Research Program
Nanoscale Cell Biology

Objectives
Release of neurotransmitter and hormones by neuronal cell types as well as histamine and other mediators by mast cells occurs from vesicles via a fusion pore that forms a connection between the intravesicular and the extracellular space. An essential role in fusion is attributed to three so-called SNARE proteins that collaborate with accessory proteins. We investigate the structure and properties of the fusion pore complex in plasma membranes of live cells and release of single vesicles in neuronal cell types and mast cells combining electrochemical measurements, microfabricated devices, and fluorescence imaging with molecular manipulations of the SNARE complex.

Methods
Release of catecholamines from single vesicles in chromaffin cells or serotonin in mast cells is studied by  amperometry using a carbon fiber electrode (CFE) while the fusion pore conductance is determined by time-resolved cell-attached patch clamp admittance analysis [1]. For electrochemical imaging of single vesicle exocytosis platinum electrodes [2] as well as transparent ultrathin gold or indium tin oxide (ITO) electrodes [3, 4]. are patterned on the surface of a microscope cover glass. SNARE proteins are genetically modified to study the influence on fusion pore properties and to attach fluorescent tags to study their interactions and conformational changes. Plasma membrane sheets from PC12 cells [5] are also used for fluorescence imaging of labeled SNAREs. For chemical surface modification surface initiated atom transfer radical polymerization (SI-ATRP) was used to grow polymer brushes on gold electrode surfaces and silica insulator surfaces.

Summary
Surface patterned platinum electrodes [2] were successfully applied to measure serotonin release from mast cells stimulated by surface patterned poly-D-lysine (Fig. 1). The Pt electrodes were also found to be applicable for cyclic voltammetry. They exhibited redox peaks with full width at half maximum of ~45 mV, much sharper than those of CFE recordings, allowing determination of the detected compound [6]. Random walk simulations of catecholamine release and diffusion revealed that the time course of voltammetric signals is inconsistent with rapid diffusion confirming previous results with our ECD arrays [2]. Transparent planar microelectrodes for amperometry fabricated from both indium tin oxide (ITO) and ultrathin (12-17nm thick) gold were compared. We found that ITO is less sensitive detecting only about half as much charge as gold electrodes, with peaks roughly half the height as those detected with ultrathin gold electrodes (1.11±0.24pC for gold, 0.48±0.10pC for ITO) [4]. Apparently, some of the electrons generated through catecholamine oxidation at the ITO surface are lost in reactions [7, 8] that reduce the ITO material. Deleting the C-terminal 9 amino acids of SNAP-25 led to reduced conductance of the early fusion pore suggesting a shortened fusion pore and a fusion pore structure that directly involves SNAP-25 (Fang et al, in revision for PNAS). To monitor SNAP-25 conformation, SNAP25 derivatives were made containing a cyan fluorescent protein as FRET donor. As a FRET acceptor, monkCSNAC contained a tetracysteine (C4) motif that could be post-translationally labeled in vitro with the biarsenical dye, FlAsH but could not be labeled in plasma membrane sheets. SCORE contained Venus, a yellow fluorescent protein, as FRET acceptor [11] and a FRET signal increase was observed in plasma membrane sheets when the SNARE domain of Syntaxin was added (Fig. 2). Pharmacological manipulations were used to investigate the role of actin-myosin II interactions. We found that myosin II does not accelerate fusion pore expansion but specifically accelerates release at late stages when the fusion pore is expanded (Fig. 3) (Berberian et al, revised manuscript submitted).

Accomplishments

• Microfabricated Platinum electrodes applied for the first time to measure catecholamines by cyclic voltammetry
• C-terminus of SNAP-25 has a role in fusion pore structure indicating a longer fusion pore when this domain is deleted consistent with the zipper model of SNARE mediated fusion
• Actin plays a role in fusion pore expansion independent of myosin II but myosin II accelerates release when fusion pore is expanded.