High-Resolution Subunit Detection of Glutamate Receptor by Ultrasmall Gold Nanoparticles ALEXANDRE R. LOUKANOV 1* AND HRISTO GAGOV 2 1 Laboratory of Engineering Nanobiotechnology, Department of Engineering Geoecology, University of Mining and Geology ‘‘St. Ivan Rilski,’’ Stoyan Edrev Str., Sofia 1700, Bulgaria 2 Department of Animal and Human Physiology, Faculty of Biology, Sofia University ‘‘St. Kliment Ohridski,’’ 8 Dragan Tzankov Blvd., Sofia 1164, Bulgaria KEY WORDS glutamate D2 receptors; Purkinje cells; subunit co-localization; nanogold particles; dark-field STEM ABSTRACT In this study, we aimed to increase the sensitivity of protein labeling using 1.4 nm gold nanoparticles and glutamate d2 receptor (GluD2) from the postsynaptic membrane of the Pur- kinje cells. The very small marker size of the particles reduces the steric hindrance between anti- bodies leading to a higher labeling efficiency of more than one subunit per single receptor molecule. The nanoparticles are visible in 200 kV dark-field scanning transmission electron microscope on freeze-fractured carbon replica of nervous tissue after plasma cleaning treatment. The different elemental composition of nanoparticles as Au nanogold or CdS quantum dot can be distinguished by energy dispersive X-ray spectroscopy. This method ensures detection of an average of three sub- units per GluD2 and often labels all four of them with 1.4 nm Au nanoparticles. It is concluded that this high-resolution microscopic method is useful for exploring the quaternary structure of mem- brane proteins. Microsc. Res. Tech. 75:1159–1164, 2012. V V C 2012 Wiley Periodicals, Inc. INTRODUCTION Several approaches monitor the quaternary struc- ture of membrane proteins: atomic force microscopy, co-immunoprecipitation, various types of fluorescence and bioluminescence resonance energy transfer, neutron scattering experiments, covalent cross linking, co-processing, binding studies, pharmacological and functional complementation (Milligan and Bouvier, 2005; Vincente et al., 2006). Nevertheless, the tissue distribution of homo- and heteromeric proteins-like ion channels (Kerr et al., 2001; Plane et al., 2005) and iono- tropic receptors (Mayer and Armstrong, 2004; Torres et al., 1999) remains generally unclear. A controversy still surrounds the G protein-coupled receptor (GPCR) oligomerization as well (Bouvier, 2001; Chabre et al., 2009; Ferre ´ et al., 2009). Recently, we reported a new electron microscopy (EM) method for immunolabeling (Loukanov et al., 2010), where two approaches are per- formed to distinguish yet unattainable spatial resolu- tion: (i) for the first time as small as 1.4 nm colloidal gold nanoparticles were applied and observed and (ii) the scanning transmission electron microscope (STEM) equipped with an energy dispersive X-ray (EDX) detec- tor was used to distinguish equal in size labels as metal Au nanoparticle or semiconductor CdS quantum dot. Glutamate d2 receptor (GluD2) (Collingridgeet al., 2009) was referred to as an orphan receptor because of its unknown endogenous ligand(s). Recently, it has been found that Cbln1, a C1q tumor necrosis factor superfamily member, when secreted from cerebellar granule cells formed Cbln1-GluD2 complex, is neces- sary and sufficient to induce new synapses in vitro and in vivo (Matsuda et al., 2010). GluD2 have two main functions in Purkinje cells (PC) 2 to participate in the formation of synapses with parallel fibers (PF) and to regulate the long-term depression (LTD) type of synap- tic plasticity, responsible for motor learning (Kakegawa et al., 2009). The induction of LTD and motor learning require C-terminal PDZ-binding motive of GluD2, which further interacts with several proteins, includ- ing delphilin and a protein tyrosine phosphatase, which are the most probable candidates for LTD regu- lators (Kakegawa et al., 2008). The number and density of AMPA receptors at single postsynaptic site in rat PC were estimated by electro- physiological measurements. Single synapse stimula- tion by a two-photon uncaging of glutamate was used, and a nonstationary fluctuation analysis of ion currents was performed together with reconstruction of ultra thin sections for synapse size measurements (Tanaka et al., 2005). The number of counted 5 nm immunogold particles was almost equal to the number of functionally calculated AMPA receptors that suggested one label per AMPA channel. The same technique reveals that GluD2 are constantly and homogeneously distributed in postsy- naptic membranes of dendritic spines of PC where they form synapses with PF (Masugi-Tokita et al., 2007). The vast majority of GluD2 exist as homomeric receptors in vivo (Yuzaki, 2009), and therefore an improved immuno- staining method increase the binding efficiency up to four labeling nanoparticles per GluD2 molecule if anti- bodies bind all available subunits. In this study, single subunits of GluD2 receptor were detected by immunolabeling with 1.4 nm nanogold *Correspondence to: Alexandre R. Loukanov, Laboratory of Engineering Nano- biotechnology, Department of Engineering Geoecology, University of Mining and Geology ‘‘St. Ivan Rilski,’’ Stoyan Edrev Str., Sofia 1700, Bulgaria. E-mail: alexandre_loukanov@abv.bg Received 4 January 2012; accepted in revised form 26 February 2012 DOI 10.1002/jemt.22043 Published online 28 March 2012 in Wiley Online Library (wileyonlinelibrary.com). V V C 2012 WILEY PERIODICALS, INC. MICROSCOPY RESEARCH AND TECHNIQUE 75:1159–1164 (2012)