Actin-based motility: from molecules to movement

Extensive progress has been made recently in understanding the mechanism by which cells move and extend protrusions using site-directed polymerization of actin in response to signalling. Insights into the molecular mechanism of production of force and movement by actin polymerization have been provided by a crosstalk between several disciplines, including biochemistry, biomimetic approaches and computational studies. This review focuses on the biochemical properties of the proteins involved in actin-based motility and shows how these properties are used to generate models of force production, how the predictions of different theoretical models are tested using a biochemically controlled reconstituted motility assay and how the changes in motility resulting from changes to the concentrations of components of the assay can help understand diverse aspects of the motile behavior of living cells.     

MEK kinase activity is not necessary for Raf-1 function

Raf-1 protein kinase has been identified as an integral component of the Ras/Raf/MEK/ERK signalling pathway in mammals. Activation of Raf-1 is achieved by RAS:GTP binding and other events at the plasma membrane including tyrosine phosphorylation at residues 340/341. We have used gene targeting to generate a 'knockout' of the raf-1 gene in mice as well as a rafFF mutant version of endogenous Raf-1 with Y340FY341F mutations. Raf-1(-/-) mice die in embryogenesis and show vascular defects in the yolk sac and placenta as well as increased apoptosis of embryonic tissues. Cell proliferation is not affected. Raf-1 from cells derived from raf-1(FF/FF) mice has no detectable activity towards MEK in vitro, and yet raf-1(FF/FF) mice survive to adulthood, are fertile and have an apparently normal phenotype. In cells derived from both the raf-1(-/-) and raf-1(FF/FF) mice, ERK activation is normal. These results strongly argue that MEK kinase activity of Raf-1 is not essential for normal mouse development and that Raf-1 plays a key role in preventing apoptosis.     

A family of gamma-like calcium channel subunits

The gamma subunit was initially identified as an auxiliary subunit of the skeletal muscle calcium channel complex. Evidence for the existence of further gamma subunits arose following the characterization of a genetic defect that induces epileptic seizures in stargazer mice. We present here the first account of a family of at least five putative gamma subunits that are predominantly expressed in brain. The gamma-2 and gamma-4 subunits shift the steady-state inactivation curve to more hyperpolarized potentials upon coexpression with the P/Q type alpha(1A) subunit. The coexpression of the gamma-5 subunit accelerates the time course of current activation and inactivation of the alpha(1G) T-type calcium channel.     

Plasma and vessel wall lipoprotein lipase have different roles in atherosclerosis

Lipoprotein lipase (LPL) is a key enzyme in lipoprotein metabolism, and has been hypothesized to exert either pro- or anti-atherogenic effects, depending on its localization. Decreased plasma LPL activity is associated with the high triglyceride (TG);-low HDL phenotype that is often observed in patients with premature vascular disease. In contrast, in the vessel wall, decreased LPL may be associated with less lipoprotein retention due to many potential mechanisms and, therefore, decreased foam cell formation. To directly assess this hypothesis, we have distinguished between the effects of variations in plasma and/or vessel wall LPL on atherosclerosis susceptibility in apoE-deficient mice. Reduced LPL in both plasma and vessel wall (LPL(+/-)E(-/-)) was associated with increased TG and increased total cholesterol (TC) compared with LPL(+/+)E(-/-) sibs. However despite their dyslipidemia, LPL(+/-)E(-/-) mice had significantly reduced lesion areas compared to the LPL(+/+)E(-/-) mice. Thus, decreased vessel wall LPL was associated with decreased lesion formation even in the presence of reduced plasma LPL activity. In contrast, transgenic mice with increased plasma LPL but with no increase in LPL expression in macrophages, and thus the vessel wall, had decreased TG and TC and significantly decreased lesion areas compared with LPL(+/+)E(-/-) mice. This demonstrates that increased plasma LPL activity alone, in the absence of an increase in vessel wall LPL, is associated with reduced susceptibility to atherosclerosis.Taken together, these results provide in vivo evidence that the contribution of LPL to atherogenesis is significantly influenced by the balance between vessel wall protein (pro-atherogenic) and plasma activity (anti-atherogenic).     

Nitric oxide: a trigger for classic preconditioning?

