Fig. 5: Septin5 depletion increases SGs dynamics and its access to PM and regulates MT stability.

a, b Spatiotemporal analysis of SG dynamics during glucose stimulation in WT (a) and Septin5-depleted (b) human β-cells by TIRFM. Representative TIRFM images (left), sum of SG trajectories (right), and time-lapse images of typical SG dynamics before fusion (bottom). c, d Histogram of the granule diffusion coefficient D measured from the trajectory of individual granules in WT (c) and Septin5 knockdown (d) human β-cell after glucose stimulation. Diffusion coefficient D is a measure of how constrained diffusion of the particles, smaller values indicate more restricted movement. The raw data (dots) are fitted with a three-component Gaussian function (red line), corresponding to the nearly immobile, restricted and highly dynamic SG population, respectively (from left to right). n = 6 cells, ~700 trajectories analyzed. e The spatial profile of SGs “exploring” the PM before fusion showed that Septin5 depletion increases SGs access to PM. To visualize the “exploring” region before fusion, time-lapse images of ~70 fusion events spatially aligned by the center coordinates of fusion site within a window of size 9×9 pixels were temporally averaged during the 5-s preceding fusion pore opening (aligning point illustrated on left). Spatial profiles of the average signal of NPY-EGFP at fusion sites showed in middle, single-granule profile included as reference. Dashed lines represent raw data, and the histogram was fitted with Gaussian function (right). Width, FWHM. f Septin5 depletion decreased the stability of MT after high glucose stimulation as indicated by the ratio (fire) of glu-tubulin (turquoise) to total tubulin (vermillion). The higher magnification (right) of regions as the dashed box (left) indicated showed example of region with decreased MT stability (bottom) compared to control (top). Representative images of human islets treated with high glucose for 30 min and then stained for stable MT marker, Glu-tubulin and total MT marker tubulin (n = 3 independent experiments, quantitative analysis of the experiments shown in Supplementary Fig. 6).