Figure 3. Lacrt-ELP fusion proteins are prosecretory in LGACs.
A) A cartoon depicts structure for ex vivo clusters of LGACs obtained from rabbits. These primary cultures form an apical lumen (L) that is bounded by a thick network of actin filaments. SV: secretory vesicles. B) Rabbit secretory vesicles (SV) release β-hexosaminidase in a dose dependent manner in response to secretagogues. The percentage of cellular secretion for each treatment (Eq. 2) has been normalized to a positive control defined by CCh-stimulation, which is defined as 100 %. LGACs were treated with 0.1 to 20 μM of LV96, Lacrt, V96, or no treatment for 1 h at 37 °C. 10 and 20 μM LV96 significantly enhanced secretion compared to the V96 group (**p<0.01), and a similar effect was found with 20 μM Lacrt (*p<0.05). Data were shown as mean ± S.D. and analyzed by ANOVA followed by Bonferroni’s Post-Hoc Test. C) Live-cell confocal microscopy was performed using LGACs labeled for F-actin (red). Actin-RFP is enriched beneath the apical membrane surrounding the lumen. Shortly after 10 min, increased irregularity of apical actin filaments and actin-coated secretory vesicle (SV) formation beneath the apical membrane (white arrows) were observed in CCh treated cells (100 μM). Similarly LV96 (20 μM) induced time-dependent actin remodeling after 20 min, which increased the irregularity of apical actin filaments and formation of secretory vesicles (white arrows). No significant remodeling of actin filaments was observed in a V96 treated control group. Apical lumen are indicated (white asterisk *). Scale bar: 10 μm.