Home Research Project Details A2 - Mapping neuronal ion channel distributions with single channel resolution
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A2 - Mapping neuronal ion channel distributions with single channel resolution

Jörg Enderlein and Andreas Neef

Ion channels are the key players of electrical signal integration, action potential generation and propagation in neurons. Biophysical multi-compartment models of neuronal dynamics rely, in principle, on the precise knowledge of the spatial distribution and local density of the different types of ion channels within the neuronal compartments. Such detailed knowledge, however, is often missing as can be seen for instance in the long-standing controversy on the local density of sodium channels in the axon-initial segment of pyramidal cells: here, functional data obtained by patch-clamp suggest a cannel density comparable to that of the somatic membrane, whereas immuno-histochemistry hints at a rather large density [Colbert et al. 2002, Kole et al. 2008]. In this project, advanced single-molecule imaging will be used for studying the spatial distribution, local concentration, and organization of different types of ion channels in neurons. Our goal is to map channel distributions in the cell membrane with a spatial lateral resolution of approximately 30 nm using photoactivated localization microscopy [Betzig et al. 2006] (PALM) or, equivalently, stochastic optical reconstruction microscopy [Rust et al. 2006] (STORM). In PALM/STORM, fluorescent markers are photophysically switched between fluorescent and dark states, such that at any moment in time only a few spatially wellseparated markers are in a fluorescent state. As individual molecules can be localized with a much higher spatial resolution than the optical resolution limit of a wide-field fluorescence microscope, see e.g. Ref. [Enderlein et al. 2006], this allows for a pointillistic reconstruction of fluorophore distribution within a sample. Recent advances allow for using virtually any fluorescent dye for PALM/STORM applications [Heilemann et al. 2008]. This offers the ability to label membrane proteins of interest with standard fluorescent dyes (via toxins or antibodies [Ando et al. 2004]) and to optically read out their local concentration and distribution. In the project, three key methodological concepts will be pursued:

(1) finding efficient ways of specifically labelling ion channels of interest, which involves covalently linking toxins with fluorescent dyes;

(2) improving PALM/STORM by using dedicated redox photochemistry [Heilemann et al. 2008, Van de Linde et al. 2008] for tuning photo-switching efficiency and improving photon yield; and

(3) developing algorithms that allow for a correct quantification of fluorescent label (and thus channel) density on a dozen nanometre scale. The quantification of ion channel distributions will be complemented by functional imaging of channel activity with SICM (A4). The expected results on detailed morphology and ion channel density are key parameters needed to model neuronal activity and will be integrated in the modelling efforts of the groups ABC.

Belongs to Group(s):
Bernstein Fellows, Single Molecule Spectroscopy and Imaging

Is part of  Section A 

Members working within this Project:
Wang, Ting 
Neef, Andreas 
Enderlein, Jörg 

Selected Publication(s):

Geissbuehler, S, Sharipov, A, Godinat, A, Bocchio, NL, Sandoz, PA, Huss, A, Jensen, NA, Jakobs, S, Enderlein, J, van der Goot, FG, Dubikovskaya, EA, Lasser, T, and Leutenegger, M (2014).
Live-cell multiplane three-dimensional super-resolution optical fluctuation imaging
Nature Communications 5:5830:1-7.