Journal: PLoS Pathogens

Article Title: Toll-Like Receptor 3 (TLR3) Plays a Major Role in the Formation of Rabies Virus Negri Bodies

doi: 10.1371/journal.ppat.1000315

Figure Lengend Snippet: (A) RABV-infected Ntera-2clD/1 cells were co-stained with Ab directed against the viral nucleocapsid (NC green), TLR3 (Ab Sc-C20, red), and Hoechst (nuclei, blue). In all infected cells TLR3–positive aggregates colocalize with viral NC forming perinuclear structures (Merge, lower panel). Bar = 10 µm. (B) RABV-infected Ntera-2clD/1 cells were co-stained with Ab directed against the viral nucleocapsid (NC green), TLR3 (Ab Sc-C20, red), and Hoechst (nuclei in blue). T is the transmission picture. The merged picture shows that NC accumulates with some perinuclear TLR3–positive aggregates (arrow). These perinuclear aggregates are visible in the transmission image. M is the 3D rendering (Imaris, Bitplane AG) after deconvolution (Huygens, Scientific Volume, Imaging). TLR3 constitutes the core of the structure (red internal core), surrounded by a coating of viral NC (green halo). Nuclear material was not present in these structures. Bar = 5 µm. (C) Enlargement of a 3D rendering of a TLR3–containing aggregate, showing the typical organisation of RABV–induced inclusions bodies composed of an inner core (TLR3 in red) surrounded by a viral NC protein cage (green halo). Diameter of the aggregate is 2.7 µm.

Article Snippet: Modelling of deconvoluted confocal images with 3D Imaris® software confirmed the particular spatial organisation of TLR3/NC aggregates ( ).

Techniques: Infection, Staining, Transmission Assay, Imaging

Journal: PLoS Pathogens

Article Title: Toll-Like Receptor 3 (TLR3) Plays a Major Role in the Formation of Rabies Virus Negri Bodies

doi: 10.1371/journal.ppat.1000315

Figure Lengend Snippet: (A) RABV–infected cells were immunostained with Ab against viral NC (1 and 4) and co-stained with either anti-pan-tubulin (2 and 3) or anti-vimentin Ab (5 and 6). Merged images are shown in 3 and 6. NBs (arrows) are associated with tubulin fibres (3); however, in contrast to canonical aggresomes, NBs are not surrounded by a ‘cage’ of the intermediate filament protein vimentin (6). N = nucleus. Bars = 10 µm. (B) RABV–infected cells were co-stained with Abs directed against viral NC (1) and anti Hsp70 (2). NBs are associated with the chaperone Hsp70, as shown by confocal analysis and 3D modelling [3 is the merged image; 4 is a 3D rendering (Imaris®, Bitplane AG) after deconvolution (Huygens, Scientific Volume, Imaging) of the perinuclear area (white square) from 3]. Bar = 5 µm. (C) NBs (green) are not formed at the MTOC/centrosome, detected using an antibody directed against γ-tubulin (arrows). N = nucleus. Bar = 10 µm. (D) RABV–infected cell lysates were separated after detergent treatment, into soluble (S) and insoluble (IS) fractions. The IS fraction contained the insoluble cytoskeletal protein vimentin but not tubulin or calnexin (Western Blot, upper panel). The IS fraction also contained NBs, which showed positively with anti-NC (green) and anti-TLR3 Ab (red). Bars = 2 µm.

Article Snippet: Modelling of deconvoluted confocal images with 3D Imaris® software confirmed the particular spatial organisation of TLR3/NC aggregates ( ).

