JGP_201711842 921..934

Upon fusion of the secretory granule with the plasma membrane, lumenal constituents are discharged at very different rates. This is explained in some cases by molecular size. For example, a low molecular weight neurotransmitter such as epinephrine is usually discharged in fewer than 100 ms, whereas co-stored proteins can be released over many seconds. Specific proteins can be discharged at widely different rates independently of cell type. GFP-tagged neuropeptide Y (NPY) and tissue plasminogen activator (tPA) have contrasting behaviors. NPY usually discharges within several hundred milliseconds of fusion, whereas tPA discharges after many seconds in primary chromaffin cells (Perrais et al., 2004), PC12 cells (Taraska et al., 2003), and insulin-secreting cells (Tsuboi et al., 2004). This large difference is unlikely to reflect simply a difference in the molecular weights of the proteins (tPA-GFP, ∼100 kD; NPY-GFP, ∼40 kD). Indeed, there is another explanation. By measuring the orientation of a fluorescent probe within the plasma membrane with polarized total internal reflection fluorescence (pTIRF) microscopy, we found that more than two-thirds of the fusion events of tPA-cerulean–containing granules maintain curvature for greater than 10 s (Weiss et al., 2014a). The maintained curvature reflects a narrow fusion pore. This conclusion is consistent with the finding using a fluorescent cytosolic probe that tPA-containing granules maintain long-lived, volume-enclosing structures on the surface of PC12 cells (Taraska et al., 2003). Such events are uncommon upon fusion of fluorescent-labeled NPY-containing granules. Indeed, pTIRF microscopy (Anantharam et al., 2010a; Weiss et al., 2014a) and real-time imaging of invaginations on the cell surface (Chiang et al., 2014) reveal that curvature changes and volume-filling omega figures resulting from fusion of NPY-containing granules have a much shorter duration, often no longer than several hundred milliseconds. tPA initiates an autocrine/paracrine pathway through its proteolytic enzymatic activity that locally regulates subsequent exocytosis within the adrenal medulla (Parmer et al., 1997, 2000). Thus, the slow postfusion discharge of tPA at the cell surface likely influences the kinetics of the pathway. The ability of tPA to almost freeze the fusion pore may have effects in addition to slowing its own release. Our experiments explore the notion that the inhibition of fusion pore expansion creates a novel compartment on the cell surface in which undiluted lumenal proteins are suddenly exposed to a pH shift from 5.5 to 7.4. We explore the implications of this concept in the context of the biochemistry of tPA. tPA is best known as a circulating serine protease that converts plasminogen into plasmin, which in turn breaks down fibrin clots by proteolysis. The activity of tPA in the plasma is regulated by plasminogen activator inhibitor 1 (PAI), a protein that acts as a suicide substrate to covalently inhibit the proteolytic activity of A lumenal secretory granule protein, tissue plasminogen activator (tPA), greatly slows fusion pore dilation and thereby slows its own discharge. We investigated another outcome of the long-lived narrow fusion pore: the creation of a nanoscale chemical reaction chamber for granule contents in which the pH is suddenly neutralized upon fusion. Bovine adrenal chromaffin cells endogenously express both tPA and its primary protein inhibitor, plasminogen activator inhibitor 1 (PAI). We found by immunocytochemistry that tPA and PAI are co-packaged in the same secretory granule. It is known that PAI irreversibly and covalently inactivates tPA at neutral pH. We demonstrate with zymography that the acidic granule lumen protects tPA from inactivation by PAI. Immunocytochemistry, total internal reflection fluorescence (TIRF) microscopy, and polarized TIRF microscopy demonstrated that co-packaged PAI and tPA remain together in granules for many seconds in the nanoscale reaction chamber, more than enough time to inhibit tPA and create a new secreted protein species. Slow fusion pore expansion creates a unique reaction chamber for co-packaged cargo