Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep integral membrane proteins (MPs) water soluble. In this review, we discuss their structure and solution behavior; the way they associate with MPs; and the structure, dynamics, and solution properties of the resulting complexes. All MPs tested to date form water-soluble complexes with APols, and their biochemical stability is in general greatly improved compared with MPs in detergent solutions. The functionality and ligand-binding properties of APol-trapped MPs are reviewed, and the mechanisms by which APols stabilize MPs are discussed. Applications of APols include MP folding and cell-free synthesis, structural studies by NMR, electron microscopy and X-ray diffraction, APol-mediated immobilization of MPs onto solid supports, proteomics, delivery of MPs to preexisting membranes, and vaccine formulation.

The lateral organization of a prototypical G protein-coupled receptor, the neurokinin-1 receptor (NK1R), was investigated in living cells by fluorescence resonance energy transfer (FRET) microscopy, taking advantage of the recently developed acyl carrier protein (ACP) labeling technique. The NK1R was expressed as fusion protein with ACP to which small fluorophores were then covalently bound. Our approach allowed the recording of FRET images of receptors on living cells with unprecedented high signal-to-noise ratios and a subsequent unequivocal quantification of the FRET data owing to (i) the free choice of optimal fluorophores, (ii) the labeling of NK1Rs exclusively on the cell surface, and (iii) the precise control of the donor-acceptor molar ratio. Our single-cell FRET measurements exclude the presence of constitutive or ligandinduced homodimers or oligomers of NK1Rs. The strong dependence of FRET on the receptor concentration further reveals that NK1Rs tend to concentrate in microdomains, which are found to constitute Ϸ1% of the cell membrane and to be sensitive to cholesterol depletion.ACP labeling ͉ G protein-coupled receptor (GPCR) oligomerization G protein-coupled receptors (GPCRs) were for a long time presumed to be distributed in the plasma membrane exclusively in a monomeric form (1, 2), but recent reports have unveiled a more complex behavior; in particular, dimeric structures have been found for several GPCRs using biochemical and biophysical methods (3-9). Dimerization can occur between receptors of the same subtype (homodimerization) or of different subtypes (heterodimerization). Some GPCRs remain dimeric all of the time, whereas others cycle between monomeric and dimeric states in a ligand-regulated process (7). Although GPCR homodimerization seems to be important for receptor ontology and trafficking, heterodimerization might result in altered ligand selectivity and distinctive coupling to signal transduction pathways, providing an additional possibility for the fine tuning of cellular signaling.In addition to dimerization, the lateral distribution of GPCRs in cell membranes has been extensively debated recently. Several reports based on biochemical (10), plasmon-resonance spectroscopy (11), single-molecule microscopy (12), and fluorescence recovery after photobleaching experiments (13) propose that GPCRs are localized in microdomains, but a clear demonstration of the existence and nature of such microdomains in living cells remains elusive, in particular because the interpretation of biochemical data can be rather equivocal (14-17). Compartmentalization in form of microdomains was proposed to explain the efficiency of signal transduction at the low physiological surface concentrations of the signaling partners by their enrichment inside specialized signaling platforms (10, 18).Bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) experiments have gained increasing interest to investigate these two central questions on GPCR signaling. (i) They can be p...

The perspectives offered by vertical arrays of nanowires for biosensing applications in living cells depend on the access of individual nanowires to the cell interior. Recent results on electrical access and molecular delivery suggest that direct access is not always obtained. Here, we present a generic approach to directly visualize the membrane conformation of living cells interfaced with nanowire arrays, with single nanowire resolution. The method combines confocal z-stack imaging with an optimized cell membrane labelling strategy which was applied to HEK293 cells interfaced with 2-11 μm long and 3-7 μm spaced nanowires with various surface coatings (bare, aminosilane-coated or polyethyleneimine-coated indium arsenide). We demonstrate that, for all commonly used nanowire lengths, spacings and surface coatings, nanowires generally remain enclosed in a membrane compartment, and are thereby not in direct contact with the cell interior.

Arrays of nanowires (NWs) are currently being established as vehicles for molecule delivery and electrical- and fluorescence-based platforms in the development of biosensors. It is conceivable that NW-based biosensors can be optimized through increased understanding of how the nanotopography influences the interfaced biological material. Using state-of-the-art homogenous NW arrays allow for a systematic investigation of how the broad range of NW densities used by the community influences cells. Here it is demonstrated that indium arsenide NW arrays provide a cell-promoting surface, which affects both cell division and focal adhesion up-regulation. Furthermore, a systematic variation in NW spacing affects both the detailed cell morphology and adhesion properties, where the latter can be predicted based on changes in free-energy states using the proposed theoretical model. As the NW density influences cellular parameters, such as cell size and adhesion tightness, it will be important to take NW density into consideration in the continued development of NW-based platforms for cellular applications, such as molecule delivery and electrical measurements.