Techniques: Infection, Staining, Imaging, Western Blot

Journal: eLife

Article Title: AMP-activated protein kinase fortifies epithelial tight junctions during energetic stress via its effector GIV/Girdin

doi: 10.7554/eLife.20795

Figure Lengend Snippet: ( A ) Subconfluent monolayers of MDCK cells grown on cover slips were fixed and stained for α-Tubulin (green), pS245-GIV (red) and DAPI (blue; nuclei) and analyzed by confocal microscopy. Image A shows an xy-plane maximum projection of a deconvolved stack of images, acquired at 20-nm z-intervals using a Olympus cellSens deconvolution (constrained iterative) and restoration system (Olympus FV3000) and a Apochromat 60XOSC2, NA 1.4 objective. The total thickness of the image stack is 4.8 mm. Two regions of interest (ROI), 1 and 2, are indicated with white boxes. ( B–D ) 3D reconstruction of each ROI shows the close proximity of pS245-GIV (red) to connecting microtubules (green) running along the cell-cell junctions (arrowheads). Little or no pS245-GIV staining (red pixels) is seen on microtubules at the 'free' cell border in panel D (arrows). ( E – H ) Maximum projected ROI‘s 1 and 2 are magnified in E and G , respectively. Individual Z-stacks of each ROI is shown in . Single Z-stacks of ROI‘s 1 and 2 are displayed in F and H , respectively. The insets in panels F and H show red, green and merged channels of respective figures. The white line indicates the pixels used for generating the RGB profile plots shown in I and J . RGB profiles show that pS245-GIV (red pixels) often co-localizes either completely with microtubule tracks (green pixels; I ) or lay between two microtubule tracks ( J ) at the cell-cell junctions. DOI: http://dx.doi.org/10.7554/eLife.20795.015

Article Snippet: Images were processed using the 3D deconvolution and 3D reconstructions tools of the Olympus cellSens (version Dimension Desktop 1.15) software.

Techniques: Staining, Confocal Microscopy

Journal: Molecular Therapy

Article Title: Directing Integrin-linked Endocytosis of Recombinant AAV Enhances Productive FAK-dependent Transduction

doi: 10.1038/mt.2011.295

Figure Lengend Snippet: Manganese treatment increases adeno-associated virus (AAV) clustering. (a, b) Treatment with Mn++ increases clustering of rAAV2 and sensitivity of detecting fluorescently labeled virions. Primary mouse embryonic fibroblasts (PMEF) were infected with Alexa Fluor568–rAAV2 (Alexa568-AAV2) for 1 hour with or without Mn++ treatment. To accurately image both small and large clusters of rAAV, both low (i.e., high sensitivity) and high (i.e., low sensitivity) thresholds were used when capturing confocal microscopic images. The high threshold was chosen to highlight the increased quantity of large and bright AAV clusters in Mn++ treated cells (b), while the low threshold panel demonstrates the presence of AAV on untreated cells (a). (c) The average size and intensity of AAV clusters increases with Mn++ treatment. Stacks of confocal images taken through cells infected with Alexa568-rAAV2 for 1 hour, as in (a, b), were 3D deconvoluted with MetaMorph software. The average intensity and volume of AAV clusters from randomly chosen cells from each condition are shown. Values represent the mean ± SEM of N = 10 cells from a representative experiment. Asterisks mark significant differences as assessed by a two-tailed Student's t-test (P < 0.05 when using averages from each of N = 10 cells in the analysis). Fold changes are also marked. (d) Effect of Mn++ treatment on the distribution of AAV object size as a function of intensity. The average size and intensity of AAV objects used for calculations in (c) is presented in an XY scatter plot. The percent of objects contained within selected ranges of AAV cluster size is indicated. For example, in untreated cells, 89.8% of AAV2 objects are between 0 and 0.25 µm3, which is significantly lower in Mn++-treated cells (75.8%). Statistical comparison between these two groups was significant using at two-tailed Mann–Whitney test (P < 0.0001). The number of objects quantified in each panel is indicated.

Article Snippet: The images displayed are a single slice from a stack of images that were deconvoluted with Metamorph 3D software.