To determine whether nitric oxide (NO) is involved in classic preconditioning (PC), the effect of NO donors as well as inhibition of the L-arginine-NO-cGMP pathway were evaluated on 1) the functional recovery during reperfusion of ischemic rat hearts and 2) cyclic nucleotides during both the PC protocol and sustained ischemia. Tissue cyclic nucleotides were manipulated with NO donors [S-nitroso-N-penicillamine (SNAP), sodium nitroprusside (SNP), or L-arginine] and inhibitors of nitric oxide synthase (N(omega)-nitro-L-arginine methyl ester or N-nitro-L-arginine) or guanylyl cyclase (1H-[1,2,4]oxadiazolol-[4,3-a]quinoxaline-1-one). Pharmacological elevation in tissue cGMP levels by SNAP or SNP before sustained ischemia elicited functional improvement during reperfusion comparable to that by PC. Administration of inhibitors before and during the PC protocol partially attenuated functional recovery, whereas they had no effect when given after the ischemic PC protocol and before sustained ischemia only, indicating a role for NO as a trigger but not as a mediator. Ischemic PC, SNAP, or SNP caused a significant increase in cGMP and a reduction in cAMP levels after 25 min of sustained ischemia that may contribute to the protection obtained. The results obtained suggest a role for NO (and cGMP) as a trigger in classic PC.     

The mutational burden of acral melanoma revealed by whole‐genome sequencing and comparative analysis

Acral melanoma is a subtype of melanoma with distinct epidemiological, clinical and mutational profiles. To define the genomic alterations in acral melanoma, we conducted whole‐genome sequencing and SNP array analysis of five metastatic tumours and their matched normal genomes. We identified the somatic mutations, copy number alterations and structural variants in these tumours and combined our data with published studies to identify recurrently mutated genes likely to be the drivers of acral melanomagenesis. We compared and contrasted the genomic landscapes of acral, mucosal, uveal and common cutaneous melanoma to reveal the distinctive mutational characteristics of each subtype.     

Rotor architecture in the yeast and bovine F1-c-ring complexes of F-ATP synthase

The F₁F_O-ATP synthase is a rotary molecular nanomotor. F₁ is a chemical motor driven by ATP hydrolysis while F_O is an electrical motor driven by the proton flow. The two stepping motors are mechanically coupled through a common rotary shaft. Up to now, the three available crystal structures of the F₁c₁₀ sub-complex of the yeast F₁F_O-ATP synthase were isomorphous and then named yF₁c₁₀(I). In this crystal form, significant interactions of the c₁₀-ring with the F₁-head of neighboring molecules affected the overall conformation of the F₁-c-ring complex. The symmetry axis of the F₁-head and the inertia axis of the c-ring were tilted near the interface between the F₁-central stalk and the c-ring rotor, resulting in an unbalanced machine. We have solved a new crystal form of the F₁c₁₀ complex, named yF₁c₁₀(II), inhibited by adenylyl-imidodiphosphate (AMP-PNP) and dicyclohexylcarbodiimide (DCCD), at 6.5 Å resolution in which the crystal packing has a weaker influence over the conformation of the F₁-c-ring complex. yF₁c₁₀(II) provides a model of a more efficient generator. yF₁c₁₀(II) and bovine bF₁c₈ structures share a common rotor architecture with the inertia center of the F₁-stator close to the rotor axis.     

Global indicators of biological invasion: species numbers, biodiversity impact and policy responses

Aim Invasive alien species (IAS) pose a significant threat to biodiversity. The Convention on Biological Diversity’s 2010 Biodiversity Target, and the associated indicator for IAS, has stimulated globally coordinated efforts to quantify patterns in the extent of biological invasion, its impact on biodiversity and policy responses. Here, we report on the outcome of indicators of alien invasion at a global scale. Location Global. Methods We developed four indicators in a pressure‐state‐response framework, i.e. number of documented IAS (pressure), trends in the impact of IAS on biodiversity (state) and trends in international agreements and national policy adoption relevant to reducing IAS threats to biodiversity (response). These measures were considered best suited to providing globally representative, standardized and sustainable indicators by 2010. Results We show that the number of documented IAS is a significant underestimate, because its value is negatively affected by country development status and positively by research effort and information availability. The Red List Index demonstrates that IAS pressure is driving declines in species diversity, with the overall impact apparently increasing. The policy response trend has nonetheless been positive for the last several decades, although only half of countries that are signatory to the Convention on Biological Diversity (CBD) have IAS‐relevant national legislation. Although IAS pressure has apparently driven the policy response, this has clearly not been sufficient and/or adequately implemented to reduce biodiversity impact. Main conclusions For this indicator of threat to biodiversity, the 2010 Biodiversity Target has thus not been achieved. The results nonetheless provide clear direction for bridging the current divide between information available on IAS and that needed for policy and management for the prevention and control of IAS. It further highlights the need for measures to ensure that policy is effectively implemented, such that it translates into reduced IAS pressure and impact on biodiversity beyond 2010.     

Studies on the primary sequence requirements for PKC-alpha, -beta 1 and -gamma peptide substrates.

The substrate specificity of purified PKC-alpha, -beta and -gamma has been investigated. A series of synthetic peptides based upon the sequence surrounding serine-7 in glycogen synthase were generated and used to determine the basic residue requirements of these PKC isotypes. While PKC-alpha and -beta are indistinguishable in their phosphorylation of these peptides, PKC-gamma shows a distinct specificity profile for these synthetic substrates.