Figure 3.
Sep7-GFP intensity increases with time at IR septin rings but not at branch rings. Single SEP7-GFP (AG127) cells were observed using 3D time-lapse fluorescence microscopy. Image stacks were acquired every 20 min and were processed identically. (A and E) Images show examples of an IR ring (A) and a branch ring (E) that were measured. The GFP fluorescence (green) and phase contrast cell outlines (red) are shown. Bars, 5 μm. (B and F) Cumulative change in fluorescence intensity of Sep7-GFP at IR rings (B; N = 50) and branch rings (F; N = 30) was measured for 4 h after establishment. Change in fluorescence intensity was calculated by subtracting the intensity of the ring immediately after establishment (t = 0) from the intensity measured at a given time point. Intensity values were corrected for background fluorescence of the cytosol. Values are given in arbitrary fluorescence units (au). The black lines represent the median cumulative change for the population. Fifty percent of the measured rings are in the dark gray regions, and 80% of the rings fall in the combined light gray and dark gray regions. (C and G) The box and whisker plots show the distribution of slopes of the linear regression of fluorescence change at individual rings. The black line denotes the median value and the asterisk shows the mean value. The box is the middle 50% of the data, and the whiskers extend to the most extreme data point, no more than 1.5 times the interquartile range. Rates are given in arbitrary fluorescence units per minute (au/min). (D and H) The slopes of the linear regression of fluorescence changes for a ring are graphed against the initial fluorescence at that ring for each individual septin ring analyzed. Values are given in arbitrary fluorescence units and rates are given in arbitrary fluorescence units per minute. In both cases, R values are insignificant.