Imaging renin granule exocytosis in Juxtaglomerular cells by Total internal reflection (TIRF) microscopy
Mendez M. Imaging renin granule exocytosis in Juxtaglomerular cells by Total internal reflection (TIRF) microscopy. FASEB Journal 2017; 31(1 Suppl):701.3.
Renin is essential for angiotensin I generation and blood pressure regulation. Renin is stored in dense core granules in juxtaglomerular (JG) cells. Renin release is highly regulated with only a small percentage (2 to 5%) of the total renin content being released after maximal stimulation with agonists that increase cAMP. In vivo, stimulation of renin release resulted in rare events of renin granule disappearance as shown by electron microscopy. We found that stimulated-renin release is in part mediated by exocytic proteins present in the renin granules. However, real-time visualization of renin granule exocytosis has not been done. We hypothesize that renin exocytosis from JG cells is due to controlled exocytic events, termed kissand- run exocytosis, where fusion of the granule with the plasma membrane is transient and granule integrity is maintained. To study this we generated a new construct in which full length mouse renin was tagged with yellow fluorescent protein in its Carboxyl-terminus (Renin-YFP). First, we packed the Renin-YFP construct in adenoviruses (Ad-Ren-YFP) and characterized its proper expression and activity in Att20 cells, an endocrine pituitary cell line that does not express endogenous renin. By Western Blot, we observed a band at the expected molecular mass of renin-YFP (70 kDa), detected with antibodies against YFP or renin (n=4). Then, we tested whether Ad-Renin-YFP retains its enzymatic activity (rate of angiotensin I production from angiotensinogen) in Att20 cells. Renin activity was only detected in Att20 cells transduced with renin-YFP (n=3; p<0.01). To monitor exocytosis of renin-YFP, we transduced primary cultures of mouse JG cells. After 24 hs, we transferred JG cells to a 37C chamber and imaged YFP-containing granules by TIRF microscopy. TIRF allows imaging of granule movements and changes in fluorescence intensity within 250 nm of the plasma membrane. Under baseline conditions we observed an average of 16±4 granules per cell docked with the plasma membrane (n=12). Translational movement (X-Y planes) of docked granules was negligible and most granules remained docked during 20 minutes of imaging. TIRF events were quantified over 10 minutes by measuring: A) recruitment of new granules, B) full fusion followed by granule disappearance and C) rapid axial movement (Z-axis) of docked granules (observed as rapid bursts of fluorescence followed by return to baseline intensity). In the absence of cAMP, the total number of events per cell was low and no full fusion events were detected (1.5 ±0.5 total events per cell, n=4). After stimulation with cAMP, the number and frequency of events increased 4 fold to 5.3±1.0 events per cell (n=8, p<0.05). Only one full fusion event was detected (n=8 cells, 152 granules imaged). Most of the events caused by cAMP (73.5%) were due to fluorescence bursts of docked granules and 36.5% of events were caused by recruitment of newcomer granules to the TIRF field. We conclude that in JG cells, full fusion of granules is not the main mechanism of renin exocytosis. The rapid bursts in fluorescence intensity of docked granules suggest that kiss-and-run is the main mechanism of stimulatedrenin exocytosis. This is the first time that a renin-YFP construct was characterized and renin exocytosis is imaged in JG cells.