1. Vimentin reorganisation requires actin and microtubules.
Vimentin reorganisation requires actin and microtubules. (A) Representative confocal images showing the vimentin cytoskeleton in primary B cells in the absence (upper panel) and presence (lower panel) of WFA. Scale bar = 5 μm. (B) Quantification showing the degree of vimentin reorganisation in stimulated primary B cells treated with or without withaferin. (C) Representative confocal images showing distribution of the vimentin cytoskeleton in resting primary B cells where the microtubule cytoskeleton was perturbed. Primary B cells were purified and microtubule cytoskeleton was depolymerised using either nocodazole or incubation at low temperature. The cells were then fixed onto poly-lysine–coated coverslips and processed for confocal microscopy. Scale bar = 2 μm. Data are representative of three independent experiments. (D) Quantification of the degree of vimentin reorganisation in response to BCR stimulation in the control cells and in cells treated with nocodazole. t test was used to determine statistical significance. (E) Representative images of Airyscan confocal microscopy showing the distribution of intracellular vimentin in B cells treated with latrunculin A. Quantification shows the proportion of B cells with reorganised vimentin after BCR stimulation in the absence or presence of Lat A. Scale bar = 2 μm. (F) Quantification of the degree of antigen localisation in response to BCR stimulation in the control cells and in cells treated with Lat A. t test was used to determine statistical significance. (G) Representative single z-stack image of Airyscan confocal microscopy showing the distribution of intracellular vimentin and antigen in B cells treated with latrunculin A. Scale bar = 2 μm. (H) Representative confocal images and quantification showing vimentin distribution after BCR stimulation in WT and Cdc42−/− B cells. Scale bar = 2 μm. (I) Representative confocal images and quantification showing the distribution of antigen-containing and LAMP1+ compartments after BCR stimulation in WT and Cdc42−/− B cells. Scale bar = 2 μm. (J) Representative confocal images and analysis of colocalisation showing the distribution of antigen-containing and LAMP1+ compartments after BCR stimulation in WT and Cdc42−/− B cells. Scale bar = 2 μm.
Carlson Tsui et al. LSA 2018;1:e
© 2018 Tsui et al.