VAMP2 and synaptotagmin mobility in chromaffin granule membranes: implications for regulated exocytosis

Granule-plasma membrane docking and fusion can only occur when proteins that enable these reactions are present at the granule-plasma membrane contact. Thus, the mobility of granule membrane proteins may influence docking, and membrane fusion. We measured the mobility of vesicle associated membrane protein 2 (VAMP2), synaptotagmin 1 (Syt1), and synaptotagmin 7 (Syt7) in chromaffin granule membranes in living chromaffin cells. We used a method that is not limited by standard optical resolution. A bright flash of strongly decaying evanescent field produced by total internal reflection (TIR) was used to photobleach GFP-labeled proteins in the granule membrane. Fluorescence recovery occurs as unbleached protein in the granule membrane distal from the glass interface diffuses into the more bleached proximal regions, enabling the measurement of diffusion coefficients. We found that VAMP2-EGFP and Syt7-EGFP are mobile with a diffusion coefficient of approximately 3 × 10-10 cm2/s. Syt1-EGFP mobility was below the detection limit. Utilizing these diffusion parameters, we estimated the time required for these proteins to arrive at docking and nascent fusion sites to be many tens of milliseconds. Our analyses raise the possibility that the diffusion characteristics of VAMP2 and Syt proteins could be a factor that influences the rate of exocytosis.

VAMP2 AND SYNAPTOTAGMINS ARE RELATIVELY IMMOBILE ON CHROMAFFIN GRANULE MEMBRANES: IMPLICATIONS FOR MEMBRANE FUSION AND FUSION PORE EXPANSION

Although many of the proteins of secretory granules have been identified, little is known about their molecular organization and diffusion characteristics. Granule-plasma membrane fusion can only occur when proteins that enable fusion are present at the granule-plasma membrane contact. Thus, the mobility of granule membrane proteins may be an important determinant of fusion pore formation and expansion. To address this issue, we measured the mobility of (fluorophore-tagged) vesicle associated membrane protein 2 (VAMP2), synaptotagmin 1 (Syt1), and synaptotagmin 7 (Syt7) in chromaffin granule membranes in living chromaffin cells. We used a method that is not limited by standard optical resolution. A bright flash of strongly decaying evanescent field (∼80 nm exponential decay constant) produced by total internal reflection (TIR) was used to photobleach GFP-labeled proteins in the granule membrane. Fluorescence recovery occurs as unbleached protein in the granule membrane distal from the glass interface diffuses into the more bleached proximal regions, thereby enabling the measurement of diffusion coefficients. The studies revealed that VAMP2, Syt1, and Syt7 are relatively immobile in chromaffin granules membranes with diffusion constants of ≤ 3 × 10−10 cm2/s. Utilizing these diffusion parameters and the known density of VAMP2 and Syt 1 on synaptic vesicles, we estimated the time required for these proteins to arrive at a nascent fusion site to be tens of milliseconds. We propose that the mobilities of secretory granule SNARE and Syt proteins, heretofore unappreciated factors, influence the kinetics of exocytosis and protein discharge.Significance StatementIn eukaryotic cells, secretory vesicles fuse with the plasma membrane to secrete chemical transmitters, hormones and proteins that enable diverse physiological functions including neurotransmission. Fusion proteins need to be assembled at the fusion site in sufficient number in order to enable membrane fusion. However, the diffusion characteristics of fusogenic proteins on secretory vesicles remained unknown. Here we used a novel method not limited by standard optical resolution to measure the diffusion of VAMP2 and synaptotagmins on chromaffin granule membranes. We found they have limited mobility. The time required for these proteins to reach the granule-plasma membrane contact site suggests that their limited mobility likely influences the kinetics of membrane fusion and subsequent fusion pore expansion.

Chromogranin A, the major lumenal protein in chromaffin granules, controls fusion pore expansion

Upon fusion of the secretory granule with the plasma membrane, small molecules are discharged through the immediately formed narrow fusion pore, but protein discharge awaits pore expansion. Recently, fusion pore expansion was found to be regulated by tissue plasminogen activator (tPA), a protein present within the lumen of chromaffin granules in a subpopulation of chromaffin cells. Here, we further examined the influence of other lumenal proteins on fusion pore expansion, especially chromogranin A (CgA), the major and ubiquitous lumenal protein in chromaffin granules. Polarized TIRF microscopy demonstrated that the fusion pore curvature of granules containing CgA-EGFP was long lived, with curvature lifetimes comparable to those of tPA-EGFP–containing granules. This was surprising because fusion pore curvature durations of granules containing exogenous neuropeptide Y-EGFP (NPY-EGFP) are significantly shorter (80% lasting <1 s) than those containing CgA-EGFP, despite the anticipated expression of endogenous CgA. However, quantitative immunocytochemistry revealed that transiently expressed lumenal proteins, including NPY-EGFP, caused a down-regulation of endogenously expressed proteins, including CgA. Fusion pore curvature durations in nontransfected cells were significantly longer than those of granules containing overexpressed NPY but shorter than those associated with granules containing overexpressed tPA, CgA, or chromogranin B. Introduction of CgA to NPY-EGFP granules by coexpression converted the fusion pore from being transient to being longer lived, comparable to that found in nontransfected cells. These findings demonstrate that several endogenous chromaffin granule lumenal proteins are regulators of fusion pore expansion and that alteration of chromaffin granule contents affects fusion pore lifetimes. Importantly, the results indicate a new role for CgA. In addition to functioning as a prohormone, CgA plays an important role in controlling fusion pore expansion.