Technical Reference #1622

1622.

Detection of Ligand-Induced CNTF Receptor Dimers in Living Cells By Fluorescence Cross Correlation Spectroscopy Felix Neugart b;1; Andrea Zappe b;1; Deborah M. Buk a;1; Inna Ziegler a; Steffen Steinert b; Monika Schumacher a; 4 Eva Schopf a; Ralph Bessey a; Kathrin Wurster a; Carsten Tietz b; Michael Börsch b; 5 Jörg Wrachtrup b; Lutz Graeve a;⁎, Universitat Hohenheim, Biochimica et Biophysica Acta, 1788(1622), (2009)

Abstract:
Ciliary neurotrophic factor (CNTF) signals via a receptor complex consisting of the specific CNTF receptor (CNTFR) and two promiscuous signal transducers gp130 and leukemia inhibitory factor receptor (LIFR). Whereas earlier studies suggested that the signaling complex is a hexamer more recent analyses strongly support a tetrameric structure. However all studies so far analyzed the stoichiometry of the CNTF receptor complex in vitro and not in the context of living cells. We generated and expressed in mammalian cells acyl carrier protein-tagged versions of both CNTF and CNTFR. After labeling CNTF and CNTFR with different dyes we analyzed their diffusion behavior at the cell surface. Fluorescence (cross) correlation spectroscopy (FCS/FCCS) measurements reveal that CNTFR diffuses with a diffusion constant of about 2 × 10− 9 cm2 s− 1 independent of whether CNTF is bound or not. FCS and FCCS measurements detect the formation of receptor complexes containing at least two CNTFs and CNTFRs. In addition we measured Förster-type fluorescence resonance energy transfer between two differently labeled CNTFs within a receptor complex indicating a distance of 5–7 nm between the two. These findings are not consistent with a tetrameric structure of the CNTFR complex suggesting that either hexamers and or even higher-order structures (e.g. an octamer containing two tetramers) are formed.

Keywords:
CNTRF; fluorescence cross correlation spectroscopy; IL-6-type cytokine; receptor complex; stoichiometry; dimer

Materials & Methods:
HeLa ATCC cells were maintained in RPMI 1640/5–10% fetal calf 151 serum/1% penicillin/streptomycin. HeLa cells were transfected with 152 Nanofectin 153 ® (PAA Laboratories Pasching Austria) or Effectene (Qiagen Hilden Germany) for fluorescence correlation spectroscopy 154 (FCS) and fluorescence cross correlation spectroscopy (FCCS) mea- 155 surements according to the manufacturer's instructions. HeLa cells 156 were seeded in 30-mm glass bottom culture dishes (MatTek 157 Corporation Ashland USA) the day before transfection. The spectro- 158 scopic analyses were done 24 h after transfection. Collection of 159 mediumcontaining ACP-CNTF or analysis of expression of ACP-CNTFR 160 respectively was carried out 24–48 h after transfection. Parental 161 MDCK cells or cells expressing CNTFRwt were used as controls.

Microscopic Technique
Confocal Miscroscopy, Dual Color Excitation

Cell Type(s)
HeLa