Figure 2

Expression of PHM-GFP allows quantification of LDCV dynamics and secretion. (A) Dissociated trigeminal ganglion neurons were transfected with vector encoding PHM-GFP and grown in culture for 2 days. Cells were fixed and processed for immunocytochemistry; PHM-GFP fluorescence was visualized in puncta throughout the cell soma and processes (green) along with CGRP (red). (B) A growth cone (labeled) and the preceding region of its axon are shown with immunofluorescent staining of CGRP (green) and βIII tubulin (blue); filamentous actin was visualized with a fluorescent phalloidin conjugate (red). F-actin enriched stress fibers in a non-neuronal cell are seen at the bottom of the image. (C) Duplicate cultures were analyzed at room temperature (23°C) and at 30°C; 30 minute medium collections were made under basal conditions and during stimulation with 1 μM PMA. Secretion was quantified by measuring units of PHM activity (pmol product/h) in basal and stimulated media and in cell extracts. (D) Cultures were stimulated for 5, 15, or 30 minutes at 37°C with 1 μM PMA or 50 mM KCl. Secretion was quantified by assaying units of PHM activity in basal and stimulated media; stimulated secretion was calculated by subtracting basal from total secretion (gray line); separate experiments with cultures of different densities were performed for PMA and KCl.