Plasma membrane stress induces relocalization of Slm proteins and activation of TORC2 to promote sphingolipid synthesis

The plasma membrane delimits the cell, and its integrity is essential for cell survival. Lipids and proteins form domains of distinct composition within the plasma membrane. How changes in plasma membrane composition are perceived, and how the abundance of lipids in the plasma membrane is regulated to balance changing needs remains largely unknown. Here, we show that the Slm1/2 paralogues and the target of rapamycin kinase complex 2 (TORC2) play a central role in this regulation. Membrane stress, induced by either inhibition of sphingolipid metabolism or by mechanically stretching the plasma membrane, redistributes Slm proteins between distinct plasma membrane domains. This increases Slm protein association with and activation of TORC2, which is restricted to the domain known as the membrane compartment containing TORC2 (MCT; ref.?). As TORC2 regulates sphingolipid metabolism, our discoveries reveal a homeostasis mechanism in which TORC2 responds to plasma membrane stress to mediate compensatory changes in cellular lipid synthesis and hence modulates the composition of the plasma membrane. The components of this pathway and their involvement in signalling after membrane stretch are evolutionarily conserved.     

The Synaptonemal Complex Protein ZYP1 Is Required for Imposition of Meiotic Crossovers in Barley

In many cereal crops, meiotic crossovers predominantly occur toward the ends of chromosomes and 30 to 50% of genes rarely recombine. This limits the exploitation of genetic variation by plant breeding. Previous reports demonstrate that chiasma frequency can be manipulated in plants by depletion of the synaptonemal complex protein ZIPPER1 (ZYP1) but conflict as to the direction of change, with fewer chiasmata reported in Arabidopsis thaliana and more crossovers reported for rice (Oryza sativa). Here, we use RNA interference (RNAi) to reduce the amount of ZYP1 in barley (Hordeum vulgare) to only 2 to 17% of normal zygotene levels. In the ZYP1^RNAi lines, fewer than half of the chromosome pairs formed bivalents at metaphase and many univalents were observed, leading to chromosome nondisjunction and semisterility. The number of chiasmata per cell was reduced from 14 in control plants to three to four in the ZYP1-depleted lines, although the localization of residual chiasmata was not affected. DNA double-strand break formation appeared normal, but the recombination pathway was defective at later stages. A meiotic time course revealed a 12-h delay in prophase I progression to the first labeled tetrads. Barley ZYP1 appears to function similarly to ZIP1/ZYP1 in yeast and Arabidopsis, with an opposite effect on crossover number to ZEP1 in rice, another member of the Poaceae.     

Synthesis of N,N'-disubstituted 3-aminobenzo[c] and [d]azepin-2-ones as potent and specific farnesyl transferase inhibitors

A structure-activity study was performed by synthesis on N,N'-disubstitution of 3-aminobenzo[c] and [d]azepin-2-one 2 and 3 to afford potent and specific farnesyl transferase inhibitors with low nM enzymatic and cellular activities.     

The metabolism of a stable N-hydroxymethyl derivative of a N-methylamide

N-Formylbenzamide and benzamide were characterised by high pressure liquid chromatography and mass spectrometry as products of the metabolism of N-hydroxymethylbenzamide in incubation mixtures with mouse liver preparations and isolated hepatocytes. This biotransformation occurred predominantly in 9000g and microsomal supernatant fractions and was also catalyzed by horse liver alcohol dehydrogenase fortified with NAD and could be inhibited by pyrazole. Unlike N-hydroxymethylbenzamide, which is very stable, N-formylbenzamide degraded rapidly to benzamide in buffer at pH 7.4 with a half-life of 7.8 min. The instability of N-formylbenzamide and the time course of its metabolic generation together with benzamide suggest that benzamide is a chemical breakdown product of N-formylbenzamide. N-Formylbenzamide was also tentatively identified as a urinary metabolite of N-hydroxymethylbenzamide. This is the first time that an N-hydroxymethyl compound has been shown to undergo metabolism either in vitro or in vivo.     

Cell cycle specific induction of HL-60 cell differentiation and apoptosis by mycophenolic acid

HL-60 cells undergo terminal differentiation and apoptosis in response to different types of sub-toxic and toxic perturbations respectively. The mechanism by which cells sense different amounts of perturbation to activate pathways that lead to the engagement of a relevant biological response is not known. The response of HL-60 cells to treatment with the immunosuppressant mycophenolic acid (MPA), a specific inhibitor of dGTP/GTP-synthesis, allowed quantitation of a metabolic perturbation which triggered a cellular response. 1.5 microM MPA induced 38% terminal differentiation to CD14 positive, early monocyte-like cells and 22% cell death by apoptosis, whereas 3 microM MPA induced 70% apoptosis but no differentiation. Despite the difference in biological outcomes, 72 h exposure to both 1.5 microM and 3 microM MPA caused a similar ( approximately 75%) depletion of total GTP levels. Cells synchronized by centrifugal elutriation were treated with MPA. Elutriated cells were overall less sensitive to the effects of MPA but 3 microM MPA induced significantly less apoptosis and more differentiation in an elutriation-enriched G1-population than in a population normally distributed in the cell cycle, suggesting that the effects of MPA in S-phase may subsequently lead to cell death. However, analysis of apoptosis by using a terminal deoxynucleotidyltransferase assay and measurement of bromodeoxyuridine incorporation showed that apoptosis was engaged in G1. Analysis of the phosphorylation status of the retinoblastoma protein demonstrated that Rb was hypophosphorylated prior to apoptosis and that in apoptotic cells, separated by flow cytometry, Rb protein was absent, presumably due to proteolysis. The loss of Rb protein did not appear to permit transit to S-phase, and was not accompanied by an expression of c-Myc. Surprisingly, therefore, an antimetabolite inducing a loss of GTP brought about cell death by apoptosis in the G1 phase of the cell cycle.