GPI-Anchor synthesis: Ras takes charge

A new study shows that Ras2 regulates GPI-anchor synthesis in the ER. Reciprocally, the targeted enzyme GPI-GlcNAc transferase regulates Ras2 signal output. This novel intersection of Ras2 signaling and an ER-localized protein complex has interesting implications for Ras function.

Three separable domains regulate GTP-dependent association of H-ras with the plasma membrane

The microlocalization of Ras proteins to different microdomains of the plasma membrane is critical for signaling specificity. Here we examine the complex membrane interactions of H-ras with a combination of FRAP on live cells to measure membrane affinity and electron microscopy of intact plasma membrane sheets to spatially map microdomains. We show that three separable forces operate on H-ras at the plasma membrane. The lipid anchor, comprising a processed CAAX motif and two palmitic acid residues, generates one attractive force that provides a high-affinity interaction with lipid rafts. The adjacent hypervariable linker domain provides a second attractive force but for nonraft plasma membrane microdomains. Operating against the attractive interaction of the lipid anchor for lipid rafts is a repulsive force generated by the N-terminal catalytic domain that increases when H-ras is GTP loaded. These observations lead directly to a novel mechanism that explains how H-ras lateral segregation is regulated by activation state: GTP loading decreases H-ras affinity for lipid rafts and allows the hypervariable linker domain to target to nonraft microdomains, the primary site of H-ras signaling.

Individual Palmitoyl Residues Serve Distinct Roles in H-ras Trafficking, Microlocalization, and Signaling

H-ras is anchored to the plasma membrane by two palmitoylated cysteine residues, Cys181 and Cys184, operating in concert with a C-terminal S-farnesyl cysteine carboxymethylester. Here we demonstrate that the two palmitates serve distinct biological roles. Monopalmitoylation of Cys181 is required and sufficient for efficient trafficking of H-ras to the plasma membrane, whereas monopallmitoylation of Cys184 does not permit efficient trafficking beyond the Golgi apparatus. However, once at the plasma membrane, monopalmitoylation of Cys184 supports correct GTP-regulated lateral segregation of H-ras between cbolesterol-dependent and cholesterol-independent microdomains. In contrast, monopallmitoylation of Cys181 dramatically reverses H-ras lateral segregation, driving GTP-loaded H-ras into cholesterol-dependent microdomains. Intriguingly, the Cys181 monopalmitoylated H-ras anchor emulates the GTP-regulated microdomain interactions of N-ras. These results identify N-ras as the Ras isoform that normally signals from lipid rafts but also reveal that spacing between palmitate and prenyl groups influences anchor interactions with the lipid bilayer. This concept is further supported by the different plasma membrane affinities of the monopalmitoylated anchors: Cys181-palmitate is equivalent to the dually palmitoylated wild-type anchor, whereas Cys184-pahnitate is weaker. Thus, membrane affinity of a pallmitoylated anchor is a function both of the hydrophobicity of the lipid moieties and their spatial organization. Finally we show that the plasma membrane affinity of monopahnitoylated anchors is absolutely dependent on cholesterol, identifying a new role for cholesterol in promoting interactions with the raft and nonraft plasma membrane.

Mechanistic modelling of pilling. Part I: Detailing of mechanisms

Mechanistic models of pilling are discussed in general terms, and a framework for pilling simulations is thereby created. A fundamental flaw in earlier models of pilling is revealed. A more comprehensive model of fibre diffusion and withdrawal from the fabric is proposed, and this is solved in general terms to find the rate of fuzz growth. Fuzz wear-off and entanglement into pills are discussed. Fibre fatigue is introduced, and it is demonstrated that this potentially increases the rate of withdrawal of anchor fibres.

Legislative and regulatory measures for preventing alcohol-related drownings and near-drownings

Objective: Alcohol contributes to about 30% of drowning fatalities associated with recreational aquatic activity and to 35% of drownings associated with boating. We consider regulatory and legislative strategies for preventing such deaths. Methods: We contacted water police in each Australian State and Territory to identify legislation creating alcohol-related offences for operators of recreational boats in their jurisdiction and to determine whether they conducted random breath testing (RBT). We also sought information from all 152 (81 urban and 71 rural) local government councils in NSW regarding restrictions on consumption of alcohol in public places within their shires. Results: Four Australian States (New South Wales, Queensland, Victoria and South Australia) have legislation prescribing maximum blood alcohol concentrations (BACs) for operators of recreational boats; all support this with RBT Western Australia, Tasmania and the Australian Capital Territory define more general offences for operating vessels while under the influence, of alcohol. Prohibitions or restrictions on consumption of alcohol in public places exist in 78 of the 86 shires in NSW that responded: 69 councils had alcohol-free zones, 53 restricted consumption of alcohol in public parks and reserves, and 33 had prohibitions or restrictions in some aquatic environments. Conclusions/implications: Legislation restricting BACs for recreational boat operators should be adopted in all Australian States and Territories. Optimal legislation would require that all occupants of recreational boats are required to comply with prescribed BAC levels, including when vessels are at anchor. Extension of by-laws prohibiting or restricting the consumption of alcohol specifically in aquatic environments warrants consideration.