a b s t r a c tAdrenergic receptors are a key component of nanoscale multiprotein complexes that are responsible for controlling the beat rate in a mammalian heart. We demonstrate the ability of near-field scanning optical microscopy (NSOM) to visualize b 2 -adrenergic receptors (b 2 AR) fused to the GFP analogue Venus at the nanoscale on HEK293 cells. The expression of the b 2 AR-Venus fusion protein was tightly controlled using a tetracycline-induced promoter. Both the size and density of the observed nanoscale domains are dependent on the level of induction and thus the level of protein expression. At concentrations between 100 and 700 ng/ml of inducer doxycycline, the size of domains containing the b 2 AR-Venus fusion protein appears to remain roughly constant, but the number of domains per cell increase. At 700 ng/ml doxycycline the functional receptors are organized into domains with an average diameter of 150 nm with a density similar to that observed for the native protein on primary murine cells. By contrast, larger micron-sized domains of b 2 AR are observed in the membrane of the HEK293 cells that stably overexpress b 2 AR-GFP and b 2 AR-eYFP. We conclude that precise chemical control of gene expression is highly advantageous for the use b 2 AR-Venus fusion proteins as models for b 2 AR function. These observations are critical for designing future cell models and assays based on b 2 AR, since the receptor biology is consistent with a relatively low density of nanoscale receptor domains.Ó 2009 Elsevier Inc. All rights reserved. IntroductionThe beating rate in the mammalian heart is strongly influenced by the binding of catecholamines to b-adrenergic G-protein coupled receptors (bARs) in cardiac myocytes, initiating an adrenergic response [1][2][3]. The signaling cascade initiated by catecholamine binding requires the association of bARs into multiprotein complexes called signalosomes [4] that include the a, b and c G-protein subunits. Changes in the molecular composition of signalosomes are associated with receptor desensitization, sequestration of the receptor to subcellular membranous compartments and internalization, all of which strongly influence bAR function [5][6][7][8][9][10][11]. Signaling has also been shown to depend on differential interactions with scaffolding proteins in signaling complexes [12]. Recently we have used near field scanning optical microscopy (NSOM) to demonstrate that functional receptors are organized into multi-protein domains of $140 nm average diameter on murine neonatal and embryonic cardiac myocytes [13]. Colocalization experiments in these primary cells at the nanometer scale show that 15-20% of receptors are pre-associated in caveolae, an important component of signalosomes [13]. bAR signaling represents an important pharmacological target [14] and many assays have been developed to aid in the drug discovery process [15][16][17][18]. Herein we investigate the conditions required for mimicking the nanoscale distributions and local environment observed in primary cel...
A novel and versatile DNA packaging approach was developed by grafting DNA-binding oligopeptides onto a polymer scaffold to combinatively self-assemble with DNA into compact nanostructures.
DNA condensation in-vitro has been studied as a model system to reveal common principles underlying gene packaging in biology, and as the critical first step towards the development of non-viral gene delivery vectors. In this study, we use a bio-inspired approach, where small DNA-binding peptides are controllably clustered by an amphiphilic block copolymer scaffold, to reveal the effect of clustered peptide binding on the energetics, size, shape and physical properties of DNA condensation in-vitro. This provides insights into the general architectural effect of gene-binding proteins on DNA condensation process. Moreover, the versatility afforded by regulating the clustering density and composition of peptides may provide a novel design platform for gene delivery applications in the future.
We reveal the vital role of DNA topology and conformation in directing the combinative self-assembly and condensation pathway and morphology.
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