0000000000414127
AUTHOR
Philippe I. H. Bastiaens
Configurable low-cost plotter device for fabrication of multi-color sub-cellular scale microarrays.
We report on the construction and operation of a low-cost plotter for fabrication of microarrays for multiplexed single-cell analyses. The printing head consists of polymeric pyramidal pens mounted on a rotation stage installed on an aluminium frame. This construction enables printing of microarrays onto glass substrates mounted on a tilt stage, controlled by a Lab-View operated user interface. The plotter can be assembled by typical academic workshops from components of less than 15 000 Euro. The functionality of the instrument is demonstrated by printing DNA microarrays on the area of 0.5 squared centimeters using up to three different oligonucleotides. Typical feature sizes are 5 μm diam…
A Protein-Interaction Array Inside a Living Cell
Cell phenotype is determined by protein network states that are maintained by the dynamics of multiple protein interactions.1 Fluorescence microscopy approaches that measure protein interactions in individual cells, such as by Forster resonant energy transfer (FRET), are limited by the spectral separation of fluorophores and thus are most suitable to analyze a single protein interaction in a given cell. However, analysis of correlations between multiple protein interactions is required to uncover the interdependence of protein reactions in dynamic signal networks. Available protein-array technologies enable the parallel analysis of interacting proteins from cell extracts, however, they can …
Biochips for cell biology by combined dip-pen nanolithography and DNA-directed protein immobilization.
A general methodology for patterning of multiple protein ligands with lateral dimensions below those of single cells is described. It employs dip pen nanolithography (DPN) patterning of DNA oligonucleotides which are then used as capture strands for DNA-directed immobilization (DDI) of oligonucleotide-tagged proteins. This study reports the development and optimization of PEG-based liquid ink, used as carrier for the immobilization of alkylamino-labeled DNA oligomers on chemically activated glass surfaces. The resulting DNA arrays have typical spot sizes of 4-5 μm with a pitch of 12 μm micrometer. It is demonstrated that the arrays can be further functionalized with covalent DNA-streptavidi…
Activation of the p75 neurotrophin receptor through conformational rearrangement of disulphide-linked receptor dimers.
Ligand-mediated dimerization has emerged as a universal mechanism of growth factor receptor activation. Recent structural studies have shown that neurotrophins interact with dimers of the p75 neurotrophin receptor (p75NTR), but the actual mechanism of receptor activation has remained elusive. Here we show that p75NTR forms disulphide-linked dimers independently of neurotrophin binding through the highly conserved Cys257 in its transmembrane domain. Mutation of Cys257 abolished neurotrophin-dependent receptor activity but did not affect downstream signaling by the p75NTR/NgR/Lingo-1 complex in response to MAG, indicating the existence of distinct, ligand-specific activation mechanisms for p7…