410 research outputs found
Systems analysis of the spatial regulation of oncogenic Ras signalling
No full textRas (Rat sarcoma) isoforms are small GTP-binding proteins that play a major role in the signalling networks controlling cell growth and survival. The Kras isoform is of particular interest as in many severe kinds of cancer the presence of oncogenic Kras mutations is associated with a poor prognosis. Kras is associated with the plasma membrane due to its farnesyl moiety and a polybasic motif and functions as a signalling hub. If Kras gets lost from the plasma membrane due to spontaneous dissociation or endocytosis, it will equilibrate over the extensive endomembrane system inside the cell. With Kras no longer present at the plasma membrane, its activation and the following activation of subsequent pathways can no longer take place. However, to remain on the plasma membrane, Kras has to be constantly enriched there. This enrichment must be actively maintained in the cell by an energy-driven mechanism involving the solubilising factor PDEδ. Consequently, inhibition or down-modulation of PDEδ results in mislocalisation of Kras, making PDEδ an interesting target for anti-cancer drug development. In 2013 a small molecule, Deltarasin, was identified as a potent inhibitor of PDEδ causing a redistribution of Kras from the plasma membrane towards endomembranes when applied to cells. This work investigates whether small molecule PDEδ inhibitors such as Deltarasin affect (K) Ras localization in space and time. It demonstrates that PDEδ inhibition causes Ras relocalisation from the plasma membrane towards the endomembranes in different human cancer cell lines and in murine small intestine organoids, which express endogenous levels of oncogenic Kras. Nonetheless, it has been shown that Deltarasin has certain side effects, e.g. it becomes cytotoxic at higher concentrations. Hence, a new PDEδ inhibitor, Deltazinone 1, which is supposed to be less cytotoxic in comparison to Deltarasin, was synthesized. In order to determine whether it represents a viable alternative to Deltarasin, its ability to relocalise Kras in a panel of cancer cell lines was tested and it was indeed possible to mislocalise Kras with this inhibitor in Kras-dependent PancTuI cells. Deltazinone 1 and Deltarasin both had a demonstrable effect on cell growth/survival, respectively. In this way it appears that PDEδ constitutes a valid target for the pharmacological therapy of Kras-dependent tumours. This work demonstrates that two specific PDEδ inhibitors with completely different lead structures are capable of mislocalising Kras to endomembranes. In sum, it demonstrates that the availability of PDEδ is essential to ensure (K) Ras localization at the plasma membrane in Kras-dependent cancer cells and thus that the survival of those cells are ultimately dependent on PDEδ
A Protein-Interaction Array Inside a Living Cell
No full textProtein-interaction arrays were generated in living cells by the interaction of bait-presenting artificial receptor constructs (bait-PARCs) with micrometer-scaled antibody surface patterns (see figure). This method was applied to simultaneously monitor the interaction kinetics of a prey protein with two distinct bait proteins in individual living cells
Multiplexed Sub-Cellular Scale Microarrays from direct DNA Nanolithography
No full textThe multiplexed, high-throughput fabrication of microarrays is of vital importance for many applications in life sciences, including drug screening, medical diagnostics and cell biology. In single cell investigations, features smaller than 10 μm are needed for functional manipulation of sub-cellular structures. Several top-down methodologies like electron beam lithography and microcontact printing can be employed for indirect surface patterning at this scale, however those approaches often require clean rooms and multiplexing of several different biomolecules on the same surface is limited [1]. To overcome these obstacles, we combined Dip-pen nanolithography (DPN) and DNA-directed immobilization (DDI) of proteins to fabricate cell-compatible functionalized glass surfaces [2]. We optimized ink formulation for ssDNA printing and the produced arrays were then functionalized with epidermal growth factor (EGF) taking advantage of covalent ssDNA-streptavidin conjugates as adaptor molecules. The surface-immobilized EGF was used for recruiting EGFR in the plasma membrane of MCF7 cells. Via this bottom-up structuring approach, we were able to analyse multiple protein-protein interactions simultaneously in individual living cells [3].
To improve the efficiency of multiplexed surface patterning, we developed a prototype of a robust custom plotter based on 2D polymer-pen lithography (2D-PPL) [4]. This device enables rapid fabrication of microarrays at ambient conditions in a multiplexed direct-writing mode. The printing process was carried out by polymeric pyramidal pens onto which multiple (up to 36) ssDNA solutions can be loaded through a microfluidic inkwell device. Subsequent to optimization of ink viscosity and surface tension by glycerol and tween-20, DNA arrays were plotted and used for DDI of EGF-bearing ssDNA-streptavidin conjugates. The resulting microarrays covered areas of about 0.5 cm2, and were capable of recruiting and activating EGF receptors in sub-cellular regions within human MCF7 cells [4].
References
[1] G. Arrabito, B. Pignataro. 2012. Solution Processed Micro- and Nano- Bioarrays for Multiplexed Biosensing. Anal. Chem. 84:5450–5462.
[2] G. Arrabito et al. 2013. Biochips for Cell Biology by Combined Dip-Pen Nanolithography and DNA-Directed Protein Immobilization. Small. 9:4243-4249.
[3] S. Gandor et al. 2013. A Protein-Interaction Array Inside a Living Cell. Angew. Chem. Int. Ed. Engl. 52:4790–4794.
[4] G. Arrabito, et al. 2014. Low-cost Plotter Device for Sub-Cellular Scale Microarray Fabrication. Small. DOI: 10.1002/smll.201303390
Configurable Low-Cost Plotter Device for Fabrication of Multi-Color Sub-Cellular Scale Microarrays
No full textWe 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 diameter with a pitch of 15 μm, leading to densities of up to 10(4) – 10(5) spots/squared millimeters. The fabricated DNA microarrays were used to produce subcellular scale arrays of bioactive epidermal growth factor peptides by means of DNAdirected
immobilization. The suitability of these biochips for cell biological studies is
demonstrated by specifi c recruitment, concentration and activation of EGF receptors
within the plasma membrane of adherent living cells. Our work illustrates that the
presented plotter gives access to bio-functionalized arrays usable for fundamental
research in cell biology, such as the manipulation of signal pathways in living cells at
subcellular resolution
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