Modulation of Cargo Release from Dense Core Granules by Size and Actin Network

During regulated fusion of secretory granules with the plasma membrane, a fusion pore first opens and then dilates. The dilating pore allows cargo proteins from the dense core to be released into the extracellular space. Using real‐time evanescent field fluorescence microscopy of live PC12 cells, it was determined how rapidly proteins of different sizes escape from single granules after fusion. Tissue plasminogen activator (tPA)‐Venus is released 40‐fold slower than the three times smaller neuropeptide Y [NPY‐monomeric GFP (mGFP)]. An NPY bearing two mGFPs in tandem [NPY‐(mGFP) 2 ] as an intermediate‐sized fusion probe is released most slowly. Although, the time–course of release varies substantially for a given probe. Coexpression of β‐actin, actin‐related protein 3 or mAbp1 slowed the release of the two larger cargo molecules but did not affect release of NPY‐mGFP or of the granule‐membrane‐bound probe Vamp‐pHluorin. Additionally, high concentrations of cytochalasin D slowed release of the tPA‐Venus. Together these results suggest that fusion pore dilation is not the only determinate of release time–course and that actin rearrangements similar to those mediating actin‐mediated motility influences the time–course of release without directly interfering with the granule membrane to cell membrane connection.