The effect of hydrostatic pressure on lipid membrane lateral structure.

High pressure is both an environmental challenge to which deep sea biology has to adapt, and a highly sensitive thermodynamic tool that can be used to trigger structural changes in biological molecules and assemblies. Lipid membranes are amongst the most pressure sensitive biological assemblies and pressure can have a large influence on their structure and properties. In this chapter, we will explore the use of high pressure small angle X-ray diffraction and high pressure microscopy to measure and quantify changes in the lateral structure of lipid membranes under both equilibrium high pressure conditions and in response to pressure jumps.

Metabonomics study of the effects of single copy mutant KRAS in the presence or absence of WT allele using human HCT116 isogenic cell lines.

INTRODUCTION: KRAS was one of the earliest human oncogenes to be described and is one of the most commonly mutated genes in different human cancers, including colorectal cancer. Despite KRAS mutants being known driver mutations, KRAS has proved difficult to target therapeutically, necessitating a comprehensive understanding of the molecular mechanisms underlying KRAS-driven cellular transformation. OBJECTIVES: To investigate the metabolic signatures associated with single copy mutant KRAS in isogenic human colorectal cancer cells and to determine what metabolic pathways are affected. METHODS: Using NMR-based metabonomics, we compared wildtype (WT)-KRAS and mutant KRAS effects on cancer cell metabolism using metabolic profiling of the parental KRAS G13D/+ HCT116 cell line and its isogenic, derivative cell lines KRAS +/- and KRAS G13D/-. RESULTS: Mutation in the KRAS oncogene leads to a general metabolic remodelling to sustain growth and counter stress, including alterations in the metabolism of amino acids and enhanced glutathione biosynthesis. Additionally, we show that KRASG13D/+ and KRASG13D/- cells have a distinct metabolic profile characterized by dysregulation of TCA cycle, up-regulation of glycolysis and glutathione metabolism pathway as well as increased glutamine uptake and acetate utilization. CONCLUSIONS: Our study showed the effect of a single point mutation in one KRAS allele and KRAS allele loss in an isogenic genetic background, hence avoiding confounding genetic factors. Metabolic differences among different KRAS mutations might play a role in their different responses to anticancer treatments and hence could be exploited as novel metabolic vulnerabilities to develop more effective therapies against oncogenic KRAS.

Metabolic characterization of colorectal cancer cells harbouring different KRAS mutations in codon 12, 13, 61 and 146 using human SW48 isogenic cell lines.

INTRODUCTION: Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) mutations occur in approximately one-third of colorectal (CRC) tumours and have been associated with poor prognosis and resistance to some therapeutics. In addition to the well-documented pro-tumorigenic role of mutant Ras alleles, there is some evidence suggesting that not all KRAS mutations are equal and the position and type of amino acid substitutions regulate biochemical activity and transforming capacity of KRAS mutations. OBJECTIVES: To investigate the metabolic signatures associated with different KRAS mutations in codons 12, 13, 61 and 146 and to determine what metabolic pathways are affected by different KRAS mutations. METHODS: We applied an NMR-based metabonomics approach to compare the metabolic profiles of the intracellular extracts and the extracellular media from isogenic human SW48 CRC cell lines with different KRAS mutations in codons 12 (G12D, G12A, G12C, G12S, G12R, G12V), 13 (G13D), 61 (Q61H) and 146 (A146T) with their wild-type counterpart. We used false discovery rate (FDR)-corrected analysis of variance (ANOVA) to determine metabolites that were statistically significantly different in concentration between the different mutants. RESULTS: CRC cells carrying distinct KRAS mutations exhibited differential metabolic remodelling, including differences in glycolysis, glutamine utilization and in amino acid, nucleotide and hexosamine metabolism. CONCLUSIONS: Metabolic differences among different KRAS mutations might play a role in their different responses to anticancer treatments and hence could be exploited as novel metabolic vulnerabilities to develop more effective therapies against oncogenic KRAS.

Dual direction CRISPR transcriptional regulation screening uncovers gene networks driving drug resistance.

Pooled CRISPR-Cas9 knock out screens provide a valuable addition to the methods available for novel drug target identification and validation. However, where gene editing is targeted to amplified loci, the resulting multiple DNA cleavage events can be a cause of false positive hit identification. The generation of nuclease deficient versions of Cas9 has enabled the development of two additional techniques - CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) - that enable the repression or overexpression, respectively, of target genes. Here we report the first direct combination of all three approaches (CRISPRko, CRISPRi and CRISPRa) in the context of genome-wide screens to identify components that influence resistance and sensitivity to the BRAF inhibitor, vemurafenib. The pairing of both loss- and gain-of-function datasets reveals complex gene networks which control drug response and illustrates how such data can add substantial confidence to target identification and validation analyses.

n-Alcohol Length Governs Shift in Lo-Ld Mixing Temperatures in Synthetic and Cell-Derived Membranes.

