1991 © 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com Localization and Dynamics of Glucocorticoid Receptor at the Plasma Membrane of Activated Mast Cells Emmanuel Oppong, Per Niklas Hedde, Sylwia Sekula-Neuner, Linxiao Yang, Falko Brinkmann, René M. Dörlich, Michael Hirtz, Harald Fuchs, Gerd Ulrich Nienhaus, and Andrew C. B. Cato* these actions by binding to a glucocorticoid receptor (GR) that is localized in the cytoplasm of target cells in complex with molecular chaperones. Upon hormone binding, the GR undergoes a conformation change, dissociates from the In addition to their actions in the cell nucleus, glucocorticoids exhibit rapid non- nuclear responses that are mechanistically not well understood. To explain these effects, the localization of a glucocorticoid receptor (GR) expressed in mast cells as a GFP fusion was analyzed after activation of the cells on allergenic lipid arrays. These arrays were produced on glass slides by dip-pen nanolithography (DPN) and total internal reflection (TIRF) microscopy was used to visualize the GR. A rapid glucocorticoid-independent and -dependent recruitment of the GR-GFP to the plasma cell membrane was observed following contact of the cells with the allergenic array. In addition, the mobility of the GR at the membrane was monitored by fluorescence recovery after photobleaching (FRAP) and shown to follow binding kinetics demonstrating interactions of the receptor with membrane-bound factors. Furthermore the recruitment of the GR to the cell membrane was shown to result in a glucocorticoid-mediated increase in Erk phosphorylation. This is evidenced by findings that destruction of the membrane composition of the mast cells by cholesterol depletion impairs the membrane localization of the GR and subsequent glucocorticoid-mediated enhancement of Erk phosphorylation. These results demonstrate a membrane localization and function of the GR in mast cell signaling. Nanolithography 1. Introduction Glucocorticoids modulate a large variety of physiological processes ranging from the control of intermediary metab- olism to the mediation of stress response. [1,2] They exert small 2014, 10, No. 10, 1991–1998 DOI: 10.1002/smll.201303677 Dr. E. Oppong, [+] Prof. G. U. Nienhaus, Prof. A. C. B. Cato Karlsruhe Institute of Technology Institute of Toxicology and Genetics 76344, Eggenstein-Leopoldshafen, Germany E-mail: Andrew.Cato@kit.edu Dr. S. Sekula-Neuner, F. Brinkmann, Dr. M. Hirtz, Prof. H. Fuchs Karlsruhe Institute of Technology Institute of Nanotechnology (INT) & Karlsruhe Nano Micro Facility (KNMF) Karlsruhe Institute of Technology (KIT) 76344, Eggenstein-Leopoldshafen, Germany F. Brinkmann, Prof. H. Fuchs Physical Institute and Center for Nanotechnology (CeNTech) University of Münster 48149, Münster, Germany Dr. P. N. Hedde, [+] L. Yang, R. M. Dörlich, Prof. G. U. Nienhaus Karlsruhe Institute of Technology Institute of Applied Physics and Centre for Functional Nanostructures 76128, Karlsruhe, Germany Prof. G. U. Nienhaus Department of Physics University of Illinois at Urbana-Champaign Urbana, IL 61801, USA [+] These authors contributed equally to this work.