Results
- Showing results for:
- Reset all filters
Search results
-
Journal articleRodgers U, Lanyon-Hogg T, Masumoto N, et al., 2016,
Characterization of hedgehog acyltransferase inhibitors identifies a small molecule probe for hedgehog signaling by cancer cells
, ACS Chemical Biology, Vol: 11, Pages: 3256-3262, ISSN: 1554-8937The Sonic Hedgehog (Shh) signaling pathway plays a critical role during embryonic development and cancer progression. N-terminal palmitoylation of Shh by Hedgehog acyltransferase (Hhat) is essential for efficient signaling, raising interest in Hhat as a novel drug target. A recently identified series of dihydrothienopyridines has been proposed to function via this mode of action; however, the lead compound in this series (RUSKI-43) was subsequently shown to possess cytotoxic activity unrelated to canonical Shh signaling. To identify a selective chemical probe for cellular studies, we profiled three RUSKI compounds in orthogonal cell-based assays. We found that RUSKI-43 exhibits off-target cytotoxicity, masking its effect on Hhat-dependent signaling, hence results obtained with this compound in cells should be treated with caution. In contrast, RUSKI-201 showed no off-target cytotoxicity, and quantitative whole-proteome palmitoylation profiling with a bioorthogonal alkyne-palmitate reporter demonstrated specific inhibition of Hhat in cells. RUSKI-201 is the first selective Hhat chemical probe in cells and should be used in future studies of Hhat catalytic function.
-
Journal articleWard JA, McLellan L, Stockley M, et al., 2016,
Quantitative Chemical Proteomic Profiling of Ubiquitin Specific Proteases in Intact Cancer Cells
, ACS Chemical Biology, Vol: 11, Pages: 3268-3272, ISSN: 1554-8937Deubiquitinating enzymes play an important role in a plethora of therapeutically relevant processes and are emerging as pioneering drug targets. Herein, we present a novel probe, Ubiquitin Specific Protease (USP) inhibitor, alongside an alkyne-tagged activity-based probe analogue. Activity-based proteome profiling identified 12 USPs, including USP4, USP16, and USP33, as inhibitor targets using submicromolar probe concentrations. This represents the first intact cell activity-based profiling of deubiquitinating enzymes. Further analysis demonstrated functional inhibition of USP33 and identified a synergistic relationship in combination with ATR inhibition, consistent with USP4 inhibition.
-
Journal articleChan CL, Bolognesi G, Bhandarkar A, et al., 2016,
DROPLAY: laser writing of functional patterns within biological microdroplet displays
, Lab on a Chip, Vol: 16, Pages: 4621-4627, ISSN: 1473-0197In this study, we introduce an optofluidic method for the rapid construction of large-area cell-sized droplet assemblieswith user-defined re-writable two-dimensional patterns of functional droplets. Light responsive water-in-oil dropletscapable of releasing fluorescent dye molecules upon exposure were generated and self-assembled into arrays in amicrofluidic device. This biological architecture was exploited by the scanning laser of a confocal microscope to ‘write’ userdefined patterns of differentiated (fluorescent) droplets in a network of originally undifferentiated (non-fluorescent)droplets. As a result, long lasting images were produced on a droplet fabric with droplets acting as pixels of a biologicalmonitor, which can be erased and re-written on-demand. Regio-specific light-induced droplet differentiation within a largepopulation of droplets provides a new paradigm for the rapid construction of bio-synthetic systems with potential as tissuemimics and biological display materials.
-
Journal articleDent MR, López-Duarte I, Dickson CJ, et al., 2016,
Imaging plasma membrane phase behaviour in live cells using a thiophene-based molecular rotor
, Chemical Communications, Vol: 52, Pages: 13269-13272, ISSN: 1364-548XMolecular rotors have emerged as versatile probes of microscopic viscosity in lipid bilayers, although it has proved difficult to find probes that stain both phases equally in phase-separated bilayers. Here, we investigate the use of a membrane-targeting viscosity-sensitive fluorophore based on a thiophene moiety with equal affinity for ordered and disordered lipid domains to probe ordering and viscosity within artificial lipid bilayers and live cell plasma membranes.
-
Journal articleFriddin MS, Bolognesi G, Elani Y, et al., 2016,
Optically assembled droplet interface bilayer (OptiDIB) networks from cell-sized microdroplets
, Soft Matter, Vol: 12, Pages: 7731-7734, ISSN: 1744-6848We report a new platform technology to systematically assemble droplet interface bilayer (DIB) networks in user-defined 3D architectures from cell-sized droplets using optical tweezers. Our OptiDIB platform is the first demonstration of optical trapping to precisely construct 3D DIB networks, paving the way for the development of a new generation of modular bio-systems.
