. The number of cells analyzed is indicated. ns P > 0.05, **** P < 0.0001. ** P < 0.005, **** P < 0.0001 in ( A ), ( C ), ( E ), and ( G ).

Article Snippet: To detect interactions between NS4B-GFP and ACBD3, the samples were incubated with primary antibodies against GFP (mouse monoclonal, JL-08, Clontech) and ACBD3 (rabbit polyclonal, Atlas Antibodies) at two dilution combinations: anti-GFP 1:200 with anti-ACBD3 1:500 and anti-GFP 1:100 with anti-ACBD3 1:1000.

Techniques: Infection, Expressing, Transfection, Construct, Fluorescence, Staining, MANN-WHITNEY

Journal: eLife

Article Title: MagIC-Cryo-EM, structural determination on magnetic beads for scarce macromolecules in heterogeneous samples

doi: 10.7554/eLife.103486

Figure Lengend Snippet: ( A ) Models of potential cell cycle-dependent H1.8 dynamic binding mechanisms ( B ) Experimental flow of MagIC-cryo-EM analysis for GFP-H1.8 containing complexes isolated from chromosomes assembled in interphase and metaphase Xenopus egg extract. Fluorescence microscopy images indicate localization of GFP-H1.8 to interphase and metaphase chromosomes. DNA and GFP-H1.8 were detected either by staining with Hoechst 33342 or GFP fluorescence, respectively. ( C ) Native PAGE of fragmented interphase and metaphase chromosome sucrose gradient fractions. GFP-H1.8 and DNA were detected with either GFP fluorescence or SYTO-60 staining, respectively. ( D ) Western blot of GFP-H1.8 in interphase and metaphase chromosome sucrose gradient fractions. GFP-H1.8 was detected using anti-GFP antibodies. ( E ) SDS-PAGE of the sucrose gradient fractions 4 and 5 shown in ( C ), demonstrating heterogeneity of the samples. Proteins were stained by gel code blue. Red arrows indicate the H1.8-GFP bands. The full gel image is shown in . ( F ) In silico 3D classification of interphase and metaphase H1.8-bound nucleosomes isolated from chromosomes in Xenopus egg extract. To assess the structural variations and their population of H1.8-bound nucleosomes, ab initio reconstruction and heterogenous reconstruction were employed twice for the nucleosome-like particles isolated by the decoy classification. The initial round of ab initio reconstruction and heterogenous reconstruction classified the particles into three nucleosome-containing 3D models ( A, B, C ). Subsequent ab initio reconstruction and heterogenous reconstruction on the class A, which has weak H1.8 density, yielded three new nucleosome-containing structures, A1, A2, and A3. 3D maps represent the structural variants of GFP-H1.8-bound nucleosomes. Red arrows indicate extra densities that may represent H1.8. Green densities indicate on-dyad H1.8. The bar graphs indicate the population of the particles assigned to each 3D class in both interphase and metaphase particles (gray), interphase particles (blue), and metaphase particles (red). The pipeline for structural analysis is shown in . ( G ) Structures of H1.8-bound nucleosomes isolated from interphase and metaphase chromosomes. Figure 3—source data 1. Full images of gels and membranes shown in . (A) Full gel images used in . (B) Full membrane image used in . (C) Full gel image used in . Figure 3—source data 2. Raw images of gels and membranes shown in .

Article Snippet: For , as primary antibodies, mouse monoclonal antibody against GFP (Santa Cruz Biotechnology, # sc-9996, 1:1000 dilution) and rabbit polyclonal antibody against X. laevis H1.8 ( ; final: 1 μg/mL) were used.

Techniques: Binding Assay, Cryo-EM Sample Prep, Isolation, Fluorescence, Microscopy, Staining, Clear Native PAGE, Western Blot, SDS Page, In Silico, Membrane