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Biophys J. 2014 Jul 1;107(1):26-33. doi: 10.1016/j.bpj.2014.04.064.

Lumenal protein within secretory granules affects fusion pore expansion.

Author information

1
Department of Pharmacology, University of Michigan Medical School, Ann Arbor Michigan. Electronic address: annweiss@umich.edu.
2
Department of Biological Sciences, Wayne State University, Detroit, Michigan.
3
Department of Pharmacology, University of Michigan Medical School, Ann Arbor Michigan.
4
Department of Pharmacology, University of Michigan Medical School, Ann Arbor Michigan; Department of Physics and LSA Biophysics, University of Michigan, Ann Arbor, Michigan.
5
Department of Pharmacology, University of Michigan Medical School, Ann Arbor Michigan. Electronic address: holz@umich.edu.

Abstract

It is often assumed that upon fusion of the secretory granule membrane with the plasma membrane, lumenal contents are rapidly discharged and dispersed into the extracellular medium. Although this is the case for low-molecular-weight neurotransmitters and some proteins, there are numerous examples of the dispersal of a protein being delayed for many seconds after fusion. We have investigated the role of fusion-pore expansion in determining the contrasting discharge rates of fluorescent-tagged neuropeptide-Y (NPY) (within 200 ms) and tissue plasminogen activator (tPA) (over many seconds) in adrenal chromaffin cells. The endogenous proteins are expressed in separate chromaffin cell subpopulations. Fusion pore expansion was measured by two independent methods, orientation of a fluorescent probe within the plasma membrane using polarized total internal reflection fluorescence microscopy and amperometry of released catecholamine. Together, they probe the continuum of the fusion-pore duration, from milliseconds to many seconds after fusion. Polarized total internal reflection fluorescence microscopy revealed that 71% of the fusion events of tPA-cer-containing granules maintained curvature for >10 s, with approximately half of the structures likely connected to the plasma membrane by a short narrow neck. Such events were not commonly observed upon fusion of NPY-cer-containing granules. Amperometry revealed that the expression of tPA-green fluorescent protein (GFP) prolonged the duration of the prespike foot ∼2.5-fold compared to NPY-GFP-expressing cells and nontransfected cells, indicating that expansion of the initial fusion pore in tPA granules was delayed. The t1/2 of the main catecholamine spike was also increased, consistent with a prolonged delay of fusion-pore expansion. tPA added extracellularly bound to the lumenal surface of fused granules. We propose that tPA within the granule lumen controls its own discharge. Its intrinsic biochemistry determines not only its extracellular action but also the characteristics of its presentation to the extracellular milieu.

Comment in

PMID:
24988338
PMCID:
PMC4119268
DOI:
10.1016/j.bpj.2014.04.064
[Indexed for MEDLINE]
Free PMC Article
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