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 beta-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.
Felmy
Felix
F
Vollum Institute, Oregon Health & Science University, Portland, Oregon 97239, USA. felmy@zi.biologie.uni-muenchen.de
D000199:Actins / Q000502:physiology*; D000818:Animals; D003594:Cytoplasmic Granules / Q000502:physiology*; D016716:PC12 Cells; D051381:Rats; D022142:Secretory Vesicles / Q000502:physiology*; D010959:Tissue Plasminogen Activator / Q000235:genetics*
D000199:Actins; D010959:Tissue Plasminogen Activator