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: The EMBO Journal

Article Title: Shelterin promotes tethering of late replication origins to telomeres for replication‐timing control

doi: 10.15252/embj.201898997

Figure Lengend Snippet: Representative images of AT2088‐LacI‐GFP (green) and Ish1‐mCherry (red) obtained in 3‐min intervals before and after the onset of anaphase. Optical section images were projected by the maximum intensity method using the SoftWoRx 5.5 software on the DeltaVision Elite system. The scale bar indicates 1 μm.

Article Snippet: Optical section images were processed with 3D deconvolution and analyzed using the SoftWoRx 5.5 software on the DeltaVision Elite system (GE Healthcare Life Science).

Techniques: Software