Vesicle Motion and Fusion are Altered in Chromaffin Cells with Increased SNARE Cluster Dynamics

Abstract

The expression of SNAP-25 fused to green fluorescent protein (GFP) has been instrumental in demonstrating SNARE role in exocytosis. The wild-type GFP-SNAP-25 and a D9 form, product of botulinum neurotoxin A activity, the main ingredient in the BOTOX preparation, were employed here to study SNARE implication in vesicle mobility and fusion in cultured bovine chromaffin cells, a neuroendocrine exocytotic model. Using total internal reflection fluorescent microscopy, we have identified membrane microdomains of 500-600 nm diameter that contain both SNAP-25 and syntaxin-1 and associate with synaptobrevin-2. Interestingly, while the SNAP-25 D9 formed similar clusters, they displayed increased mobility both laterally and in the axis perpendicular to the plasmalemma, and this correlates with the enhanced dynamics of associated chromaffin granules. SNARE cluster-enhanced motion is reversed by elevation of the intracellular calcium level. Furthermore, single vesicle fusion was unlikely in the highly mobile vesicles present in the cells expressing SNAP-25 D9, which, in addition, displayed in average slower fusion kinetics. Consequently, SNARE cluster dynamics is a new aspect to consider when determining the factors contributing to the mobility of the vesicles in close vicinity to the plasma membrane and also the probability of exocytosis of this granule population. Exocytosis is a key event in the regulated release of neurotransmitters in neurons and neuroendocrine cells. The characterization of various proteins (SNAREs) that form the core complex involved in the specific docking and fusion of neurotransmitter-containing vesicles has stimulated unprecedented progress toward elucidating the molecular bases of exocytosis (1-4). The assembly of plasma membrane proteins (t-SNAREs) such as syntaxin (2) and SNAP-25 (5), as well as vesicle-associated proteins (v-SNAREs) such as synaptobrevin-2 (6), provides the specificity required for vesicle docking and probably the basic machinery for membrane fusion (7). In addition to the use of classical tools such clostridial neurotoxins (8), the generation of engineered SNARE constructs fused to the green fluorescent protein (GFP) has proven instrumental in defining the involvement of these proteins in specific steps of exocytosis. For instance, the use of a functional GFP-SNAP-25 chimera demonstrated the importance of specific amino acids in the C-terminal domain of this protein for core complex assembly and normal vesicle fusion in chromaffin cells, an accepted model of neuroendocrine exocytosis (9).Subsequently, this and other constructs were employed to define the different stages of SNARE complex formation, its interaction with other proteins or factors and the recruitment of different vesicle reservoirs (10-13). Together, these studies demonstrated that ternary complex formation constitutes a late stage in membrane fusion. However, the chain of interactions between SNAREs and other proteins that controls this process (involving SNAPs, synaptotagmin, munc-18, etc.) essentially remain ...

The F-actin cortical network is a major factor influencing the organization of the secretory machinery in chromaffin cells

“…Vesicles were visualized using GFP-synaptobrevin-II, as described previously (Gil et al, 2002). In other experiments, vesicles were stained with 2 M Acridine Orange, and the granule position was assessed by the red Acridine Orange fluorescence of mature acidic vesicles; their fusion was followed by detection of the green flashes produced after matrix neutralization during exocytosis (Lopez et al, 2009). To combine cytoskeletal images with TIRFM, the light from the halogen lamp used for light transmission was filtered to give red light (LP590 filter) for the simultaneous visualization of the labelled vesicles.…”

Super-resolution Imaging Reveals the Internal Architecture of Nano-sized Syntaxin Clusters

“…A previous nanoscopy study based on stimulated emission depletion (16), as well as other studies (17)(18)(19), have investigated the distribution of syntaxin and other synaptic proteins on the membrane and have indicated syntaxin partitioning into nano-sized clusters (16, 17, 19 -23). Sieber et al (16) suggested the existence of two pools of syntaxin: clustered and as single freely diffusing molecules.…”

The organization of the secretory machinery in chromaffin cells as a major factor in modeling exocytosis

“…Therefore, it is suggested that even at the time of experiencing exocytosis, granules interact with cytoskeletal elements. In order to prove such interaction we studied the precise localization of exocytosis in relation to cytoskeletal structures by in vivo observation of the exocytotic events using TIRFM observation of acridin orange (AO) loaded granules (López et al, 2009) and the cytoskeletal structures using transmitted light. As can be seen in the example of Fig.…”

“…Expression of DsRed and GFP-SNAP-25 was performed as described recently (López et al, 2009). The cDNA corresponding to the SNAP-25a isoform (Bark and Wilson, 1994) was cloned into the XhoI and BamHI sites of pDsRed-C3 or pEGFP-C3 (Clontech, Palo Alto, California) to express this protein fused in-frame at the C-terminus.…”

“…DsRed-SNAP-25 was expressed in chromaffin cells as described recently (López et al, 2009) and its fluorescence in the evanescent field imaged with simultaneous acquisition of transmitted light images. As can be observed in the two examples displayed in Figs.…”