Mission Administration Events News Publications Seminar Series Training & Employment
Biomolecular Devices Cell-Surface Interactions Nanoscale Cell Biology
Academia Industry Government Sciencenter
Nanobiotechnology Course K-12 Undergraduates Graduates
Cornell University Princeton University Wadsworth Center Oregon Health Sciences U. Clark Atlanta University Howard University
NBTC
nbtc logo  Program  †  Research Areas     Partners     Education     Members     Facilities 
Project Title
Electrochemical Imaging of Exocytosis Using Microfabricated Devices
 

Research Program
Nanoscale Cell Biology
 
Project #
 NCB8
 
Participating Faculty: M. Lindau, W. Almers, H. Craighead, B. Baird, C. K. Ober
NBTC Students/Postdocs: K. Kisler, O. Varmalov, A. Torres, R. Dong, K. Berberian
Other Students/Postdocs:  Q.Fang, A.Ngatchou, B.Kim, R.Molloy
 

Objectives
Release of neurotransmitter and hormones by neuronal cell types as well as histamine and other mediators by mast cells occurs from membrane bound vesicles by the mechanism of exocytosis via a fusion pore that forms a connection between the intravesicular and the extracellular space. We investigate the mechanisms of fusion and release of single vesicles in neuronal cell types and mast cells combining electrochemical measurements, microfabricated devices, fluorescence imaging and FRET constructs indicating protein conformational changes in the SNARE complex.

Methods
The relation between fusion pore properties and transmitter release is studied by patch amperometry, a technique where a carbon fiber electrode inserted into a patch pipette is used as electrochemical detector of catecholamines released from single vesicles in chromaffin cells while the fusion pore properties are measured by admittance analysis [1, 2]. For electrochemical imaging of single vesicle exocytosis platinum electrodes [3] as well as transparent ultrathin gold or indium tin oxide (ITO) electrodes [4] are patterned on the surface of a microscope cover glass. These devices detect the opening of singe fusion porees electrochemically while fluorescent markers are observed simultaneously by total internal reflection fluorescence (TIRF) microscopy [5]. Fluorescent tags are attached to proteins implicated in fusion to correlate changes in their interactions with fusion pore dynamics. Electrochemical imaging of exocytosis is also applied to study the spatio-temporal dynamics of stimulus secretion coupling in mast cells measuring release of preloaded serotonin. For chemical modification allowing detection of molecules that are not per se oxidizable such as glutamte, surface initiated atom transfer radical polymerization (SI-ATRP) is used to grow poly(sodium methacrylate)-b-poly (2-hydroxyethyl methacrylate) block copolymer brushes on gold electrode surfaces.

Summary
Combined amperometry and patch amperometry experiments showed that in contrast to current belief [6, 7], release of catecholamines through the early fusion pore is not associated with cation flux through channels in the vesicle membrane but with Na+ influx through the fusion pore following electrodiffusion [2]. It was shown that transparent 5 nm thin electrodes are capable of detecting amperometric foot signals, and thus single fusion pore openings, while fluorescence changes can be imaged through the electrode (Fig.1). Block copolymer brushes were successfully grown on gold electrode surfaces. The hydroxyl groups on poly(2-Hydroxyethyl methacrylate)  can be activated by tosyl chloride and then used to immobilize proteins. Initial fluorescent images showed that high density of proteins can be immobilized onto brushes by this method.

The protein SNAP-25  plays a key role in exocytosis [8].  To investigate its structural changes in the SNARE complex the CSNAC construct with a FRET donor (CFP) at the N terminal of the first SNARE motif and a FRET acceptor at the N terminal of the second (using a tetracysteine (C4) - motif that was post-translationally labeled with the biarsenical dye FlAsH) [9]. The endogenous cysteines of SNAP-25 were replaced with alanines. A large FRET increase was observed when it forms a binary complex with the SNARE syntaxin, or a tertiary complex with syntaxin and the SNARE VAMP.  The effect is not seen when CSNAC2 lacks either of the two SNARE motifs, nor when CSNAC2 is incubated with VAMP alone (Fig.2).

Mast cell exocytosis was detected with ECD arrays using poly-D-lysine (PDL) stimulation surface patterned employing parylene dry lift-off. Placing individual mast cells on ECDs with PDL (but not without PDL) produced amperometric serotonin signals at multiple electrodes (Fig.3).

Accomplishments

  • Amperometric detection of exocytic release with transparent 5 nm gold electrodes
  • Improved SNAP-25 FRET construct using FlAsh as acceptor probes SNAP-25 conformation change during complex formation with syntaxin.
  • Surface patterned poly-D-lysine stimulates mast cell exocytosis detected as amperometric spikes by  planar electrode arrays

Fig.1: Amperometric detection of fusion pore openings with transparent 5 nm gold electrodes in a chromaffin cell. (A) Fluorescently labeled granules are visible between as well as through the electrodes (red outlines). (B) Amperometric signals with low noise show clear foot signals indicating fusion pore expansion. (C) Amperometric charge detected per vesicle with gold electrodes is about twice that detected by ITO electrodes.

Fig.2: CSNAC and its mutants.  A.  The SNARE motifs SN1 (green) and SN2 (red) are connected by a linker (white) that targets SNAP25 to the plasma membrane. The donor fluorescent protein Cerulean (cyan) is at its N terminal and the tetracysteine motif C4 (yellow) at different positions along the linker (at amino acids R142, G132, E125 and A99 in CSNAC 1-4.  B.  The SNARE domain of syntaxin was added to purified CSNAC mutants for 30 min at the concentrations given on the abscissa, and the fluorescence was measured.  FRET ratio was calculated as the emission at 530 nm divided by that at 475 nm.

Fig.3: Electrochemical detection of serotonin release from a rat peritoneal mast cell placed on top of a surface patterned poly-D-lysine stimulus locared between the ECD electrodes. (A) Bright field microscope image. (B) Integrated amperometric charge detected by the 4 individual electrodes imdicating time course and position of release in a single fusion event. The measured amperometric spike is shown in the inset.

This material is based upon work supported in part by the STC Program of the National Science Foundation under Agreement No. ECS-9876771. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

 Home    Program    Research Areas    Partners    Education    Members    Facilities