Journal: Molecular Biology of the Cell

Article Title: The Drosophila Afadin and ZO-1 homologues Canoe and Polychaetoid act in parallel to maintain epithelial integrity when challenged by adherens junction remodeling

doi: 10.1091/mbc.E19-04-0209

Figure Lengend Snippet: Ena localizes adjacent to leading edge AJs and moderate Cno knockdown disrupts this uniform localization. Embryos, stage 13–14, genotypes indicated. All images show Ecad and Ena. (A–D) Wild type. (A) At the onset of dorsal closure, Ena localizes to all epidermal tricellular junctions (e.g., yellow arrow), and is especially enriched in segmental groove cells (red arrow). Enrichment near the leading edge AJs begins (blue arrows). (B, C) As closure proceeds, enrichment next to leading edge AJs increases. (D) Airyscan superresolution image. Leading edge “dots” sometimes resolve into two dots on the two sides of the Ecad. (E–H) cnoM-RNAi . (E) Early in closure Ena localization to epidermal tricellular junctions (e.g., yellow arrow) and segmental groove cells (red arrow) are relatively unchanged from wild type, while localization near the leading edge AJs is less uniform (blue arrows). (F) As closure proceeds, leading edge Ena localization is less focused at tricellular junctions than in wild type (arrows; compare with B), even when cell shapes are relatively normal. (G, H) In most embryos, leading edge Ena localization becomes much more irregular. Distinct leading edge dots remain in some cells (yellow), but at many tricellular junctions, Ena localization at leading edge dots is diminished (blue arrows) or broadened (red arrows). (I) Quantification of Ena puncta area at leading edge tricellular junctions. Images were binarized using ImageJ, and areas were measured using the wand tool. Scale bars in A, B, E, F, G = 20 µm. Scale bars in C, D, H = 5 µm.

Article Snippet: Superresolution embryos were imaged on Nikon N-SIM, using SR APO TIRF 100 ×/1.49 NA oil objective.

Techniques: Knockdown

Journal: Molecular Biology of the Cell

Article Title: The Drosophila Afadin and ZO-1 homologues Canoe and Polychaetoid act in parallel to maintain epithelial integrity when challenged by adherens junction remodeling

doi: 10.1091/mbc.E19-04-0209

Figure Lengend Snippet: SIM superresolution microscopy of the leading edge (LE) actin cable and tricellular junctions. SIM images of wild-type embryos mid–stage 13 (mid–dorsal closure), antigens indicated. Directional arrows indicate X (red), Y (green), and Z (blue) axes. (A–E) Ena and Ecad. (A) Ena puncta (e.g., arrows) flank Ecad at LE tricellular junctions. (B–B”) Two tricellular junctions at the LE. SIM resolves two separate Ena puncta (e.g., arrows) that are juxtaposed and surround Ecad at tricellular junctions. Maximum-intensity projection (M.I.P.) of z -stacks. (C) 3D reconstruction of M.I.P. from panel B, using alpha blending. This allows visualization of the LE 3D space. (D) Tilted view of 3D reconstruction. (E) 3D reconstruction from the vantage point of the amnioserosa (rotated 90° from panel C; apical up). Ena and Ecad are in the same plane on the apical–basal axis. (F–I) Cno and Ena. (F) Cno’s relationship to Ena parallels that of Ena and Ecad. (G–G”) Ena is juxtaposed to Cno at tricellular junctions. (H) 3D reconstruction. (I) Tilted view of 3D. (J–M) Ena and actin (visualized with phalloidin). (J) Lower magnification view of the LE. (K–K”) SIM resolves Ena localization to two separate puncta (blue arrows) located at the ends of each cell’s actin cable (magenta arrows). (L) 3D reconstruction. (M) 3D reconstruction showing view from the lateral epidermis (rotated 90° from L; apical up). Ena and Actin are parallel along the apical–basal axis. (N–R) Zipper (myosin II heavy chain) and actin. (N) Actin and myosin are arranged in an alternating pattern across the LE of the lateral epidermis. (O–O”) Two LE cells. Myosin is enriched in the central portion of each cell’s actin cable. (P) 3D view. (Q) Tilted view of 3D reconstruction. (R) 3D reconstruction side view from the lateral epidermis (rotated 90° from panel P; apical up). Myosin and actin are parallel along the apical–basal axis. Scale bars in A, F, J, and N = 20 µm. All others = 2 µm.

Article Snippet: Superresolution embryos were imaged on Nikon N-SIM, using SR APO TIRF 100 ×/1.49 NA oil objective.

Techniques: Microscopy

Journal: Journal for immunotherapy of cancer

Article Title: Single-chain variable fragment affinity tuning can optimize anti-AML CAR-NK cell functionality.

doi: 10.1136/jitc-2024-010763

Figure Lengend Snippet: Figure 5 Higher affinity 7G3 CAR-NK cells demonstrate increased clustering and cytotoxicity in a short-term time lapse. (A) Representative high-resolution confocal images of the immunological synapse formed between primary CAR-NK cells (untransduced (UTD) or expressing 7G3WT or 7G3L CARs) and GFP-tagged MOLM13 target cells (green). Phalloidin-555 was used to stain actin (red) and Pacific Blue was used to stain cytotoxic perforin granules (blue). Images were captured with a Leica Spinning Disk Confocal microscope at 60X in oil. (B–E) Representative final images from a 30 min time lapse demonstrating LysoTracker blue-stained NK cells (blue) (B) and DRAQ7-stained dead cells (white) (D) after co-culture with MOLM13 target cells, with quantification of normalized mean gray value for each channel (C, E). MOLM13 target cells without NK cell co-culture (No NK) were included as an additional control. Each dashed line represents an individual donor with the mean value from n=3 donors depicted by the solid curve. (F–I) Mean speed (F), maximum speed (G), total distance (H), and maximum distance (I) traveled by NK cells from each treatment group after co-culture with MOLM13 target cells following a 30 min time lapse, as determined through single cell tracking. Individual dots represent individual NK cells from pooled experiments with n=3 donors. Images were captured with a Nikon Eclipse Ti2-E fully motorized inverted microscope with a Yokogawa CSU-W1 SoRa super- resolution spinning disk confocal and 20×/0.45 s Plan Fluor objective. All imaging analysis was performed in Fiji Distribution of ImageJ software47 with use of the TrackMate plugin48 for single cell tracking experiments (C). Detailed statistical data are provided in online supplemental table 2. ****p<0.0001.

Article Snippet: Images were captured with a Nikon Eclipse Ti2- E fully motorized inverted microscope with a Yokogawa CSU- W1 SoRa superresolution spinning disk confocal and 20×/0.45 s Plan Fluor objective.

Techniques: Expressing, Staining, Microscopy, Co-Culture Assay, Control, Single Cell Tracking, Inverted Microscopy, Imaging