Fig. 4: Septin5 assembles at MT-PM contact sites in a MT-dependent manner.

a Septin5 depletion in human islets increases GSIS by affecting MT rather than actin microfilaments. Insulin secretion over 30 min upon 16.7 mM glucose stimulation as measured by ELISA. Human islets after two-round infection with control or Ad-Septin5 shRNA were incubated with DMSO, nocodazole, latrunculin or taxol. Data are presented as box-plots where the central line represents the median value, the box shows percentiles 25–75 and the whiskers include the most extreme values (two-tailed Student’s t-test, n.s. denotes not significant). b–e Representative 3D structured illumination microscopy images of human β-cells infected with granule marker NPY-EGFP and Ad-shRNA-Control (b, c) or Ad-shRNA-Septin5 (d, e) for 48 h and then immunostained with tubulin. Pseudo-color encodes the depth. Color corresponds to the depth from the coverslip surface, with red being closest to the PM surface and Cyan being in cell interior. b 3D reconstructed Super-resolution imaging shows Septin5 is assembled at MT-PM contact sites (bottom, white arrows, higher magnification view of white dashed rectangular region) as rod and punctate structures, some granules were observed anchoring to Septin5-rich sites along the MT (bottom, white circles, higher magnification view of white dashed rectangular region). c The TIRF optical section images of 3D SIM highlighted the MT-PM contact sites of the region as the dashed box indicated in (b). d Septin5 deletion does not alter the MT meshwork or the anchoring of granules (right, white circles). Image is depth coded as indicated. e The TIRF optical section images of 3D SIM highlighted the MT-PM contact sites of the region as the dashed box indicated in (d). Representative results of three independent experiments.