Figure 6

Septin2 forms highly dynamic filaments and rings. (A) YFP-Septin2 has the same distribution like endogenous Septin2 and it does not alter the organisation of endogenous Septin2. Bar, 20 μm (B) Photobleach experiment. NRK cells were transfected with either YFP-Septin2 or GFP-actin. Part of the filaments was bleached and recovery monitored over time. The ratio between mean fluorescence intensity of the prebleached box and the mean fluorescence of the whole cell was normalised to the prebleach ratio and expressed as a function of time (Materials and Methods). The graph shows representative recovery curves, which were fitted to a single exponential curve (solid lines) to calculate t_Ds and amount of recovery. n = 5. Images show a single confocal section of YFP-Septin2 filaments in NRK cells. Bar, 4 μm. (C) Images of a time-laps movie of YFP-Septin2 in the cell body of ruffling NRK-cells. Arrows indicate the formation cycle of a ring. Bar, 2 μm. (D,E) Photobleach experiments of YFP-Septin2 rings (also see additional file 1 for lysine/ruffling cells and additional file 2 for latrunculin). NRK cells growing on lysine-coated coverslips to induce ruffles were transfected with YFP-Septin2. Rings formed in the cell body were bleached and recovery monitored over time. For bleaching of Septin2 rings upon latrunculin treatment, normal NRK cells were transfected with YFP-Septin2 and treated with Latrunculin B for 20–25 Min before photobleaching. The data were analysed as in B to calculate t_Ds of recovery (D) and amount of recovery (E). n = 5. Images show a single confocal section of YFP-Septin2 rings in ruffling NRK cells. (F) Quantitative comparison of outer diameters of Septin2 rings in ruffling cells (lysine, empty circles) and upon latrunculin treatment (filled circles). Measurements were done on images of cells transfected with YFP-Septin2.