The release of hormones and neurotransmitters, mediated by regulated exocytosis, can be modified by regulation of the fusion pore. The fusion pore is considered stable and narrow initially, eventually leading to the complete merger of the vesicle and the plasma membranes. By using the high-resolution patch-clamp capacitance technique, we studied single vesicles and asked whether the Secl/Muncl8 proteins, interacting with the membrane fusion-mediating SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, affect fusion pore properties. Muncl8-1 mutants were transfected into lactotrophs to affect the interaction of Muncl8-1 with syntaxinl (Synt1) (R39C), Rab3A (E466K), and Mints (P242S). Compared with wild-type, Muncl8-1 E466K increased the frequency ofthe fusion event. The latter two mutants increased the fusion pore dwell-time. All the mutants stabilized narrow fusion pores and increased the amplitude of fusion events, likely via preferential fusion oflarger vesicIes, since overexpression of Munc18-1 R39C did not affect the ave rage size of vesicIes, as determined by stimulated emission depletion (STED) microscopy. Single-molecule atomic force microscopy experiments revealed that wild-type Munc18-1, but not Munc18-1 R39C, abrogates the interaction between synaptobrevin2 (Syb2) and Synt1 binary trans-complexes. However, neither form of Muncl8-1 affected the interaction of Syb2 with the preformed binary cis-Synt1A SNAP25B complexes. This indicates that Munc18-1 performs a proofing function by inhibiting tethering of Syb2-containing vesicles solely to Synt1 at the plasmalemma and favoring vesicular tethering to the preformed binary cis-complex of Synt1A-SNAP25B. The association of Munc18-1 with the ternary SNARE complex leads to tuning of fusion pores via multiple and converging mechanisms involving Muncl8-1 interactions with Synt1A, Rab3A, and Mints.
COBISS.SI-ID: 28521433
We used a continuum model based on the Helfrich free energy to investigate the binding dynamics of a lipid bilayer to a BAR domain surface of a crescent-like shape of positive (e.g. I-BAR shape) or negative (e.g. F-BAR shape) intrinsic curvature. According to structural data, it has been suggested that negatively charged membrane lipids are bound to positively charged amino acids at the binding interface of BAR proteins, contributing a negative binding energy to the system free energy. In addition, the cone-like shape of negatively charged lipids on the inner side of a cell membrane might contribute a positive intrinsic curvature, facilitating the initial bending towards the crescent-like shape of the BAR domain. In the present study, we hypothesize that in the limit of a rigid BAR domain shape, the negative binding energy and the coupling between the intrinsic curvature of negatively charged lipids and the membrane curvature drive the bending of the membrane. To estimate the binding energy, the electric potential at the charged surface of a BAR domain was calculated using the Langevin-Bikerman equation. Results of numerical simulations reveal that the binding energy is important for the initial instability (i.e. bending of a membrane), while the coupling between the intrinsic shapes of lipids and membrane curvature could be crucial for the curvature-dependent aggregation of negatively charged lipids near the surface of the BAR domain. In the discussion, we suggest novel experiments using patch clamp techniques to analyze the binding dynamics of BAR proteins, as well as the possible role of BAR proteins in the fusion pore stability of exovesicles.
COBISS.SI-ID: 8510036
In multicellular organisms signaling is a necessity and an important mode of communication between cells is mediated by neurotransmitters, hormones and other chemical messengers that are stored in secretory vesicles. In stimulated conditions secretory vesicles, which are trafficked to be docked at the plasma membrane, enter exocytosis, characterized by vesicle and plasma membrane merger. Due to repulsive forces of negatively charged membrane surfaces, it was long believed that the fusion pore is merely a short lived intermediate state leading irreversibly to a complete merger of both membranes. However, recent results show that the fusion pore is a rather stable structure, which can reversibly reopen to subnanometer diameters; dimensions too narrow to permit the exit of the cargo into the extracellular space. The aim of this chapter is to first review how can such a structure attain stability and compare two models in which membrane constituents are either isotropic or anisotropic in nature. Then we address the molecular nature of such a stable, release-unproductive fusion pore. We conclude that membrane constituents of the stable fusion pore membrane, being made of proteins and/or lipids, very likely consist of architectural elements that exhibit anisotropicity. The dynamics of fusion pore diameter is then determined by the density and architectural properties of these membrane constituents at fusion pore locales.
COBISS.SI-ID: 8665940