Techniques: Labeling, Infection, Software, Two Tailed Test, MANN-WHITNEY

Journal: Molecular Therapy

Article Title: Directing Integrin-linked Endocytosis of Recombinant AAV Enhances Productive FAK-dependent Transduction

doi: 10.1038/mt.2011.295

Figure Lengend Snippet: Manganese induces recruitment of intracellular vinculin to sites of recombinant adeno-associated virus 2 (rAAV2) clustering on integrins. (a, b) AAV colocalization with α5 integrins is increased with Mn++ treatment. Primary mouse embryonic fibroblasts (PMEFs) expressing green fluorescent protein (GFP)-tagged α5 integrin were infected with Alexa568-AAV2 for 1 hour with or without Mn++ treatment. The images displayed are a single slice from a stack of images that were deconvoluted with Metamorph 3D software. Combined channels are given on the left in color and signal channel images for integrin (green) and rAAV2 (red) are given in black and white in the middle panels. In the right panels labeled “Colocalized Mask,” a mask was created using Zeiss LSM software that depicts only colocalized pixels, but does not change the relative intensity of either AAV or integrin staining. A high threshold was used for these images so Mn++-induced clustering could be better appreciated. (c, d) PMEFs were infected with Alexa568-AAV2 for 1 hour with or without Mn++ treatment and stained for vinculin (green). A series of confocal slices were then taken through each cell. The first panel on the left is a slice from the bottom of the cell to depict focal adhesions. These images demonstrate that rAAV2 does not efficiently recruit to focal adhesions that contain vinculin (arrows), even in the presence of Mn++. In the second panel from the left, a colocalization mask was applied to each slice throughout the entire cell and then projected onto a single 2D-image to demonstrate total rAAV2/vinculin colocalization in the cell. The third and fourth panels from the left are magnifications of the area marked in the left panel. “Colocalization mask: XYZ” refers to the Z-stacked masked sections throughout the cell projected into a single 2D image, while “Colocalization mask: XY” refers a mask of a single confocal slice. (e) The effect of integrin-binding peptide RGDS on Mn++-induced aggregation of rAAV2. Transformed control MEFs were infected with Alexa568-rAAV2 for 1 hour in the presence of Mn++ and Mn++ with RGDS peptide. Confocal images are shown with rAAV2 in white and nucleus in blue. Boxed region is enlarged in the inset of each panel.

Article Snippet: The images displayed are a single slice from a stack of images that were deconvoluted with Metamorph 3D software.

Techniques: Recombinant, Expressing, Infection, Software, Labeling, Staining, Binding Assay, Transformation Assay

Journal: The Journal of Cell Biology

Article Title: Arp2/3-dependent endocytosis ensures Cdc42 oscillations by removing Pak1-mediated negative feedback

doi: 10.1083/jcb.202311139

Figure Lengend Snippet: Pak1 removal from the plasma membrane is associated with endocytic patch internalization. (A) Fim1-mCherry and punctate Pak1-mEGFP localization at cell ends. Middle slice shows a single frame of a cell end expressing Pak1-mEGFP and Fim1-mCherry. Arrowhead marks overlapping Fim1-mCherry and Pak1-mEGFP puncta (scale bar = 2 µm). Bull’s eye view shows 3D reconstructed image of the same cell end. A dashed circle marks the outline of the cell end (scale bar = 6 µm). Center panel shows a whole cell with a white dashed outline (scale bar = 10 µm). Red box indicates the region shown as kymographs in the right panels. Yellow circles mark Pak1-mEGFP loss from the membrane while red arrows mark the onset of Fim1-mCherry internalization at the membrane (scale bar = 800 nm). Kymograph brightness is adjusted for ease of visibility for changes in Pak1 intensity. Representative images of the cell end in the left panel are deconvolved, clarified, and denoised with Nikon NIS elements. (B) Super-resolution images of cells expressing Pak1-mEGFP and Fim1-mCherry using Airyscan microscopy. Top row shows a maximum intensity projection. The dotted ROI indicates the cell that is presented in 3-D below). The bottom row shows 3D view of Pak1-mEGFP and Fim1-mCherry localization in the indicated cell. Magenta outline indicates the location of the cell (scale bar = 10 µm). Arrows indicate examples of Pak1-mEGFP at Fim1-mCherry patches. Airyscan images are processed and presented in 3D using Zeiss Zen Blue Automatic processing and 3D deconvolution. (C) Kymograph of Pak1-mEGFP and Fim1-mCherry as captured in a 3-min timelapse movie with 1-s intervals. Red box marks the region quantified in C (scale bar = 800 nm; frame rates = 1 s per frame [SPF]). (D) Quantification of Pak1-mEGFP and Fim1-mCherry intensities at the membrane over time. Red dashed lines indicate the peak and subsequent drop of Fim1-mCherry intensity. (E) Quantification of the time taken for dissipation of Pak1-mEGFP relative to Fim1-mCherry internalization ( n ≥ 6 endocytic events per kymograph, N = 21 kymographs from 3 replicates).