-
Journal articleKuimova MK, Mika JT, Thompson AJ, et al., 2016,
Measuring the viscosity of the Escherichia coli plasma membrane using molecular rotors
, Biophysical Journal, Vol: 111, Pages: 1528-1540, ISSN: 1542-0086The viscosity is a highly important parameter within the cell membrane, affecting the diffusion ofsmall molecules and, hence, controlling the rates of intra-cellular reactions. There is significantinterest in the direct, quantitative assessment of membrane viscosity. Here we report the use offluorescence lifetime imaging microscopy (FLIM) of the molecular rotor BODIPY C10 in themembranes of live Escherichia coli (E. coli) bacteria to permit direct quantification of the viscosity.Using this approach we investigated the viscosity in live E. coli cells, spheroplasts and liposomesmade from E. coli membrane extracts. For live cells and spheroplasts the viscosity was measured atboth room temperature (23o C) and the E. coli growth temperature (37o C), while the membraneextract liposomes were studied over a range of measurement temperatures (5-40o C). At 37o C werecorded a membrane viscosity in live E. coli cells of 950 cP, which is considerably higher than thatpreviously observed in other live cell membranes (e.g., eukaryotic cells, membranes of Bacillusvegetative cells). Interestingly, this indicates that E. coli cells exhibit a high degree of lipid orderingwithin their liquid-phase plasma membranes.
-
Journal articleWarren SC, Kim Y, Stone JM, et al., 2016,
Adaptive multiphoton endomicroscopy through a dynamically deformed multicore optical fiber using proximal detection
, Optics Express, Vol: 24, Pages: 21474-21484, ISSN: 1094-4087This paper demonstrates multiphoton excited fluorescenceimaging through a polarisation maintaining multicore fiber (PM-MCF)while the fiber is dynamically deformed using all-proximal detection.Single-shot proximal measurement of the relative optical path lengths of allthe cores of the PM-MCF in double pass is achieved using a Mach-Zehnderinterferometer read out by a scientific CMOS camera operating at 416 Hz.A non-linear least squares fitting procedure is then employed to determinethe deformation-induced lateral shift of the excitation spot at the distal tip ofthe PM-MCF. An experimental validation of this approach is presented thatcompares the proximally measured deformation-induced lateral shift infocal spot position to an independent distally measured ground truth. Theproximal measurement of deformation-induced shift in focal spot position isapplied to correct for deformation-induced shifts in focal spot positionduring raster-scanning multiphoton excited fluorescence imaging.
-
Journal articleMessager L, Burns JR, Kim J, et al., 2016,
Biomimetic hybrid nanocontainers with selective permeability
, Angewandte Chemie International Edition, Vol: 55, Pages: 11106-11109, ISSN: 1521-3757Chemistry plays a crucial role in creating synthetic analogues of biomacromolecular structures. Of particular scientific and technological interest are biomimetic vesicles that are inspired by natural membrane compartments and organelles but avoid their drawbacks, such as membrane instability and limited control over cargo transport across the boundaries. In this study, completely synthetic vesicles were developed from stable polymeric walls and easy‐to‐engineer membrane DNA nanopores. The hybrid nanocontainers feature selective permeability and permit the transport of organic molecules of 1.5 nm size. Larger enzymes (ca. 5 nm) can be encapsulated and retained within the vesicles yet remain catalytically active. The hybrid structures constitute a new type of enzymatic nanoreactor. The high tunability of the polymeric vesicles and DNA pores will be key in tailoring the nanocontainers for applications in drug delivery, bioimaging, biocatalysis, and cell mimicry.
-
Conference paperWalker R, Madej B, Lin C, et al., 2016,
Adventures in the world of lipids: Towards the routine simulation of complex membranes and membrane bound proteins
, Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727 -
Journal articleMachta BB, Gray E, Nouri M, et al., 2016,
Conditions that Stabilize Membrane Domains Also Antagonize n-Alcohol Anesthesia
, Biophysical Journal, Vol: 11, Pages: 537-545, ISSN: 1542-0086Diverse molecules induce general anesthesia with potency strongly correlated with both their hydrophobicity and their effects on certain ion channels. We recently observed that several n -alcohol anesthetics inhibit heterogeneity in plasma-membrane-derived vesicles by lowering the critical temperature (Tc) for phase separation. Here, we exploit conditions that stabilize membrane heterogeneity to further test the correlation between the anesthetic potency of n -alcohols and effects on Tc. First, we show that hexadecanol acts oppositely to n -alcohol anesthetics on membrane mixing and antagonizes ethanol-induced anesthesia in a tadpole behavioral assay. Second, we show that two previously described “intoxication reversers” raise Tc and counter ethanol’s effects in vesicles, mimicking the findings of previous electrophysiological and behavioral measurements. Third, we find that elevated hydrostatic pressure, long known to reverse anesthesia, also raises Tc in vesicles with a magnitude that counters the effect of butanol at relevant concentrations and pressures. Taken together, these results demonstrate that ΔTc predicts anesthetic potency for n-alcohols better than hydrophobicity in a range of contexts, supporting a mechanistic role for membrane heterogeneity in general anesthesia.
This data is extracted from the Web of Science and reproduced under a licence from Thomson Reuters. You may not copy or re-distribute this data in whole or in part without the written consent of the Science business of Thomson Reuters.