A persistent challenge in membrane biophysics has been to quantitatively predict how membrane physical properties change upon addition of new amphiphiles (e.g., lipids, alcohols, peptides, or proteins) in order to assess whether the changes are large enough to plausibly result in biological ramifications. Because of their roles as general anesthetics, n-alcohols are perhaps the best-studied amphiphiles of this class. When n-alcohols are added to model and cell membranes, changes in membrane parameters tend to be modest. One striking exception is found in the large decrease in liquid-liquid miscibility transition temperatures (Tmix) observed when short-chain n-alcohols are incorporated into giant plasma membrane vesicles (GPMVs). Coexisting liquid-ordered and liquid-disordered phases are observed at temperatures below Tmix in GPMVs as well as in giant unilamellar vesicles (GUVs) composed of ternary mixtures of a lipid with a low melting temperature, a lipid with a high melting temperature, and cholesterol. Here, we find that when GUVs of canonical ternary mixtures are formed in aqueous solutions of short-chain n-alcohols (n ≤ 10), Tmix increases relative to GUVs in water. This shift is in the opposite direction from that reported for cell-derived GPMVs. The increase in Tmix is robust across GUVs of several types of lipids, ratios of lipids, types of short-chain n-alcohols, and concentrations of n-alcohols. However, as chain lengths of n-alcohols increase, nonmonotonic shifts in Tmix are observed. Alcohols with chain lengths of 10-14 carbons decrease Tmix in ternary GUVs of dioleoyl-PC/dipalmitoyl-PC/cholesterol, whereas 16 carbons increase Tmix again. Gray et al. observed a similar influence of the length of n-alcohols on the direction of the shift in Tmix. These results are consistent with a scenario in which the relative partitioning of n-alcohols between liquid-ordered and liquid-disordered phases evolves as the chain length of the n-alcohol increases.

Retroviral insertional mutagenesis implicates E3 ubiquitin ligase RNF168 in the control of cell proliferation and survival.

The E3 ubiquitin ligase RNF168 is a ring finger protein that has previously been identified to play an important regulatory role in the repair of double-strand DNA breaks.  In the present study, an unbiased forward genetics functional screen in mouse granulocyte/ macrophage progenitor cell line FDCP1 has identified E3 ubiquitin ligase RNF168 as a key regulator of cell survival and proliferation. Our data indicate that RNF168 is an important component of the mechanisms controlling cell fate, not only in human and mouse haematopoietic growth factor-dependent cells, but also in the human breast epithelial cell line MCF-7. These observations therefore suggest that RNF168 provides a connection to key pathways controlling cell fate, potentially through interaction with PML nuclear bodies and/or epigenetic control of gene expression. Our study is the first to demonstrate a critical role for RNF168 in the in the mechanisms regulating cell proliferation and survival, in addition to its well-established role in DNA repair.

Increasing the performance of pooled CRISPR-Cas9 drop-out screening.

Components of the type II CRISPR-Cas complex in bacteria have been used successfully in eukaryotic cells to facilitate rapid and accurate cell line engineering, animal model generation and functional genomic screens. Such developments are providing new opportunities for drug target identification and validation, particularly with the application of pooled genetic screening. As CRISPR-Cas is a relatively new genetic screening tool, it is important to assess its functionality in a number of different cell lines and to analyse potential improvements that might increase the sensitivity of a given screen. To examine critical aspects of screening quality, we constructed ultra-complex libraries containing sgRNA sequences targeting a collection of essential genes. We examined the performance of screening in both haploid and hypotriploid cell lines, using two alternative guide design algorithms and two tracrRNA variants in a time-resolved analysis. Our data indicate that a simple adaptation of the tracrRNA substantially improves the robustness of guide loss during a screen. This modification minimises the requirement for high numbers of sgRNAs targeting each gene, increasing hit scoring and creating a powerful new platform for successful screening.

Conditions that Stabilize Membrane Domains Also Antagonize n-Alcohol Anesthesia.

Diverse 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.

Separation of liquid domains in model membranes induced with high hydrostatic pressure.

We have imaged the formation of membrane microdomains immediately after their induction using a novel technology platform coupling high hydrostatic pressure to fluorescence microscopy. After formation, the ordered domains are small and highly dynamic. This will enhance links between model lipid assemblies and dynamic processes in cellular membranes.

Pressure-temperature phase behavior of mixtures of natural sphingomyelin and ceramide extracts.

Ceramides are a group of sphingolipids that act as highly important signaling molecules in a variety of cellular processes including differentiation and apoptosis. The predominant in vivo synthetic pathway for ceramide formation is via sphingomyelinase catalyzed hydrolysis of sphingomyelin. The biochemistry of this essential pathway has been studied in detail; however, there is currently a lack of information on the structural behavior of sphingomyelin- and ceramide-rich model membrane systems, which is essential for developing a bottom-up understanding of ceramide signaling and platform formation. We have studied the lyotropic phase behavior of sphingomyelin-ceramide mixtures in excess water as a function of temperature (30-70 °C) and pressure (1-200 MPa) by small- and wide-angle X-ray scattering. At low ceramide concentrations the mixtures form the ripple gel phase (P(ß)') below the gel transition temperature for sphingomyelin, and this observation has been confirmed by atomic force microscopy. Formation of the ripple gel phase can also be induced at higher temperatures via the application of hydrostatic pressure. At high ceramide concentration an inverse hexagonal phase (HII) is formed coexisting with a cubic phase.

Electrostatic swelling of bicontinuous cubic lipid phases.

Lipid bicontinuous cubic phases have attracted enormous interest as bio-compatible scaffolds for use in a wide range of applications including membrane protein crystallisation, drug delivery and biosensing. One of the major bottlenecks that has hindered exploitation of these structures is an inability to create targeted highly swollen bicontinuous cubic structures with large and tunable pore sizes. In contrast, cubic structures found in vivo have periodicities approaching the micron scale. We have been able to engineer and control highly swollen bicontinuous cubic phases of spacegroup Im3m containing only lipids by (a) increasing the bilayer stiffness by adding cholesterol and (b) inducing electrostatic repulsion across the water channels by addition of anionic lipids to monoolein. By controlling the composition of the ternary mixtures we have been able to achieve lattice parameters up to 470 Å, which is 5 times that observed in pure monoolein and nearly twice the size of any lipidic cubic phase reported previously. These lattice parameters significantly exceed the predicted maximum swelling for bicontinuous cubic lipid structures, which suggest that thermal fluctuations should destroy such phases for lattice parameters larger than 300 Å.