Article Snippet: Images were then automatically processed and 3-D deconvolved in Zeiss Zen Blue software.

Techniques: Clinical Proteomics, Membrane, Expressing, Microscopy

Journal: Journal of visualized experiments : JoVE

Article Title: Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-Cell Microscopy

doi: 10.3791/59822

Figure Lengend Snippet: 4. Live-cell imaging 2 and processing

Article Snippet: NOTE: For details on the microscope equipment and software used in this protocol, refer to the . table ft1 table-wrap mode="anchored" t5 Name of Material/Equipment Company Catalog Number 60× Plan Apochromat objective lens Olympus AMEP4694 Adenine Sigma A-8751 Agar 7558B IB4917–10 kg Agarose Sigma A9539–500g Belly Dancer rotator Stovall US Patent #4.702.610 Biotin Sigma B-4501 Boric acid Sigma B-6768–5kg CaCl2·2H20, Sigma C3306–500G Citric acid Sigma C-0759 Convolve 3D software plug-in OptiNav, Inc. n/a CoolSnap HQ CCD camera Roper n/a Cover slip VWR 16004–302 CuSO4·5H20, END CX-2185–1 DeltaVision deconvolution fluorescence microscope GE Healthcare/Applied Precision n/a Diffraction PSF 3D software plug-in OptiNav, Inc. n/a Edinburgh minimal medium (EMM) Notrogen Sunrise 2023;1kg FeCl2·6H2O END FX9259–04 Glucose Sigma G-7021 Heat plate Barnstead Thermo Lyne Histidine Sigma H8125–100g Huygens deconvolution software SVI n/a Imaris deconvolution software Bitplane n/a Incubator Shell lab #3015 Inositol Sigma I-5125 Iterative Deconvolve 3D software plug-in OptiNav, Inc. n/a KCl Mallinckvadt 6858–04 Lanolin Sigma L7387–1kg Leucine Sigma L8912–100g Lysine Sigma L5626–500g Malt extract (ME) MP 4103–032 MgCl2·6H20 AMRESCO 0288–500G MetaMorph Molecular Devices n/a Microscope slides VWR 16004–422 MnSO4, Mallinckvadt 6192–02 Molybdic acid Sigma M-0878 Na2S04 Mallinckvadt 8024–03 Nicotinic acid, Sigma N-4126 Pantothenic acid Sigma P-5161 Paraffin wax Fisher s80119WX Pombe glutamate medium (PMG) Sunrise 2060–250 softWorx v3.3 image processing software GE Healthcare n/a Sporulation Medium powder Sunrise 1821–500 Temperature controlled centrifuge Beckman Allwgra 6KR xentrifuge Uracil AMRESCO 0847–500g Vaseline Equaline F79658 yeast extract EMD 1.03753.0500 Yeast extract plus supplements (YES) Sunrise 2011–1kg ZnSO4·7H2O J.T.Baker 4382–04 Open in a separate window 4.

Techniques: Live Cell Imaging, Software, Fluorescence, Microscopy, Paraffin Wax

Journal: Journal of visualized experiments : JoVE

Article Title: Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-Cell Microscopy

doi: 10.3791/59822

Figure Lengend Snippet: 4. Live-cell imaging 2 and processing

Article Snippet: Iterative Deconvolve 3D software plug-in , OptiNav, Inc. , n/a.

Techniques: Live Cell Imaging, Software, Fluorescence, Microscopy, Paraffin Wax