1. Rapid vimentin reorganisation upon BCR stimulation.
Rapid vimentin reorganisation upon BCR stimulation. (A–C) Resting primary B cells and B cells stimulated with 5 μg/ml anti-IgM F(ab)2 fragment for 15 min were settled onto poly-lysine–coated coverslips and fixed. The cells were labelled for vimentin and processed for (A, B) confocal microscopy or (C) SIM. (A) Representative confocal image showing the distribution of vimentin in resting (A, upper panel) and activated (A, lower panel) B cells. Scale bar = 5 μm. (B) The degree of vimentin collapse was quantitated. (C) Representative images and magnifications of the structure of vimentin obtained using SIM. Scale bar = 5 μm. (D) Primary B cells were settled onto poly-lysine–coated or anti-κ–coated coverslips and fixed. Intracellular vimentin and actin were labelled using anti-vimentin antibody and phalloidin, respectively. (D) Representative images showing lateral view of the cell from the 3D reconstruction of multiple z-stacks obtained using confocal microscopy. The white dotted lines depict the side of the coverslip. The degree of vimentin reorganisation was quantified for the two samples. (E) Representative corresponding dSTORM reconstruction of vimentin staining and the wide-field TIRF images of f-actin of cells settled on poly-lysine–coated and anti-κ–coated coverslips. (F, G) Primary B cells were stimulated with microspheres (Ø = 4.95 μm) coated with biotinylated anti-IgM. Excess microspheres were removed and the cells were settled onto poly-lysine–coated coverslips and fixed. (F) The cells were labelled for vimentin, tubulin, and LAMP1 and processed for confocal microscopy. Scale bar = 5 μm. (G) The distributions of vimentin in cells close to and distant from microspheres were quantitated. All data are representative of three independent experiments.
Carlson Tsui et al. LSA 2018;1:e
© 2018 Tsui et al.