Journal: Cell Death & Disease

Article Title: H-Ras transfers from B to T cells via tunneling nanotubes

doi: 10.1038/cddis.2013.245

Figure Lengend Snippet: GFP-H-RasG12V transfers from B721.221 to Jurkat cells through TNTs. B721.221 cells expressing GFP-H-RasG12V and Jurkat cells labeled with PKH26GL were seeded onto Lab-Tek chambered cover glasses for 90 min. Subsequently, the cells were imaged by 4D spinning-disk confocal microscopy for an additional ∼60 min. ( A ) Image from a time-lapse movie showing a long-range GFP-H-RasG12V-labeled TNT connecting between a B721.221 cell and a Jurkat cell. ( B ) Reconstructed 3D image of another similar TNT extracted from a 4D time-lapse movie. ( C ) TNT that is composed from the PM extensions of both cell types. ( D ) The images were extracted from a 4D time-lapse sequence. The sequence depicts the transfer of a GFP-H-RasG12V-enriched (green) membrane patch via the red-labeled TNT from B721.221 cells to a Jurkat cell that is eventually retained on the Jurkat cell's PM even after the tearing of the TNT (see also ). Scale bars represent 5 μ m. The images shown are from representative experiments out of >10 experiments performed ( n >10)

Article Snippet: Images were acquired using a motorized spinning-disk confocal microscope (Yokogawa CSU-22, Zeiss Axiovert 200M, Jena, Germany).

Techniques: Expressing, Labeling, Confocal Microscopy, Sequencing, Membrane

Journal: Cell Death & Disease

Article Title: H-Ras transfers from B to T cells via tunneling nanotubes

doi: 10.1038/cddis.2013.245

Figure Lengend Snippet: GFP-H-RasG12V accumulates at the junction between TNTs and the Jurkat cell body and subsequently transfers to Jurkat cells. Cells were seeded onto Lab-Tek chambered cover glasses and imaged as detailed above. Images are maximum-intensity projections of confocal z -sections, acquired during continuous (every 80 s) 4D spinning-disk confocal microscopy. The images are from a 4D time-lapse sequence depicting the continuous transfer of GFP-H-RasG12V membrane patches at the junction region (indicated by arrowheads) between a TNT derived from the PM extension of a B721.221-GFP-H-RasG12V cell (out of the frame) and the Jurkat cell body. Scale bars represent 3 μ m. Insets show with further detail the dynamic separation and transfer, over time, of discrete GFP-H-RasG12V-enriced membrane patches from the end region of the TNT to the Jurkat cell (marked by *). The results are representative of a typical experiment out of >3 experiments performed

Article Snippet: Images were acquired using a motorized spinning-disk confocal microscope (Yokogawa CSU-22, Zeiss Axiovert 200M, Jena, Germany).

Techniques: Confocal Microscopy, Sequencing, Membrane, Derivative Assay

Journal: Cell Death & Disease

Article Title: H-Ras transfers from B to T cells via tunneling nanotubes

doi: 10.1038/cddis.2013.245

Figure Lengend Snippet: H-Ras intercellular transfer depends on its farnesylation and palmitoylation. ( a ) Cells were plated on a Lab-Tek chambered cover glass, and the entire cell volume was captured through successive z -sections using spinning-disk confocal microscopy. Panels depict a confocal mid-section ( x , y dimensions, large central square) as well as x , z and y , z cross-sections (smaller lateral rectangles) of the different GFP-H-RasG12V mutants in typical B721.221 stable transfectants. For GFP-H-RasG12V and GM130 colocalization, cells were fixed and permeabilized and labeled with rabbit anti-GM130 Abs as detailed in the Materials and methods section. Images were deconvoluted with NearestNeighbors algorithm (Slidebook) for greater clarity. Scale bars represent 10 μ m. ( b ) CD3 + T cells were cocultured with the indicated B721.221 transfectants, expressing GFP-H-RasG12V with or without mutations in its C-terminal region, as detailed in the Materials and methods section. The various histogram overlays depict GFP signal detected in T cells (CD3 + singlet-cell gate) cocultured with the indicated B721.221 transfectants (black line) versus T cells cultured alone (gray-shaded histogram). ( c ) Bar chart represents the relative percentage of transfer: that is, relative mean fluorescence intensity of GFP signal detected in T cells cocultured with the various H-RasG12V mutants compared with that in T cells cocultured with the positive control B721.221-GFP-H-RasG12V cells (the latter set as 100% transfer). Bars represent mean±S.E.M., and *P <0.01 for all mutants compared with the unmodified GFP-H-RasG12V (comparing the relative GFP-H-Ras transfer values by the non-parametric Mann–Whitney U test). Data are from three independent experiments performed in duplicates or triplicates

Article Snippet: Images were acquired using a motorized spinning-disk confocal microscope (Yokogawa CSU-22, Zeiss Axiovert 200M, Jena, Germany).

Techniques: Confocal Microscopy, Labeling, Expressing, Cell Culture, Fluorescence, Positive Control, MANN-WHITNEY